Compounds and methods for inhibiting hepatitis C virus replication

Abstract

The inventors have discovered that an ATPase-deficient dominant-negative mutant NS3 protein of hepatitis C virus inhibits activity of the wild-type NS3 protein and inhibits replication of hepatitis C virus (HCV). The solved crystal structure of a multi-enzyme NS3 complex on a DNA substrate is also provided. The inventors have tested a peptide matching the sequence of a portion of NS3 that interacts with another NS3 molecule for inhibiting HCV replication. The peptide inhibits HCV replication. Accordingly, the invention provides a method of inhibiting HCV replication in cells infected with HCV involving transforming the cells with a vector expressing a dominant-negative mutant NS3 gene. The invention also provides a method of inhibiting HCV replication in cells infected with HCV involving administering to the cells a dominant-negative mutant NS3 protein. The invention also provides peptides and agents that inhibit HCV replication and methods of identifying agents that inhibit HCV replication.

Description

[0001] This patent application is a divisional application of U.S. patent application Ser. No. 11/119,587, filed May 1, 2005.

COMPACT DISC

[0003] This specification is accompanied by an original compact disc and one identical copy, the contents of which are incorporated by reference. The compact discs each contain the files 110-001US2_sequehce_listing.txt (96 kb sequence listing file) and Table-2-RTF.rtf (845 kb file of Table 2).

TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090215983A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

BACKGROUND

[0004] An estimated 3% of the world's population is seropositive for hepatitis C virus (HCV) (1, 2, 3). Approximately 70% of seropositive individuals develop a chronic infection. Infection with HCV predisposes victims to liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma (18). Most seropositive persons eventually develop hepatocellular carcinoma (4), and therefore HCV infection is also the leading cause of liver failure and the need for liver transplants in the U.S. (3,5).

[0005] HCV is a 9.6 kb positive strand RNA virus of the Flavirviradae family, genus Hepacivirus (6). The RNA comprises a 5' UTR (untranslated region) of approximately 340 nucleotides that includes an internal ribosome entry sequence (IRES), a single open reading frame (ORF) of approximately 9000 nucleotides and a 3' UTR of approximately 230 nucleotides. The internal ribosome entry sequence mediates initiation of viral RNA. The single open reading frame is translated into a polyprotein of approximately 3000 amino acid residues. This is cleaved by proteases to produce at least three structural proteins (core, E1, and E2) and six non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) (3).

[0006] The core protein forms a capsid, and E1 and E2 interact with plasma membranes of hepatocytes. NS2 is a zinc metaloprotease that cleaves the polyprotein at the NS2-NS3 junction between Leu1026 and Ala1027 (7). NS3 is a bifunctional enzyme, with its N terminus a serine protease that cleaves the rest of the polyprotein in conjunction with its cofactor, NS4a. The C terminus of NS3 is a helicase that is responsible for unwinding and separating putative double-stranded replication intermediates in the HCV life cycle (3,8). The roles of NS4b and NS5a have not been well defined, although it is postulated that NS5a may act as an interferon antagonist. NS5b is an RNA-dependent RNA polymerase that can copy the positive and negative strands of RNA.

[0007] Recently another ORF of HCV has been identified, which encodes protein F of unknown function (9).

[0008] NS3 is a helicase. Helicases are enzymes that unwind dsDNA and dsRNA in various biological processes, including replication, recombination, and repair. Helicases act by converting the chemical energy of ATP hydrolysis to the mechanical energy of unwinding. NS3 is a 67 kDa, 3'-to-5' RNA-DNA helicase, of the SFII superfamily, and is thought to unwind dsRNA and other secondary structures during HCV replication (11). The oligomeric state of NS3 has been a subject of debate, since it has been shown to be a monomer (12-14), dimer (15), and oligomer (16) in the literature.

[0009] The current treatments for HCV infection are alpha interferon (IFN-.alpha.) in combination with ribavirin or a polyethylene glycol-modified form of IFN-.alpha.. But sustained responses are only observed in about half of the treated patients, and effectiveness varies depending on the HCV genotype (Blight, K. J. et al. 2002. J. Virol. 76:13001). Thus, improved treatments for HCV infection are needed. Treatments for HCV infection would include methods of inhibiting HCV replication. Thus, compounds and methods for inhibiting HCV replication are needed. Methods of identifying compounds that inhibit HCV replication are also needed.

SUMMARY

[0010] The inventors have discovered that a mutant NS3 gene functions in a dominant-negative manner in inhibiting wild-type NS3 activity and inhibiting HCV replication. That is, the inventors have discovered that expressing an ATPase-deficient NS3 protein from a nucleic acid vector in a cell infected with wild-type HCV replicon partially or completely inhibits replication of the wild-type HCV replicon in the cell. Thus, the mutant NS3 acts in a dominant negative manner. This shows that NS3 protein is an oligomer, and incorporating mutant ATPase-deficient monomers of NS3 in the oligomer with wild-type NS3 monomers inhibits or inactivates the oligomeric enzyme complex. NS3 activity is necessary for replication of HCV, and the inventors have demonstrated that expressing a dominant-negative mutant NS3 gene in cells harboring HCV partially or completely inhibits HCV replication. These data also indicate that administering dominant-negative mutant NS3 protein to cells harboring HCV will inhibit replication of HCV.

[0011] The crystal structure of a complex containing a 16-nt DNA complexed with 3 molecules of NS3 helicase is also solved. The crystal structure shows that two NS3 molecules simultaneously bind the DNA and interact with each other. Domain 2 of one molecule and domain 3 of the other molecule interact. The residues in contact with each other include residues 545-553, 584-591, 435-453, 477-488, and 524-536 of NS3.

[0012] HCV replicons carrying mutations in NS3 in some of these interface residues were created. The replicons also carried a drug-resistance gene, and when these were transformed into Huh-7 liver cells and transformants were selected for growth in the presence of the drug, an NS3 .DELTA.543-545 deletion mutant and D543K/H545D/Q549A mutant both generated far fewer colonies. The colonies that did grow were much smaller than colonies of cells transformed with wild-type replicon. Replicon carrying an R587D/L588D/K589D/T591D NS3 mutant also supported fewer colonies than wild-type replicon. Despite the large biological effects produced by the NS3 proteins mutant in these residues, the D543K/H545D/Q549A and R587D/L588D/K589D/T591D mutant NS3 proteins had only modestly decreased ATPase and helicase activity in assay conditions measuring the activity of monomeric NS3. The decreases in activity were larger in assays depending on processivity of the NS3 enzyme on a single substrate molecule, which depends more on NS3-NS3 interactions.

[0013] These data show the importance of the 541-551 region of NS3 for interaction of NS3 monomers with each other and possibly biologically significant interactions with other proteins. A short peptide carrying the sequence of NS3 residues 541-551 coupled to a sequence that facilitates cell permeation was created. When Huh-7 cells carrying HCV replicon were exposed to this peptide, replication of the HCV was strongly inhibited.

[0014] Accordingly, one embodiment of the invention provides a method of inhibiting hepatitis C virus (HCV) replication in cells infected with HCV involving transforming the cells with a vector expressing a dominant-negative mutant NS3 gene, wherein the vector reduces replication of viral nucleic acid in the cells or spread of the virus to other cells.

[0015] One embodiment of the invention provides a method of inhibiting HCV replication in cells infected with HCV involving administering to the cells a dominant-negative mutant NS3 protein, wherein the protein reduces replication of viral nucleic acid in the cells or spread of the virus to other cells.

[0016] One embodiment of the invention provides a method of testing genetic therapy against hepatitis C virus involving: administering a vector expressing a dominant-negative mutant NS3 gene to a mammal infected with HCV; and monitoring replication of HCV in the mammal.

[0017] One embodiment of the invention provides a method of inhibiting hepatitis C virus (HCV) replication in cells infected with HCV involving: contacting the cells with an agent that inhibits NS3 enzyme activity by inhibiting NS3 oligomerization; wherein the agent reduces replication of viral nucleic acid in the cells or spread of virus to other cells.

[0018] One embodiment of the invention provides a peptide comprising at least 4 contiguous residues of HIDAHFLSQTK (SEQ ID NO:1, residues 541-551 of NS3); wherein the peptide has 100 or fewer amino acid residues; wherein the peptide inhibits hepatitis C virus replication, or inhibits NS3 enzyme activity by inhibiting NS3 oligomerization.

[0019] One embodiment of the invention provides a complex for inhibiting hepatitis C virus (HCV) replication containing: an inhibitory peptide comprising 4 or more contiguous residues of HIDAHFLSQTK (SEQ ID NO:1, residues 541-551 of NS3), complexed with a cell-entry vehicle; wherein the complex inhibits replication of HCV in mammalian cells.

[0020] One embodiment of the invention provides a compound of molecular weight 10,000 or less, wherein the compound interacts with NS3 to inhibit NS3 oligomerization and wherein the compound inhibits hepatitis C virus (HCV) replication.

[0021] Another embodiment of the invention provides a method of identifying a compound that inhibits hepatitis C virus (HCV) replication involving: (a) contacting a cell comprising an HCV replicon with a candidate compound; and (b) monitoring replication of the HCV replicon; wherein the candidate compound inhibits NS3 enzyme activity by inhibiting NS3 oligomerization.

[0022] Another embodiment of the invention provides a method of identifying a candidate compound to test for inhibiting HCV virus replication involving: (a) applying a 3-dimensional molecular modeling algorithm to spatial coordinates of a molecular interface of NS3; and (b) electronically screening stored spatial coordinates of a set of compounds against the spatial coordinates of the molecular interface of NS3 to identify at least one candidate compound that is expected to bind to the molecular interface of NS3.

[0023] Another embodiment of the invention provides a computer-assisted method for designing a candidate inhibitor compound for inhibiting hepatitis C virus (HCV) replication involving: (a) supplying to a computer modeling application a set of spatial coordinates of a molecular interface of NS3; (b) computationally building an agent represented by a set of structural coordinates; and (c) determining whether the agent is expected to bind to the molecular interface of NS3; wherein if the agent is expected to bind to the interface of NS3 it is a candidate inhibitor compound.

[0024] Another embodiment of the invention provides an isolated and purified viral vector comprising: a viral capsid; encasing viral nucleic acid comprising a dominant-negative NS3 gene operably linked to a promoter active in mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows a time course of luciferase activity in Huh-7 cells transfected with HCV-luciferase replicon.

[0026] FIG. 2 is a western blot showing detection of NS3 protein in cell lysates from cells transfected with HCV replicon (wtrep and mrep) and NS3-expressing plasmids (mNS3 and wtNS3).

[0027] FIG. 3A is a plot of luciferase activity in cells transformed with HCV-Luc and increasing concentrations of plasmid expressing mutant NS3.

[0028] FIG. 3B is a plot of luciferase activity in cells transformed with HCV-Luc and increasing concentrations of plasmid expressing wild-type NS3.

[0029] FIG. 4 is a plot of luciferase activity of cells transformed with ATPase-deficient mutant HCV-Luc (mtrep) and increasing concentrations of plasmid expressing wt-NS3 or control cells transformed with wt HCV-Luc (wtrep).

[0030] FIG. 5 shows plates of Huh-7 cells transformed with HCV S2204I replicon and varying concentrations of wild-type or mutant NS3 plasmid, and grown under G418 selection pressure and stained with crystal violet.

[0031] FIG. 6 is a plot showing luciferase activity of cells transfected with wt HCV-Luc replicon and increasing concentrations of mutant NS3 HCV-Luc replicon, or only the mutant replicon (mrep).

[0032] FIG. 7 is a plot of luciferase activity of Huh-7 cells transfected with mutant NS3 HCV-Luc replicon (mtrep) and increasing concentrations of wt HCV-Luc replicon.

[0033] FIG. 8. Growth of Huh-7 cells after transfection with wild type and mutant forms of the HCV replicon. Colony formation of Huh-7 cells was monitored over a period of two weeks following transfection by HCV replicon RNA. Colonies were stained with 0.1% crystal violet. A) S2204I RNA. B) no RNA. C) .DELTA.543-546 mutant RNA. D) D543K/H545D/Q549A mutant RNA. E) R587D/L588D/K589D/T591D mutant RNA.

[0034] FIG. 9. Western analysis of HCV-transfected Huh-7.5 cell lysates. 5.times.10.sup.5 cells from each lysate were loaded on a 10% polyacrylamide gel. The gel was blotted onto a PVDF membrane and the blot was incubated with rabbit anti-NS3. Primary antibody binding was detected by chemiluminescence with HRP-conjugated anti-rabbit IgG. The lane marked "REF" is purified NS3h. The lane marked "C" is the control transfection with no HCV RNA.

[0035] FIGS. 10A and B. Binding of mutant NS3h to fluorescein-labeled U.sub.20 RNA (FIG. 10A) or dT.sub.15 DNA (FIG. 10B). Nucleic acid binding was determined by measuring fluorescence polarization following incubation of protein and nucleic acid at 37.degree. C. Data were fit to a hyperbola using Kaleidagraph software. (A) NS3h wild type ( ) bound to RNA with a K.sub.D of 47.+-.5 nM and NS3h KDA (.smallcircle.) bound to RNA with a K.sub.D of 38.+-.4 mM. NS3h DDDD (.box-solid.) did not bind with high enough affinity to determine a binding constant under these conditions. (B) Binding to the dT.sub.15 by NS3h wild type ( ) resulted in a K.sub.D of 5.9.+-.1.4 nM whereas the NS3h KDA mutant (.smallcircle.) bound with a K.sub.D of 2.6.+-.0.8 nM.

[0036] FIG. 11. ATPase activity of mutant NS3h was measured as a function of NADH concentration in a coupled assay at varying concentrations of polyU. Data were fit to a hyperbola using Kaleidagraph software. Specific activity of NS3h wild type ( ) and NS3h KDA (.smallcircle.) were comparable at 126 s.sup.-1 and 108 s.sup.-1, respectively, in the presence of saturating polyU. Specific activity of NS3h DDDD (.box-solid.) was lower.

[0037] FIGS. 12A-C. Steady state unwinding activity of mutant versus wild type NS3h. A) The assay for measuring helicase unwinding activity is depicted. A partially duplexed substrate containing 30 bp and 15 nt of ss overhang (45:30 mer) was incubated with NS3h in the presence of ATP and Mg.sup.+2 leading to unwinding of the duplex. B) Comparison of unwinding of DNA and RNA substrates by NS3h. Otherwise identical 250 nM duplexed DNA and RNA substrates were incubated with 100 nM NS3h. Reactions were initiated by addition of 5 mM ATP and 10 mM MgCl.sub.2 and quenched by addition of 200 mM EDTA/0.7% SDS. C) Unwinding of 250 nM substrate under steady state conditions. Unwinding by 100 nM NS3h ( ), NS3h KDA (.smallcircle.), and NS3h DDDD (.box-solid.) occurred at rates of 5.3 nM min.sup.-1, 5.7 nM min.sup.-1, and 4.1 nM min.sup.-1, respectively.

[0038] FIG. 13. Graph showing ATP-independent unwinding activity of NS3h wild-type, NS3h KDA, and NS3h DDDD. NS3h, 500 nM, was incubated with a partial duplex DNA substrate containing 15 nt of single stranded DNA and 30 base pairs at 37.degree. C. Aliquots were quenched by addition of 100 .mu.M poly dT and 60 nM of a 30mer oligonucleotide that served to prevent reannealing. ssDNA was separated from dsDNA by native polyacrylamide electrophoresis and the resulting fractions were quantified by using IMAGEQUANT software. DNA melting is shown for NS3h ( ), NS3h KDA (.diamond-solid.), and NS3h DDDD (.box-solid.).

[0039] FIG. 14. Single turnover DNA unwinding by NS3h and NS3h mutant enzymes. A two-step mixing technique was used to measure single-turnover unwinding in the presence of excess NS3h. NS3h (500 nM) or mutant enzyme was rapidly mixed with substrate followed by a 10-second incubation. A second, rapid mixing step followed in which ATP, Mg.sup.+2, and protein trap (poly dT) was added. Data were fit according to equation 1 resulting in unwinding rates of 3.1.+-.0.1 s.sup.-1 and 3.7.+-.0.1 s.sup.-1 for NS3h ( ) and NS3h KDA (.diamond-solid.), respectively. The amplitudes for unwinding were 0.35.+-.0.01 nM and 0.21.+-.0.01 nM for NS3h and NS3h KDA, respectively.

[0040] FIG. 15 shows the effect of the HCV inhibitor peptide on replication of the HCV-Luc replicon. HCV inhibitor peptide was added to Huh-7 cells containing the HCV luciferase replicon. Luciferase activity was measured after 48 hours. The bar labeled C is the control containing HCV luciferase replicon with no peptide.

DETAILED DESCRIPTION

Definitions

[0041] The term "inhibiting" hepatitis C virus replication includes partial and complete inhibition of the replication.

[0042] The term "replication" of HCV refers to replication of copies of the virus or viral nucleic acid within a cell and/or spread of the virus or viral nucleic acid to other cells.

[0043] The term "hepatitis C virus" includes a wild type, mutant, or engineered hepatitis C virus (e.g., hepatitis C replicons, such as reported in references 17 and 18). Hepatitis C virus comprises a single-stranded RNA molecule, optionally encased in a capsid. If the virus is an engineered, truncated, or mutant form of the virus, the viral RNA is a substantial portion of the full-length viral RNA (e.g., at least 30%, preferably at least 50%, more preferably at least 70%, 80%, or 90% of the full-length viral RNA) and has in that portion at least 90%, more preferably at least 95%, most preferably at least 98% sequence identity with the wild-type viral RNA sequence (SEQ ID NO:6, genbank accession number AJ238799). Sequence identity is calculated using the default BLAST parameters for nucleotide sequence comparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

[0044] "Cells infected with HCV" refers to cells harboring HCV nucleic acid. "HCV nucleic acid" refers to viral RNA or to DNA encoding and capable of being transcribed into viral RNA. The infected cells may be transformed with viral RNA either as naked RNA or encased in the capsid, or the cells may be transformed by DNA (e.g. a plasmid) encoding and capable of being transcribed into viral RNA.

[0045] A vector that is a "virus" refers to a viral nucleic acid encased in a capsid.

[0046] The term "NS3 gene" refers to any nucleic acid, whether cDNA, viral RNA, or other source, that encodes an NS3 protein.

[0047] "Wild-type NS3 gene" refers to a gene that encodes the NS3 protein having SEQ ID NO:3, or another natural source homologous NS3 protein from a hepatitis C virus found in nature. One wild-type NS3 gene is nucleotides 3079-4971 of SEQ ID NO:6.

[0048] A "dominant-negative mutant NS3 gene" is an NS3 gene that when expressed in cells harboring and expressing a wild-type NS3 gene reduces the activity of the wild-type NS3 protein. The mutant NS3 gene can express a truncated, full-length, or extended NS3 protein. At least a portion of the mutant NS3 protein is homologous to wild-type NS3 protein.

[0049] A mutant NS3 protein is "ATPase deficient" if it has ATPase activity that is statistically significantly lower than the activity of the wild-type NS3 protein. Activity can be assayed by any standard method, such as the spectrophotometric coupled ATPase assay (23). Lower activity includes a lower k.sub.cat, a higher K.sub.M, or a combination of both. In particular embodiments, the ATPase-deficient mutant NS3 has less than 75%, less than 50%, less than 10%, less than 5%, or less than 1% of the ATPase activity of the wild-type NS3.

[0050] The term "vector" as used herein refers to any nucleic acid capable of transforming target cells and expressing an inserted NS3 gene. The vector may be autonomously replicating or not, double-stranded or single-stranded, and encased in viral capsid or not. Vectors include viruses comprising capsid and nucleic acid, viral nucleic acid without capsid, DNA plasmids, linear DNA molecules, and linear or circular RNA molecules.

[0051] The term "monitoring replication of HCV" includes monitoring direct effects of HCV replication, such as health effects, e.g., development of hepatocellular carcinoma.

[0052] The term "peptide" refers to a peptide of 2 to 100 amino acid residues that, if derived from a naturally occurring protein, is shorter than the naturally occurring protein. A "peptide" as used herein may include amino acids that are L stereoisomers (the naturally occurring form) or D stereoisomers. Peptides may be linear, branched, or circular. Peptides may include amino acids other than the 20 common naturally occurring amino acids, such as .beta.-alanine, ornithine, or methionine sulfoxide. The term "peptide" also includes peptides modified on one or more alpha-amino, alpha-carboxyl, or side-chain, e.g., by appendage of a methyl, formyl, acetyl, glycosyl, phosphoryl, and the like.

[0053] The term "transforming" refers to any method that results in nucleic acid being taken up into a cell. This includes, for instance, CaCl.sub.2-mediated uptake of plasmid DNA, cellular uptake of naked viral RNA, or transfection of a cell with a virus.

[0054] The term "molecular interface" of NS3 refers to a surface of NS3 exposed to solvent or otherwise available to bind with an agent.

[0055] The term "complexed" in the context of a vector "complexed" with an agent for targeting to the liver includes covalent coupling of vector to the agent and a non-covalent interaction between the vector and the agent that is sufficiently stable to facilitate targeting to the liver.

[0056] A candidate compound for inhibiting HCV replication is "expected to bind" to a molecular interface of NS3 if a free energy calculation or computerized molecular modeling application, such as is provided by the program DOCK-5, calculates based on the docking of spatial coordinates of the compound with spatial coordinates of the molecular interface that the compound will bind to the molecular interface, or that the compound has an approximately equal or greater binding affinity than a known inhibitor of NS3 oligomerization, such as peptide SEQ ID NO:1 or SEQ ID NO:2.

DESCRIPTION

[0057] One embodiment of the invention provides a method of inhibiting HCV replication in cells infected with HCV involving transforming cells with a vector expressing a dominant-negative mutant NS3 gene. In a particular embodiment of the invention, the dominant-negative mutant NS3 gene expresses an ATPase-deficient NS3 protein. For instance, an example of an ATPase-deficient NS3 protein is D290A NS3, a mutant NS3 protein in which aspartic acid residue 290 is changed to alanine. (The amino acid numbering in this case refers to the SEQ ID NO:3 NS3 protein with genbank accession number CAB4667, not to the polyprotein.)

[0058] In a particular embodiment, the dominant-negative mutant NS3 gene expresses a helicase-deficient NS3 protein. Helicase activity can be assayed, for instance, by the unwinding assay of reference 21.

[0059] In one embodiment, the dominant-negative mutant NS3 gene expresses a protease-deficient NS3 protein. Protease activity can be assayed, for instance, as described in reference 34.

[0060] In one embodiment of the invention, the mutant NS3 protein amino acid sequence is at least 90% identical to wild-type NS3. In one embodiment, the mutant NS3 protein amino acid sequence is at least 90% identical to wild-type NS3 and contains the D290A mutation. Sequence identity can be calculated using the default BLAST parameters for protein sequence comparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

[0061] In one embodiment of the invention, the infected cells are liver cells.

[0062] In one embodiment, the liver cells are Huh-7 cells.

[0063] In one embodiment, the infected cells are in vitro. In one embodiment, the infected cells are in vivo in a mammal. In particular embodiments, the mammal is a mouse, rat, rabbit, goat, guinea pig, dog, pig, cat, or chimpanzee. The mouse and chimpanzee are particularly preferred model animals. In a particular embodiment, the mammal is a human.

[0064] The vector expressing the dominant-negative mutant NS3 gene can be any appropriate vector. For instance, it can be a plasmid, virus, or viral nucleic acid. It can be naked nucleic acid, e.g., a nucleic acid with or without an origin of replication. The vector, e.g., a naked nucleic acid, can be a transposon or include a transposon.

[0065] Where the vector is a virus or viral nucleic acid, the virus can be a retrovirus, e.g., a murine leukemia virus.

[0066] The vector can be targeted to the liver. One mechanism of doing this is to complex the vector with an agent for targeting to the liver, such as asialoorosomucoid. Methods of coupling vectors to asialoorosomucoid are reviewed in reference 31.

[0067] A method of coupling a vector to asialoorosomucoid and to adenovirus particles is disclosed in reference 30. The adenovirus particles enhance expression by efficiently lysing the endosomes following receptor-mediated endocytosis.

[0068] Preferably, the NS3 protein is expressed from the vector as a separate protein (i.e., not part of a polyprotein).

[0069] In one embodiment, the NS3 gene integrates into chromosomal DNA in the transformed cells.

[0070] In one embodiment, the vector is complexed with an endosomolytic peptide. This improves transformation efficiency by lysing the endosomes containing the vector following endocytosis of the vector. (See reference 30.)

[0071] The invention also provides a method of inhibiting HCV replication in cells infected with HCV involving administering to the cells a dominant-negative mutant NS3 protein.

[0072] In a particular embodiment of the method involving protein administration, the infected cells are liver cells (e.g., Huh-7 cells).

[0073] The infected cells can be in vitro or in vivo in a mammal. The mammal can be, for instance, a mouse, rat, rabbit, goat, guinea pig, dog, pig, cat, chimpanzee, or human. In specific preferred embodiments, the mammal is a mouse or chimpanzee. In another preferred embodiment, the mammal is a human.

[0074] The protein can be complexed with an agent for targeting to the liver, such as asialoorosomucoid.

[0075] A nucleic acid vector or mutant NS3 protein can also be targeted to the liver by surgical techniques, including intraportal injection, intra-vena cava injection, intra-bile duct injection and including tail vein injection in the mouse or rat. These methods are disclosed in reference 33.

[0076] Another embodiment of the invention provides a method of testing genetic therapy against HCV involving: administering a vector expressing a dominant-negative mutant NS3 gene to a mammal infected with HCV; and monitoring replication of HCV in the mammal. The monitoring could be by monitoring direct or indirect evidence of HCV replication. For instance, HCV nucleic acid replication in infected cells could be monitored, the number of infected cells could be monitored, or effects of HCV replication, such as development of hepatic carcinoma, could be monitored.

[0077] One embodiment of the invention provides a method of inhibiting hepatitis C virus (HCV) replication in cells infected with HCV involving: contacting the cells with an agent that inhibits NS3 enzyme activity by inhibiting NS3 oligomerization; wherein the agent reduces replication of viral nucleic acid in the cells or spread of virus to other cells.

[0078] In a particular embodiment of the method of inhibiting HCV replication, the agent includes a peptide comprising the sequence HIDAHFLSQTK (SEQ ID NO:1).

[0079] In a particular embodiment, the agent is a peptide having the sequence HIDAHFLSQTKGGGYARAAARQARA (SEQ ID NO:2).

[0080] In some embodiments, the agent comprises a peptide comprising the reverse D analog of SEQ ID NO:1. This is the peptide having the reverse sequence of SEQ ID NO: 1 (or a portion thereof, e.g., at least 4 contiguous residues), where the amino acids are D isomers instead of L isomers.

[0081] In another embodiment, the agent comprises an ATPase-deficient mutant NS3 protein.

[0082] The cells infected with HCV and contacted with the agent can be in vitro or in vivo in a mammal.

[0083] Another embodiment of the invention provides a complex for inhibiting hepatitis C virus (HCV) replication that includes: (a) an inhibitory peptide comprising 4 or more contiguous residues of SEQ ID NO:1; complexed with (b) a cell-entry vehicle; wherein the complex inhibits replication of HCV in mammalian cells.

[0084] The inhibitory peptide in other embodiments, contains 5, 6, 7, 8, 9, 10, or all 11 contiguous residues of SEQ ID NO: 1.

[0085] In particular embodiments, the cell-entry vehicle is a cell-entry peptide, such as YARAAARQARA (SEQ ID NO:4), or an oligoarginine peptide (66, 67).

[0086] In a particular embodiment, the cell-entry vehicle is cholesterol. The cholesterol may be covalently attached to the inhibitor peptide, or may be non-covalently complexed with the inhibitor peptide.

[0087] In other particular embodiments, the cell-entry vehicle is a liposome.

[0088] In a particular embodiment, the complex comprises a liver-targeting entity. In a particular embodiment, the cell-entry vehicle is also a liver-targeting entity.

[0089] The liver-targeting entity may be, for instance, asialoorosomucoid.

[0090] Another embodiment of the invention provides a peptide comprising at least 4 contiguous residues of SEQ ID NO:1; wherein the peptide has 100 or fewer amino acid residues; wherein the peptide inhibits hepatitis C virus replication, or inhibits NS3 enzyme activity by inhibiting NS3 oligomerization.

[0091] Inhibiting NS3 enzyme activity by inhibiting NS3 oligomerization can be demonstrated by greater inhibition of the NS3 enzyme activities that depend more on NS3 oligomerization (NS3-NS3 contacts) than of the enzyme activities that are carried out more equally efficiently by NS3 monomers and NS3 oligomers. For instance, steady-state unwinding with an excess of double-stranded DNA substrate over NS3 enzyme reflects monomer activity because it is unlikely two molecules of NS3 bind to the same substrate molecule in the presence of a large excess of DNA. An assay is described in Example 3 with the results presented in FIG. 12. In contrast, ATP-independent unwinding under-single-turnover conditions with excess enzyme is more dependent on NS3-NS3 interactions. An assay under these conditions is described in Example 3 and the results are shown in FIG. 13. Thus, if an agent inhibits NS3 activity by inhibiting NS3 oligomerization, it will inhibit ATP-independent DNA unwinding under single-turnover conditions with excess enzyme more efficiently than it will inhibit steady-state unwinding with an excess of DNA substrate.

[0092] The ATPase activity of NS3 is also dependent on NS3 concentration. It increases with increasing NS3 concentration, indicating that the enzyme activity is dependent on NS3 oligomerization (16). An agent that inhibits NS3 activity by inhibiting NS3 oligomerization will have a lower K.sub.1 when assayed with low NS3 concentration than when assayed with a higher NS3 concentration.

[0093] In particular embodiments of the invention, the inhibitory peptide has 50 or fewer, or 30 or fewer, amino acid residues.

[0094] One embodiment of the invention provides a compound of molecular weight 10,000 or less, wherein the compound interacts with NS3 to inhibit NS3 oligomerization and wherein the compound inhibits hepatitis C virus (HCV) replication.

[0095] In particular embodiments, the structure of the compound fits a molecular interface of NS3 such that a free energy calculation (or molecular docking computer program) predicts the compound is expected to bind to the molecular interface of NS3.

[0096] That is, the compound has a structure that can be represented by spatial coordinates; wherein the spatial coordinates of the compound fit spatial coordinates of an interface of NS3 such that a free energy calculation predicts the compound binds to the interface of NS3.

[0097] In some embodiments, the molecular interface of NS3 which the compound fits includes at least one amino acid residue (preferably three or more residues) selected from residues 541-553, 584-591, 435-453, 477-488, and 524-536 of SEQ ID NO:3. A peptide comprising SEQ ID NO:1, residues 541-551 of NH3, was found to inhibit HCV replication. Residues 541-551 of chain B interact with a cleft formed by residues 477-481 and 452-453 of chain A in the crystal structure. Thus, in a particular embodiment, the interface of NS3 that the compound fits includes at least one (preferably all) of residues 477-481 and 452-453.

[0098] In particular embodiments, the compound has a molecular weight of 5,000 or less, 2,500 or less, or 1,000 or less.

[0099] One embodiment of the invention provides an isolated and purified viral vector comprising: a viral capsid; encasing viral nucleic acid comprising a dominant-negative NS3 gene operably linked to a promoter active in mammalian cells. The viral nucleic acid refers to nucleic acid of which more than 50% originates from a virus. The viral nucleic acid is typically recombinant. The viral nucleic acid can include heterologous segments from non-viral sources. In some embodiments, the viral nucleic acid may include the genes necessary for replication and spread of the virus to other cells. In some embodiments it may not include those genes.

[0100] In a particular embodiment, the viral nucleic acid is recombinant HCV nucleic acid.

[0101] In a particular embodiment, the viral capsid and viral nucleic acid are hepatitis C virus capsid and nucleic acid.

[0102] In a particular embodiment, the viral capsid and viral nucleic acid are not hepatitis C virus capsid and nucleic acid.

[0103] In a particular embodiment, the viral capsid and viral nucleic acid are adenovirus capsid and nucleic acid.

[0104] In a particular embodiment, the viral capsid and nucleic acid are adeno-associated virus capsid and nucleic acid or retroviral capsid and nucleic acid.

[0105] In a particular embodiment, the promoter is cauliflower mosaic virus promoter.

Computer-Assisted Methods of Identifying HCV Inhibitors

[0106] One subject of this invention is a computer-assisted method for identifying a potential inhibitor of NS3 oligomerization and thereby HCV replication. The method comprises providing a computer modeling application with a set of relative structural coordinates of NS3, or a molecular interface thereof; supplying the computer modeling application with a set of structural coordinates of a candidate inhibitor of NS3 oligomerization; comparing the two sets of coordinates and determining whether the candidate inhibitor is expected to bind to NS3 or to interfere with NS3 oligomerization. Binding to NS3, particularly on an interface involved in NS3 oligomerization, is indicative of inhibiting NS3 oligomerization and thereby inhibiting HCV replication. In most instances, determining whether the candidate inhibitor is expected to bind to a molecular interface of NS3 includes performing a fitting operation or comparison between the candidate inhibitor and NS3 or an NS3 molecular interface, followed by computational analysis of the outcome of the comparison in order to determine the association between the candidate inhibitor and the NS3 interface, or the interference of the candidate inhibitor with NS3-NS3 oligomerization. A candidate inhibitor identified by such methods is a candidate anti-HCV agent. Optionally, a candidate anti-HCV agent can be synthesized or otherwise obtained and further assessed (e.g., in vitro, in cells or in an appropriate animal model) for its ability to inhibit HCV replication.

[0107] Another embodiment of the invention provides a method of identifying a candidate compound to test for inhibiting HCV virus replication involving: (a) applying a 3-dimensional molecular modeling algorithm to spatial coordinates of a molecular interface of NS3; and (b) electronically screening stored spatial coordinates of a set of compounds against the spatial coordinates of the molecular interface of NS3 to identify at least one candidate compound that is expected to bind to the molecular interface of NS3. A suitable molecular modeling application is DOCK-5, available at http://dock.compbio.ucsf.edu.

[0108] In a particular embodiment, the molecular interface of NS3 comprises at least one amino acid residue (preferably at least three residues) selected from residues 541-553, 584-591, 435-453, 477-488, and 524-536 of SEQ ID NO:3.

[0109] In a particular embodiment, the molecular interface of NS3 comprises at least one amino acid residue selected from residues 477-481 and 452-453 of SEQ ID NO:3. In another embodiment, the molecular interface comprises residues 477-481 and 452-453 of SEQ ID NO:3.

[0110] In a particular embodiment, the method further involves comparing the spatial coordinates of the at least one compound to spatial coordinates of peptide SEQ ID NO:1 to determine whether the at least one compound is structurally similar to at least a portion of SEQ ID NO:1. The spatial coordinates of peptide SEQ ID NO:1 can be the spatial coordinates of the free peptide or of residues 541-551 of NS3, as is provided in Example 3 (chain B).

[0111] In a specific embodiment, the computer-assisted method of identifying a candidate inhibitor for inhibiting HCV replication that inhibits NS3 oligomerization comprises the steps of (1) supplying a computer modeling application the coordinates of a known agent that binds a molecular interface of NS3 (namely the peptide SEQ ID NO:1) and the coordinates of NS3 or an NS3 molecular interface; (2) quantifying the fit of the known agent to the NS3 molecular interface; (3) supplying the computer modeling application with a set of structural coordinates of an agent to be assessed to determine if it binds a molecular interface of NS3; (4) quantifying the fit of the test agent in the molecular interface using a fit function; (5) comparing the fit calculation for the known agent with that of the test agent; and (6) selecting a test agent that has a fit that is better than, or approximates the fit of the known agent.

[0112] Another embodiment of the invention provides a computer-assisted method for designing a candidate inhibitor compound for inhibiting hepatitis C virus (HCV) replication involving: (a) supplying to a computer modeling application a set of spatial coordinates of a molecular interface of NS3; (b) computationally building an agent represented by a set of structural coordinates; and (c) determining whether the agent is expected to bind to the molecular interface of NS3; wherein if the agent is expected to bind to the interface of NS3 it is a candidate inhibitor compound. A suitable molecular modeling application is DOCK-5, available at http://dock.compbio.ucsf.edu.

[0113] In particular embodiments, the molecular interface of NS3 includes at least one amino acid residue selected from residues 541-553, 584-591, 435-453, 477-488, and 524-536 of SEQ ID NO:3.

[0114] In a particular embodiment, the molecular interface of NS3 comprises at least one amino acid selected from residues 477-481 and 452-453 of SEQ ID NO:3. In another embodiment, the molecular interface comprises residues 477-481 and 452-453 of SEQ ID NO:3.

[0115] In a particular embodiment, the method further involves comparing the spatial coordinates of the at least one compound to spatial coordinates of peptide SEQ ID NO:1 to determine whether the at least one compound is structurally similar to at least a portion of SEQ ID NO:1. The spatial coordinates of peptide SEQ ID NO:1 can be the spatial coordinates of the free peptide or of residues 541-551 of NS3, as is provided in Example 3 (chain B).

[0116] One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a molecular interface of NS3, and more particularly with an interface involved in NS3-NS3 interactions. This process may begin, for example, by visual inspection of the molecular interface on the computer screen based on the NS3 atomic coordinates provided herein. Selected fragments or chemical entities may then be positioned relative to the interface of NS3. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.

[0117] Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: [0118] GRID (68) (available from Oxford University, Oxford, UK). [0119] MCSS (69) (available from Molecular Simulations, Burlington, Mass.). [0120] AUTODOCK (70) (available from Scripps Research Institute, La Jolla, Calif.). [0121] DOCK (71) (available from University of California, San Francisco, Calif.).

[0122] A commercially available computer database for small molecular compounds includes Cambridge Structural Database and Fine Chemical Database. For a review see reference 72.

[0123] Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or inhibitor. Assembly may be proceeded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of NS3. This would be followed by manual model building using software such as Quanta or Sybyl.

[0124] Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: [0125] CAVEAT (73) (available from the University of California, Berkeley, Calif.). [0126] 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.) This area is reviewed in reference 74. [0127] HOOK (available from Molecular Simulations, Burlington, Mass.).

[0128] Instead of proceeding to build an inhibitor of NS3 oligomerization in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other type of binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known inhibitor(s). Programs to execute these methods include: [0129] LUDI (75) (available from Biosym Technologies, San Diego, Calif.). [0130] LEGEND (76) (available from Molecular Simulations, Burlington, Mass.). [0131] LeapFrog (available from Tripos Associates, St. Louis, Mo.).

[0132] Other molecular modeling techniques may also be employed to screen for inhibitors of NS3 oligomerization. See, e.g., references 77 and 78. For example, where the structures of test compounds are known, a model of the test compound may be superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step. Any of these may be used. See, e.g., references 79-81, U.S. Pat. No. 5,331,573, and U.S. Pat. No. 5,500,807. The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

Medical Therapy and Pharmaceutical Compositions

[0133] Another embodiment of the invention provides a vector that expresses a dominant-negative mutant NS3 gene for use in medical therapy.

[0134] Another embodiment of the invention provides a use of a vector expressing a dominant-negative mutant NS3 gene to prepare a medicament effective to reduce replication of hepatitis C virus in a mammal such as a human.

[0135] Another embodiment of the invention provides a dominant-negative mutant NS3 protein for use in medical therapy. Another embodiment provides a use of a dominant-negative mutant NS3 protein to prepare a medicament effective to reduce replication of hepatitis C virus in a mammal such as a human.

[0136] Another embodiment of the invention provides an agent that inhibits HCV replication in cells infected with HCV and inhibits NS3 enzyme activity by inhibiting NS3 oligomerization for use in medical therapy. Another embodiment provides a use of an agent that inhibits NS3 enzyme activity by inhibiting NS3 oligomerization to prepare a medicament effective to reduce replication of HCV in a mammal, such as a human.

[0137] The invention also provides a pharmaceutical composition comprising an anti-HCV agent of the invention, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent. The anti-HCV agents of the invention include (1) a vector expressing a dominant-negative mutant NS3 gene, (2) an isolated and purified viral vector comprising a viral capsid encasing viral nucleic acid that comprises a dominant-negative NS3 gene operably linked to a promoter active in mammalian cells, (3) a dominant-negative mutant NS3 protein, (4) an agent that inhibits NS3 enzyme activity by inhibiting NS3 oligomerization, (5) a complex for inhibiting HCV replication comprising an inhibitory peptide comprising 4 or more contiguous residues of SEQ ID NO:1 complexed with a cell-entry vehicle, (6) a peptide comprising at least 4 contiguous residues of SEQ ID NO:1 wherein the peptide has 100 or fewer amino acid residues and inhibits hepatitis C virus replication, and (7) a compound of molecular weight 10,00 or less wherein the compound interacts with NS3 to inhibit NS3 oligomerization and inhibits HCV replication.

[0138] In cases where the anti-HCV agents are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, .alpha.-ketoglutarate, and .alpha.-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

[0139] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0140] The agents can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

[0141] Thus, the present agents may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the agents may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the agent in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0142] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the agent, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the agent may be incorporated into sustained-release preparations and devices.

[0143] The agents may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the agents can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0144] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0145] Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0146] For topical administration, the present agents may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0147] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent-pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0148] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0149] Examples of useful dermatological compositions which can be used to deliver the agents of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0150] Useful dosages of the anti-HCV agents of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0151] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0152] The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Example 1

ATPase-Deficient Mutant NS3 Protein Acts in a Dominant Negative Manner to Inhibit Wild-Type NS3 Activity and Inhibit Hepatitis C Virus Replication

Experimental Procedures:

[0153] Plasmid Construction: DNA sequences encoding wild-type NS3 or mutant NS3 having an alanine in place of aspartic acid at position 290 of NS3 (position 1361 of the polyprotein) (designated D290A NS3 or mNS3) were PCR amplified from a pET-26b plasmid carrying the NS3 gene using Pfu turbo. The PCR-amplified sequences were incorporated into pBUDCE4.1 plasmid (Invitrogen) by blunt end ligation. These plasmids drive the expression of NS3 via a mammalian expression promoter (CMV promoter) and can be used in cell culture experiments. They were sequenced to confirm incorporation of the NS3 gene and named pwtNS3 and pmNS3 for the wild type and the mutant forms, respectively.

[0154] Site-directed mutagenesis was performed on a pUC-18-NS3 plasmid, which contains all the nucleotides of the wild-type NS3 gene and serves as the shuttle vector for mutation of the replicon plasmid using the QUIK CHANGE site-directed mutagenesis kit (Stratagene) to incorporate the D290A mutation in the NS3 region of the plasmid. This mutation was confirmed by sequencing and called pUC-mNS3. The mutated NS3 gene was cut out from this plasmid using PmeI and MluI restriction enzymes and incorporated into the replicon vector HCV replb BartMan/AvaII Luciferase replicon (19), cut with the same enzymes, by ligation. The ligation mix was transformed to give the mutant replicon.

[0155] RNA synthesis: The replicon DNA having the luciferase reporter gene was digested with ScaI for 3 hrs, followed by in-vitro transcription with the AMBION MEGASCRIPT kit according to the manufacturer's directions. The RNA was stored at -80.degree. C.

[0156] HCV Replicon assays: Huh-7 cells were transiently transfected with replicon RNA having a firefly (P. pyralis) luciferase gene (0.2 .mu.g/well), and with a control plasmid pRL (0.05 .mu.g/well) having a renilla (R. renifomis) luciferase gene, using DMRIEC (Invitrogen) reagent as per the kit protocol (19). The specific additions of pmNS3, mutant replicon, pwt-NS3 etc were done concurrently. The cells were lysed after 48 hours, and luciferase activity detected using the DUAL LUCIFERASE assay kit (Promega) (24, 25).

[0157] Trans complementation assays: These assays were performed in the same manner as the regular replicon assays except that in the trans complementation assays two RNA's of different replicons (wild type and the mutant) were added along with a pRL plasmid to control for transfection efficiency.

[0158] Colony formation assays: Huh-7 cells stably transfected with S22041-mutant-containing replicon (2204 refers to the amino acid residue number in the polyprotein, genbank accession number AJ238799, SEQ ID NO:8) with a neomycin resistance gene were transfected with increasing concentration of pmNS3 and pwtNS3 and plated on 100 mm plates with 10 ml of DMEM media with 10% FBS and 1% non-essential amino acids (lipofection media) and allowed to grow for 24 hours. After 24 hours the media was changed to lipofection media containing G418 (Cellgro) at 500 .mu.g/ml concentration. The cells were kept under the selection medium for 21 days for colony formation. At the end of 21 days the plates were washed with PBS and then stained with 0.1% crystal violet Excess stain was washed with PBS and colonies were observed.

Results:

[0159] Time course of HCV-Luc RNA transfection demonstrates that replication can be detected at 48 hours in Huh-7 cells: Huh-7 cells were plated in a 12-well plate at 70-80% confluency. Cells were transfected with HCV-Luc replicon, HCV-Luc replicon incorporating a mutation in NS3 rendering it ATPase deficient (NS3 def), and HCV-Luc replicon with a mutation in NS5b rendering it polymerase deficient (Pol def), as well as with a renilla luciferase plasmid that serves as an internal control. (FIG. 1.) Cells were lysed using passive lysis buffer (Promega) for 15 minutes on ice and a dual luciferase assay was done to test for replication activity.

[0160] Replicon with an ATPase-deficient NS3 (D290A of NS3, D1361A of the polyprotein) (16) and replicon with a polymerase-deficient NS5b (G317A, D318A, D319G triple mutant NS5b) are both inactive in replication after 48 hours. In contrast, the wild-type replicon is still active at 48 hours, showing a persistent level of RNA (FIG. 1). This time course allows us to look at effects on replication at 48 hrs.

[0161] NS3 protein is detectable in all the plasmid constructs: Cell lysates from cells transformed with the plasmid expressing ATPase-deficient mutant NS3 (mNS3) or wild-type NS3, as well as cells transfected with wild-type HCV-Luc replicon or the HCV-Luc replicon with the ATPase-deficient mutant NS3 were subjected to western blot analysis. Equal amounts of cell lysates were run on a 12% polyacrylamide gel, the proteins were then transferred onto a PVDF membrane (Osmonics) for an hour. The membrane was blocked in 5% non-fat dry milk containing TBS-Tween (0.1%) for 1 hour, followed by incubation of the membrane in primary antibody overnight at 4.degree. C. The membrane was subjected to 5 washes of 5 minutes each with TBS-Tween, followed by 1 hour incubation in secondary antibody (goat anti-rabbit IgG-HRP) from Biolabs. This was followed by 5 washes of 5 minutes each with TBS-Tween. The protein was detected using ECL chemi-luminiscent kit (Amersham Pharmacia).

[0162] Western analysis of NS3 protein in both mutant and wild type form, as well as the two replicons demonstrates that NS3 protein is present in cell extracts at 48 hours post transfection (FIG. 2).

[0163] In vivo the dominant negative mNS3 (pmNS3) down regulates the activity of the HCV-Luc replicon: Huh-7 cells were plated in a 12-well plate at 0.1 million cells per well. The cells were grown to 70-80% confluency and then transfected with the replicon (HCV-Luc) RNA (0.2 .mu.g/well), with renilla luciferase plasmid (internal control), and increasing concentrations of mutant NS3 plasmid under a mammalian expression promoter using DMRIEC reagent (Invitrogen). The cells were kept in serum-free and antibiotic-free conditions for 5 hours. Thereafter, the cells were kept under 10% FBS, 0.1% non-essential amino acids in DMEM (Cellgro). After 48 hours cells were lysed using passive lysis buffer from the DUAL LUCIFERASE kit (Promega). The DUAL LUCIFERASE assay was performed as per kit instructions.

[0164] There is a 9-fold reduction in activity of the replicon on addition of 0.5 .mu.g of pmNS3 (FIG. 3A), while there is no appreciable reduction in the activity of the replicon on addition of exogenous wild-type NS3 (FIG. 3B)

[0165] Exogenous wt-NS3 is unable to rescue the activity of the mutant replicon: Huh-7 cells were transfected with HCV-Luc replicon encoding the ATPase-deficient mutant NS3 along with increasing concentration of wtNS3 plasmid and the renilla luciferase plasmid for transfection efficiency control. The cells were treated in conditions identical to those used in FIG. 3 and lysed using Promega's passive lysis buffer followed by luciferase assays at 48 hours.

[0166] Co-transfection of increasing concentration of pwtNS3 plasmid along with mutant replicon did not change the activity of the replicon (FIG. 4).

[0167] Results of colony formation assay corroborate the effect seen using HCV-Luc replicon: Huh-7 cells stably transfected by S2204I replicon, which encodes an adaptive mutation allowing continuous replication in cells under G418 selection pressure, were transfected with increasing concentration of wtNS3 or mutant NS3 plasmids and plated onto 100 mm dishes at 1 million cells per dish. After 24 hours antibiotic-free lipofection media was replaced with DMEM with 10% FBS and G418 (500 .mu.g/ml) for selection. The colonies were allowed to form over a period of 21 days. At the end of 21 days the plates were washed with PBS followed by staining with 0.1% crystal violet.

[0168] FIG. 5 shows the plates. Colony counts are in parentheses. The plates with addition of mnNS3 plasmid showed a marked reduction in the number of colonies formed while the plates containing wtNS3 showed no significant difference in the number of colonies formed (FIG. 5). Therefore, we concluded that the ATPase-deficient form of NS3 inhibits the activity of the replicon in Huh-7 cells.

[0169] The dominant negative effect does not show up upon trans-complementation of wild-type replicon with mutant replicon: Huh-7 cells were plated in a 12-well plate at 70-80% confluency. Cells were transfected with the replicon (HCV-Luc) RNA with renilla luciferase plasmid (internal control) and increasing concentrations of D290A NS3 mutant replicon, using DMRIEC reagent (Invitrogen). The cells were kept in serum-free and antibiotic-free conditions for 5 hours. Thereafter, the cells were kept under 10% FBS, 0.1% non-essential amino acids in DMEM (Cellgro). After 48 hours cells were lysed using passive lysis buffer from DUAL LUCIFERASE kit (Promega). The DUAL LUCIFERASE assay was performed as per kit instructions.

[0170] Upon co-transfection of cells with wild-type replicon (0.2 .mu.g/well) and increasing concentration of mutant replicon we did not observe an appreciable effect on the activity of wt-replicon (FIG. 6). This led us to conclude that the dominant negative effect observed in the case of mutant NS3 does not occur at the polyprotein level.

[0171] Next, the conditions were reversed. Cells were transfected with the mutant replicon (0.2 .mu.g/well) and increasing concentrations of wild-type replicon. Addition of increasing concentration of wt-replicon to the mutant replicon yielded an additive effect in replicon assays (FIG. 7). This also indicates that the dominant negative effect of mutant NS3 does not occur at the polyprotein level.

Discussion:

[0172] Previous studies have shown that the helicase domain of NS3 alone is viable as an oligomer but the oligomer is unstable (16). Also, there is evidence for subunit exchange and mixed oligomers of NS3 (16). However, the previous biochemical studies have been conducted under single-turnover conditions, under which the concentration of the enzyme is higher than the concentration of the nucleic acid substrate.

[0173] The aim of this Example was to establish the effect of NS3 on hepatitis C virus in vivo. It has previously been shown that NS3 is required for replication of the virus (8). We decided to use the HCV replicon containing a luciferase gene (19, 26) as the model system to study the effect of exogenous ATPase-deficient mutant NS3 on the replication of HCV in vivo. Earlier studies have shown that the HCV replicon system is an excellent representation of HCV replication after infection. Two assays have been used to measure replication potential of the replicon: colony formation and luciferase activity. The two measurements have been shown to give results consistent with each other (19). We tested the system by conducting a time course study on the HCV-Luc replicon in comparison with replicons incorporating mutations that are known to inhibit replication of the virus, namely the D290A mutation in NS3, which obliterates the ATPase activity of NS3 and the G317A, D318A, D319G triple mutation in NS5b, which renders it polymerase deficient (16, 17, 19). The results showed that at 48 hours post-transfection, the luciferase activity accurately reports replication of HCV or the absence of replication (FIG. 1).

[0174] We then proceeded to demonstrate that hepatitis C virus NS3 protein is expressed in our cell culture system by both the replicon and the plasmids (pmNS3 and pwtNS3) encoding the NS3 gene driven by a CMV promoter, through western blotting using an antibody specific to NS3 (FIG. 2). Once the system was in place we transfected increasing quantities of pmNS3 and pwtNS3 plasmids along with the replicon RNA and showed that the addition of exogenous ATPase-deficient NS3 under a mammalian expression promoter (pmNS3) is able to reduce replication, while an identical amount of pwtNS3 has very little effect on the luciferase activity of the replicon (FIG. 3). These data demonstrate that the cleaved NS3 proteins interact with each other to form an active oligomer.

[0175] Independent confirmation of the results of the replicon assay was obtained by performing a colony formation assay. These data substantiated the finding that mNS3 functions in a dominant negative manner and is able to inhibit replication of the replicon, as evidenced by the reduction in number of colonies formed as a factor of increasing pmNS3 concentration (FIG. 5). As a control we showed that the addition of pwtNS3 in an identical manner did not alter the number of colonies formed as compared to the untransfected cells (FIG. 5).

[0176] It has been shown that HCV RNA translates into a polyprotein which is subsequently cleaved by proteases to yield mature independent proteins. To investigate the effect of expressing mutant polyprotein as opposed to mutant NS3 single protein, we performed trans-complementation assays in which we added increasing quantities of a replicon encoding an ATPase-deficient mutant NS3 to the wild-type HCV-Luc replicon. We found no effect with the addition of increasing concentration of the mutated replicon to the wt-HCV-Luc replicon (FIG. 6). Therefore, the mutant polyprotein does not have an effect on the replicative potential of the wild-type replicon, showing that mnNS3 protein is interacting with the cleaved proteins and not the polyprotein to inhibit replication of the virus. It is also possible that the quantity of mutant NS3 protein produced by the replicon is insufficient to elicit the same response as when the protein is expressed from a plasmid. The addition of wild-type luciferase replicon has an additive effect on the activity of the replicon encoding the ATPase-deficient mutant NS3 (FIG. 7). That indicates the translation of replicon RNA is not limiting in these experiments.

[0177] This dominant negative effect of NS3 provides for an alternative mechanism of neutralizing the hepatitis C virus--by intervention with NS3 oligomerization by targeting the HCV-infected liver with mNS3 DNA or mNS3 protein.

Example 2

Helper-Dependent Adenovirus Vector for Targeting Mutant NS3 Expression to Liver

[0178] This Example describes preparation of a helper-dependent adenovirus vector, coupled to asialoorosomucoid for targeting to liver (28, 29). Helper-dependent (HD) adenovirus vectors have minimal adenovirus sequences and give more stable expression of the foreign DNA in the mammalian target cells than first generation adenoviruses, which retain almost all of the native adenovirus DNA. To replicate, helper-dependent adenoviruses require helper adenoviruses to provide necessary functions in trans (28).

[0179] A helper-dependent adenovirus vector is created containing 500 bp of cis-acting adenovirus sequences necessary for vector DNA replication (ITRs and packaging sequences), the ATPase-deficient NS3 gene under the control of a SV40 promoter, 400 bp of adenovirus sequence from the right end of the virus and containing the E4 promoter but not coding sequence (29, p. 1004-05), and stuffer sequence to bring the final vector size to 28-36 kb, preferably 28-31 kb. Stuffer DNA may be, for instance, noncoding human DNA lacking repetitive elements (29)

[0180] The helper virus is a first generation adenovirus with the E1 region deleted and with the virus packaging signal flanked by loxP sites (28, 29). An example is the H14 helper virus (29). A stuffer sequence is inserted into the E3 region to render any E1.sup.+ recombinants too large to be packaged (28). Following infection of 293Cre cells, the helper virus genome is rendered unpackageable by excision of the packaging signal by Cre-mediated site-specific recombination between the loxP sites.

[0181] Low-passage 293 and 293Cre4 cells are maintained in 150-mm dishes and split 1 to 2 or 1 to 3 when they reach 90% confluency. 293Cre4 cells are maintained under 0.4 mg/ml G418 selection (28).

[0182] The HD vector is amplified by transfecting 293Cre4 cells with the HD vector plasmid in CaCl.sub.2. After a 6-16 hour incubation, the cells are washed with fresh medium and then infected with helper virus at a multiplicity of infection (MOI) of 5 pfu/cell.

[0183] Complete cytopathic effect (>90% of the cells rounded up and detached from the dish) is observed by about 48 hours postinfection. The cells are scraped into the medium at that time. DNA is extracted from one ml for analysis to monitor vector amplification. The remainder is stored at -70.degree. C. after adding sucrose to 4% w/v.

[0184] After thawing, 0.4 ml of the lysate is used to coinfect a 60-mm dish of 90%-confluent 293 Cre4 cells with helper virus at an MOI of 1 pfu/cell.

[0185] After complete cytopathic effect at about 48 hours, the cells are scraped into the medium, DNA is extracted from 1 ml for analysis, and 0.4 ml of the remainder is used for another round of amplification by cotransfection with helper virus at 1 pfu helper virus/cell.

[0186] The vector titer is quantified with each passage to determine the optimal number of passages--the number of passages after which the increase in HD vector titer slows substantially or the number of passages after which the lysate contains the maximal amount of HD vector with a low amount of helper virus.

[0187] For large-scale preparation, 150-mm dishes of 90% confluent 293Cre4 cells (seeded 1-2 days previously in nonselective complete medium) are coinfected with 1 ml of lysate from the passage previous to the optimum passage, and with helper virus at an MOI of 1 pfu/cell. At complete cytopathic effect, about 48 hours postinfection, cells are scraped and harvested, and the cell suspension is extracted for purification of the HD vector.

[0188] HD vector can be further purified by centrifugation in a CsCl step gradient using 1.25, 1.35, and 1.5 g/ml CsCl solutions. The vector should settle at the interface between the 1.25 and 1.35 g/ml layers.

[0189] The number of HD particles per ml can be calculated as follows:

(OD.sub.260)(dilution factor)(1.1.times.10.sup.12)(36)/(size of vector in kb)

Coupling Hd Adenovirus Vector to Asialoorosomucoid for Targeting to Liver Cell Receptors (30, 31)

[0190] The HD vector particles isolated above are dialzyed against 150 mM NaCl, 20 mM Hepes-NaOH, pH 7.4. In 4 ml, 5 mg asialoorosomucoid (AsOR) and 1.2 mg poly-L-lysine is dissolved with 1.4.times.10.sup.11 HD particles, with the pH adjusted to 7.4. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) is added to 1 mM final concentration. After incubation on ice for 4 hours, the conjugated adenovirus-PLL-AsOR is separated from unreacted reagents by centrifugation (150,000.times.g) for 18 hours on a CsCl gradient at a CsCl concentration of 1.35 g/ml.

[0191] The adenovirus-PLL-AsOR conjugate is used to deliver adenovirus with high-specificity to liver cells in vitro or in vivo, by contacting the liver cells with the adenovirus conjugate cells (e.g., by intravenous administration of the adenovirus) at approximately 10.sup.3 adenovirus particles per liver cell.

Example 3

Identification of Surface Residues of Hepatitis C Virus Helicase Required for Optimal Replication

Introduction

[0192] Hepatitis C virus (HCV) is a 9.6 kb positive, single-stranded RNA virus. Many aspects of the HCV replication mechanism remain unknown, but it appears that at least five of the viral non-structural proteins are required for replication (6). Several non-structural protein-protein interactions have been identified (35, 36), and co-localization of non-structural proteins, including helicase (NS3) and polymerase (NS5B), on membrane structures within cells has been observed (37, 38), indicating formation of a multi-protein replication complex containing both polymerase and helicase enzymes. Understanding the mechanism of nucleic acid unwinding by NS3 is therefore a key step in characterizing the viral replication mechanism.

[0193] NS3 helicase (NS3h) has been expressed and purified independently of NS3 protease in a recombinant bacterial system and retains its in vitro unwinding activity (39). The NS3h structure consists of three distinct domains, with domains 1 and 2 containing all of the conserved motifs common to superfamily II helicase enzymes, including the DExH/D box motif (40, 41). ATP binds at the interface between domains 1 and 2, and nucleic acid binds within a cleft formed at the interface between domain 3 and domains 1 and 2 (41). Interactions between the protein and nucleic acid are not sequence-specific and primarily involve the phosphate backbone of the nucleic acid.

[0194] Despite extensive structural and biochemical characterization, the oligomeric state of the functional species of NS3 has not been determined conclusively. Biochemical studies have resulted in reports of monomeric (42), dimeric (43, 44), and oligomeric (16, 45) forms of the protein. Crystal structures of NS3h (40), NS3h bound to an 8-mer poly-dU substrate (41), and a full-length NS31NS4A fusion protein (46) demonstrate that a single protein monomer can form crystals. However, none of these structures includes a substrate molecule of sufficient length to accommodate binding of multiple NS3h monomers. Crosslinking experiments indicate that NS3h can form oligomeric structures in solution (16). DNA unwinding activity of full-length NS3 is optimal at concentrations high enough to allow binding of multiple NS3h monomers to each nucleic acid molecule (unpublished data). However, no cooperative effect has been observed in binding studies (48), leaving open the possibility that NS3 is a non-processive monomer. A recently reported biochemical model suggests that monomeric NS3h is functional, but that multiple NS3h molecules are required for optimal processivity (49).

[0195] We have crystallized NS3h bound to a 16-mer poly-dU nucleic acid. The structure shows two NS3h molecules bound to a single DNA molecule and reveals an apparent interface between the two protein molecules. A mutational analysis of the protein interface region was performed in order to determine its importance for helicase activity and viral replication. We have identified several amino acid residues within this region that when mutated, result in reduced viral replication as measured in an HCV sub-genomic replicon. The biochemical activities of one of the mutant proteins are consistent with the dimeric structure in the x-ray crystal structure. However, the overall results from nucleic acid binding and helicase unwinding activity in vitro do not correlate with the biological results. These data indicate that NS3 helicase is likely to exhibit biological functions that are not reflected in the known biochemical activities.

Materials and Methods

Purification of NS3h

[0196] NS3h was purified according to published procedures (20).

Crystallization and Structural Determination of NS3h

[0197] Purified NS3h was concentrated to 32.2 mg/ml for crystallization. Examination of sample purity and determination of molecular weight were performed using SDS-PAGE on a PHAST GEL system (AP Biotech, NJ). The apparent molecular weight of the helicase was approximately 50 kDa. The (dU).sub.16 oligonucleotide was synthesized using an Expedite Nucleic Acid Synthesis System and purified by polyacrylamide gel electrophoresis to a final concentration of 4.6 mg/ml. Immediately before crystallization, NS3h was mixed with the (dU).sub.16 oligonucleotide at a 2:1 molar ratio. Crystals of diffraction quality were obtained by using the hanging drop method at room temperature with 4 .mu.l of the mixture and 1 .mu.l of reservoir solution (2.4 M (NH.sub.4).sub.2SO.sub.4, 0.1 M Tris hydrochloride, pH 8.5). Diffraction data were collected at 1.5418 .ANG. by using a single crystal of 0.2 mm.times.0.4 mm.times.0.1 mm mounted in a thin-walled glass capillary at room temperature. The X-ray radiation was produced by an in-house Rigaku RU-H3RHB generator and focused by Osmic's Gutman multilayer mirrors (Woodland, Tex.). The diffraction pattern was recorded on the R-AXIS IV phosphor image plate detector. The diffraction data were reduced, integrated, and scaled with Denzo/Scalepack. Molecular replacement analysis was performed using AMORE software (49b), and structure refinements and model building/adjustment were done using CNS (50) and XTALVIEW (51) respectively.

[0198] The scaled data of resolution 3.3 .ANG. indicated that the NS3h/(dU).sub.16 crystal belongs to the orthorhombic space group (Table 1). Space group assignment between P2.sub.12.sub.12 and P2.sub.12.sub.12, could not be made due to the absence of some (001) reflections. Matthews' Coefficient suggested the number of protein molecules in one asymmetric unit (ASU) could be 3 or 4, with solvent content being 67% or 50%, respectively.

TABLE-US-00001 TABLE 1 Data Collection and Refinement Statistics Data Collection Unit Cell .alpha. = .beta. = .gamma. = 90.degree. A (.ANG.) 108.3 B (.ANG.) 109.8 C (.ANG.) 183.4 Space group P2.sub.12.sub.12.sub.1 Wavelength (.ANG.) 1.5418 Resolution (.ANG.) 3.3 Unique reflections 33594 Completeness (%) 91.1 R.sub.sym.sup.a (%) 0.34 <I/.sigma.(I)> 4.4 Refinement Resolution (.ANG.) 30-3.3 No. reflections.sup.b 27936 (1673) R.sup.b,c (%) 24.8 (27.4) Avg. B-factors (.ANG.) 34.25 Rmsd bond lengths (.ANG.) 0.010 Rmsd bong angles (.degree.) 1.495 .sup.aR.sub.sym = .SIGMA.|I - <I>|/.SIGMA. I; I, intensity. .sup.bValue for R.sub.free set containing 5% of randomly chosen reflections. .sup.cR = .SIGMA.| F.sub.obs - F.sub.calc |/.SIGMA. F.sub.obs.

[0199] We completed the structure determination by molecular replacement (MR) using the published 1A1V structure (41) as an initial model. The 1A1V structure consists of an NS3h monomer bound to a (dU).sub.8 oligonucleotide. Prior to MR, the oligonucleotide was removed from the model. The MR results from space group P2.sub.12.sub.12 did not display any reasonable correlation among the individual solutions; however, the MR solutions in space group P2.sub.12.sub.12, showed strong correlation as well as realistic crystal packing (not shown).

[0200] A composite omit map was calculated using CNS (50) to identify any missing components. The resulting electron density map showed clearly the presence of the oligonucleotide at the nucleic acid binding sites of the protein molecules. The (dU).sub.x fragments were inserted manually into the structure using XTALVIEW (51). The Maximum-Likelihood from Structure Factors (MLF) refinement on the model was done in CNS and is presented in Table 1.

Construction of Mutant Plasmids

[0201] Mutations were introduced into a pUC 18-NS3 subclone containing nucleotides 1182-4918 from the HCV replicon sequence using the QUIKCHANGE Site-Directed Mutagenesis Kit (Stratagene). Mutant plasmids were transformed into SURE cells and purified from cultured cells with the QIAPREP Spin Miniprep Kit (QIAGEN). Mutant subcloned HCV sequences were then transferred into the replicon plasmid by digestion at Pme I and Mlu I restriction sites followed by ligation. Mutant NS3 sequences were transferred to pET26b-Ub expression plasmid by PCR followed by digestion at Sac II and EcoR I restriction sites and ligation. Quality of all final plasmid products was confirmed by sequencing.

Replicon RNA Synthesis

[0202] DNA template was prepared by digestion of replicon plasmid (10 .mu.g) with Sca I restriction endonuclease at 37.degree. C. for 4 hours. Complete linearization of plasmid was confirmed by agarose gel electrophoresis. RNA was synthesized in vitro by incubating 0.5 .mu.g linear DNA template with 0.5 .mu.g T7 RNA polymerase in 350 mM HEPES pH=7.5, 32 mM magnesium acetate, 40 mM DTT, 2 mM spermidine, and 28 mM NTPs at 37.degree. C. for 3 hours. Template DNA was removed by incubation with 2 units Dnase I at 37.degree. C. for 30 min. RNA was precipitated overnight in LiCl at -20.degree. C. RNA purity and quality were verified by agarose gel electrophoresis.

Colony Formation Assays

[0203] HCV replicon RNA (1 .mu.g) and Huh-7.5 cells (2.times.10.sup.6) were incubated with DMRIE-C lipofection reagent in serum-free medium at 37.degree. C. for 60 min with gentle agitation. Transfected cells were centrifuged at 4000 rpm for 4 min, resuspended in 7.5 ml medium+10% fetal bovine serum, and transferred to a 10 cm culture plate. 0.5 mg/ml G418 was added 24 hours after transfection. Colony formation was monitored over a period of two to three weeks with replacement of growth medium every two days. Mature colonies were stained with 0.1% crystal violet.

Western Analysis

[0204] Huh-7.5 cells were transiently transfected with HCV RNA with TRANSMESSENGER Lipofectin Reagent (Qiagen) according to the manufacturer's instructions. Cells were lysed at 4 hours post-transfection in SDS-PAGE denaturing sample buffer. Lysate from 5.times.10.sup.5 cells from each transfection was run on 10% SDS gel. Proteins were transferred from gel to PVDF membrane using a BioRad electrophoretic transfer cell at 100 V for 1 hour at 4.degree. C. in transfer buffer (25 mM Tris, 192 mM glycine). Membranes were blocked in 5% dry milk/TBST for 1 hour, then washed three times for 5 min with TBST. Blocked membranes were exposed to rabbit polyclonal anti-NS3 (supplied by C.E.C.) in 5% BSA/TBST for 90 min, washed as described above, then exposed to HRP-conjugated goat anti-rabbit IgG (PerkinElmer) in 5% dry milk/TBST for 1 hour. Chemiluminescent detection was done by ECL western blotting analysis system (Amersham).

RNA Binding Assays

[0205] Varying concentrations of NS3h were incubated with 500 pM 5'-fluorescein-labeled rU.sub.20 (Integrated DNA Technologies) in 50 mM MOPS-K.sup.+ (pH 7.0), 10 mM NaCl, 50 .mu.M EDTA, 0.1 mg/ml BSA for 5 minutes at 37.degree. C. Binding was measured as a function of fluorescence polarization using a Beacon fluorescence polarization system. Data were fit to a hyperbola using Kaleidagraph software.

ATPase Assays NS3h was incubated with 5 mM ATP in 50 mM HEPES (pH=7.5), 5 mM EDTA, 10 mM MgCl.sub.2, 10 mM NaCl, 0.1 mg/ml BSA, 4 mM phosphoenolpyruvate, 10 U/ml pyruvate kinase/lactate dehydrogenase, and 0.7 mg/ml NADH. Absorbance of NADH at 380 nm was measured at 1 sec intervals for a period of 30 sec in the presence of the indicated concentrations of poly-U. Hydrolysis rates were calculated using an extinction coefficient of 1,210 M.sup.-1 cm.sup.-1 for NADH. Data were fit to a hyperbola using Kaleidagraph software.

Steady State DNA Unwinding Assays

[0206] The substrate used was a 45-mer/30-mer containing 30 base pairs of double-stranded DNA with a 15 base 3' single-stranded overhang. One strand was radiolabeled by incubation with .gamma.-.sup.32P-ATP and T4 polynucleotide kinase at 37.degree. C. for 60 min. Unincorporated ATP was removed by SEPHADEX G-25 filtration. Equimolar amounts of labeled and complementary unlabeled strands were combined, heated to 95.degree. C. for 10 min, and cooled slowly to room temperature to generate the final substrate. For steady state unwinding experiments, 100 nM NS3h was incubated with 250 nM substrate in 25 mM HEPES (pH=7.5), 0.5 mM EDTA, 10 mM MgCl.sub.2, 10 mM NaCl, 0.1 mg/ml BSA. Reactions were initiated by addition of 5 mM ATP. Aliquots were taken at specific time points and the reaction was quenched by addition of 200 mM EDTA, 0.7% SDS. Substrate and product were separated by native polyacrylamide gel electrophoresis and detected and quantified by phosphorimaging analysis.

ATP-Independent DNA Unwinding Assays

[0207] NS3h or NS3h mutant enzymes (500 nM) were mixed with 2 nM DNA substrate (described in steady state section) in 25 mM MOPS (pH 7.0), 10 mM NaCl, 0.1 mM EDTA (pH 8.0), 2 mM PME, and 0.1 mg/mL BSA at 37.degree. C. Aliquots were transferred to a `quench solution` containing 200 mM EDTA, 0.7% SDS, 5 mM ATP, 10 mM MgCl.sub.2, 60 nM annealing trap, and 100 .mu.M poly-dT protein trap. Double- and single-stranded DNA were resolved on a native 20% polyacrylamide gel. The radiolabeled substrate and product were detected using a PHOSPHORIMAGER (Molecular Dynamics, Sunnyvale, Calif.); quantitation was performed with IMAGEQUANT software. The ratio of single- to double-stranded DNA was determined and plotted as a function of time.

Single Turnover DNA Unwinding Assay

[0208] Unwinding assays were carried out using a Quench-Flow apparatus (RQF-3, KinTek Instruments, Austin, Tex.) with a two-step mixing protocol (49). Reactions were carried out in 25 mM MOPS (pH 7.0), 10 mM NaCl, 0.1 mM EDTA (pH 8.0), 2 mM .beta.ME, and 0.1 mg/mL BSA at 37.degree. C.; all concentrations are post-mixing. NS3h (500 nM) was mixed with 2 nM DNA substrate (described in steady state section) for 10 seconds before adding 5 mM ATP, 10 mM MgCl.sub.2, 60 nM annealing trap (complementary to the displaced strand), and 100 .mu.M poly-dT protein trap. The reaction was quenched after 0.1-15 seconds by ejection into a tube containing 200 mM EDTA, 0.7% SDS. Double- and single-stranded DNA were resolved on a native 20% polyacrylamide gel. The radiolabeled substrate and product were detected using a PHOSPHORIMAGER (Molecular Dynamics, Sunnyvale, Calif.); quantitation was performed with IMAGEQUANT software. The ratio of single- to double-stranded DNA was determined and plotted as a function of time. Data were fit to Equation 1, using KALEIDAGRAPH (Synergy Software, Reading, Pa.). This equation describes a 5-step mechanism for DNA unwinding that is necessary to fit the substantial lag phase associated with unwinding of the substrate (49, 52, 53).

A{1-[1+k.sub.obst+1/2(k.sub.obst).sup.2+1/6(k.sub.obst).sup.3+ 1/24(k.sub.obst).sup.4]e.sup.-kobs.sup.t} Eq. 1

Results

[0209] Crystallization and Structural Analysis of NS3h Bound to (dU).sub.16

[0210] The crystals of NS3h in the presence of (dU).sub.16 oligonucleotide belonged to space group P2.sub.12.sub.12.sub.1 with unit cell dimensions a=108.3 .ANG., b=109.8 .ANG., and c=183.4 .ANG. (Table 1). We determined the structure by the molecular replacement method (MR) using 1A1V.pdb (41) as an initial model, in which its (dU).sub.8 fragment was manually removed prior to the rotational function search. We identified three helicase molecules (chains A, B, and C) per asymmetric unit (ASU) in our structure, with two helicase molecules bound to a single (dU).sub.16 molecule. The final atomic model shows no major unfavorable steric interactions between the helicase molecules, and the crystal packing shows no conflicts between the protein molecules in adjacent ASUs. We found no apparent non-crystallographic symmetric operations among the three monomers in this helicase model, nor did we observe any dramatic differences among the monomers. All three helicase molecules retain the basic Y shape characteristic of previously reported structures, with minor conformational differences at the surface loop regions.

[0211] The atomic coordinates of the helicase molecules A, B, and C in the final atomic model are provided in Table 2.

[0212] Chains A and B of the complex are bound to a 13-nucleotide span of one (dU).sub.16 molecule, with chain B rotated 90 degrees relative to chain A. The binding mode of both chains is consistent with that of the 1A1V structure, with the binding cleft at the interface of domains 1 and 2 with domain 3 in each protein molecule. Chain C appears to be independent of the dimer-oligonucleotide complex. The nucleic acid binding cleft of chain C faces away from the dimer structure and is occupied by a second oligonucleotide molecule. Chain C does not have any evident structural or mechanistic relationship with the other two protein molecules, and there appear to be no suitable contacts between chains B and C to indicate a functional interaction between these two molecules.

[0213] To determine the degree of similarity between chains A and B, we superimposed the two using domain 1 (the NTPase domain) as an anchor (the backbone RMSD for residues 190-324 was 0.7 .ANG.). We observed only minor conformational differences between the two chains in each of the three domains. However, it appears that the relative orientation of domain 2 with respect to the anchored domain 1 is slightly different between chains A and B with a small but detectable tilting angle resulting in a slight widening of the nucleic acid binding groove in chain B. This flexibility of domain 2 relative to domain 1 is consistent with the proposed ratchet mechanism for nucleic acid translocation (41). Domain 3 displays a small degree of rotation between chains A and B, but is otherwise similar.

[0214] To assess the relationship of the apparent dimer structure to that of the monomeric NS3/oligo complex (41), we independently aligned each of the two monomer structures (chains A and B) with the 1A1V structure (data not shown). Both chains of the dimer structure align well with 1A1V, with chain A being a slightly better fit than chain B (overall RMSD .about.0.9 .ANG. and .about.1.3 .ANG., respectively). Aside from the slight widening of the groove between domains 2 and 3 in chain B of our structure, no significant structural differences exist between the structure of 1 A1V and the structures of our A and B chains.

[0215] In the dimer structure, the oligonucleotide is bound to each NS3h molecule within the groove formed at the interface of domains 1 and 2 with domain 3. The DNA interactions with chain A are virtually identical to those observed in the 1A1V structure. In the region between chains A and B, the DNA appears to be bent by nearly 90 degrees. It is possible that this bend is stabilized by the apparent base stacking between nucleotides dU.sub.8 and dU.sub.10. However, the electron density in this region is weak, and although structure validation by WHAT_CHECK (54) suggested that the bent conformation of the DNA is allowable (55), we accept the possibility that the bend may indeed exist in another conformation. Comparisons of the 3' binding regions in both chains A and B to 1A1V exhibited high similarity in nucleotide binding, suggesting that the binding mode of the oligonucleotide between chains A and B is likely to exist as presented. The overall DNA binding mode with respect to chain B is similar to that with respect to chain A. However, due to the displacement of domain 2 in chain B, domains 2 and 3 are farther apart than in chain A. In turn, the binding groove of chain B is slightly wider and causes the oligonucleotide to tilt toward domain 2, allowing domain 1 and the oligonucleotide backbone to retain the same interactions observed in chain A. The domain displacement, however, does not dramatically alter the interactions of oligonucleotide with domains 1 and 3. For example, the Trp501 side chain retains its ring-to-ring stacking position, and Thr269 remains in position to allow hydrogen bonding with the phosphate backbone of the DNA at dU.sub.11. The DNA fragment in chain B is pushed slightly out of the binding groove and shifted away from the .alpha.-helical domain. As a result, the electron density for this fragment is less clear than that in chain A.

[0216] There are numerous close interactions between chains A and B involving multiple sets of amino acid residues, including H545-A553 and C584-T591 of chain B, and T435-Q453, T477-S488, and V524-Q536 of chain A. In addition, Thr450 of chain A and Gln549 of chain B appear to be in position to allow hydrogen bond formation between the two monomers at the dimeric interface. Chain B residues 541-551 interact with a chain A cleft formed by residues 477-481 and 452-453.

[0217] To assess the strength of interaction between chains A and B, we calculated the extent of buried surface area (S.sub.AB) at the interaction site using WHATIF molecular modeling software (55b). This value is defined as S.sub.AB=A.sub.A+A.sub.B-A.sub.AB, where A is the total surface area of the folded polypeptide molecule. The surface areas of chains A and B are 5883 .ANG..sup.2 and 5839 .ANG..sup.2, respectively. The total surface area calculated for the dimer is approximately 11279 A.sup.2, leaving a difference of 443 A.sup.2 buried at the interface. This area is not sufficient to support independent dimer formation in the absence of nucleic acid, which is consistent with the observed monomeric behavior of NS3h in size exclusion chromatography (Raney and C. Chen, unpublished observations).

Biological Analysis of Surface Residues

[0218] To assess the importance of the protein-protein interface region observed in the crystal structure, we performed a mutational analysis involving two clusters of residues (Asp543/His545/Gln549 and Arg587/Leu588/Lys589/Thr591) in domain 3. These residues are situated at the interface of the two NS3h molecules and appear to be of particular importance in the protein-protein interaction. We did not introduce mutations at the domain 2 interface site because of its proximity to the conserved helicase motifs. Amino acid residues 543-545 were deleted (.DELTA.543-545), and two sets of substitution mutations (D543K/H545D/Q549A and R587D/L588D/K589D/T591D) were introduced independently into the HCV-neo-1377/NS3-3'UTR replicon (26) containing an S2204I adaptive mutation (56).

[0219] We transfected Huh-7 human hepatoma cells with mutant HCV RNA, and monitored the cells for replication-dependent growth. Cells transfected with S2204I HCV RNA formed large, densely spaced colonies after two to three weeks of growth (FIG. 8A). Cells transfected with the mutant HCV RNA showed significantly reduced colony formation, indicating that the targeted NS3 surface region is important for efficient viral replication. The .DELTA.543-545 and D543K/H545D/Q549A (NS3h KDA) mutants were of particular interest, as they supported very little cell growth (FIGS. 8C and D). Only pinpoint colonies were visible at two weeks post-transfection, and no cells remained at three weeks post-transfection. The R587D/L588D/K589D/T591D mutation (NS3h DDDD), at a different site within the protein interface than the .DELTA.543-545 and NS3h KDA mutations, had a visible but less dramatic effect on growth (FIG. 8E). Colonies were less densely spaced than in the S2204I transfection, but those that formed grew to approximately the same size as wild type colonies.

[0220] We performed a western analysis with NS3 antibody on transfected cell lysates in order to determine whether NS3 protein expression was affected by any of the mutations (FIG. 9). The .DELTA.543-545 mutant NS3 protein was present at significantly lower concentration than observed in S2204I transfected cells. However, no impairment of translation was observed for either of the two substitution mutants.

Biochemical Analysis of NS3h Mutants

[0221] In order to determine the effects of the surface mutations on the biochemical activities of NS3h, we over-expressed and purified NS3h KDA and NS3h DDDD in a prokaryotic expression system and compared the binding and enzymatic activities of the mutant NS3h proteins to those of wild type. We measured the RNA and DNA binding affinities of the mutant and wild type enzymes using fluorescein-labeled, oligonucleotide substrates (FIG. 10). The NS3h KDA mutation did not impair binding to RNA or DNA, but the NS3h DDDD mutation severely reduced binding affinity as measured by fluorescence polarization. Binding to the DNA oligonucleotide was around ten-fold tighter than to the RNA oligonucleotide. Poly-U stimulated ATP hydrolysis of the mutant and wild type enzymes was measured and no significant differences were observed between the activities of the wild type and NS3h KDA mutant (FIG. 11). The activity of the NS3h DDDD mutant was severely impaired, probably as a consequence of its reduced nucleic acid binding affinity.

[0222] Unwinding of nucleic acid was measured by using a standard helicase assay (FIG. 12A). A substrate (45:30mer) containing 30 base pairs with a 15 nt 3' single-stranded overhang was prepared by annealing appropriate oligonucleotides. We observed very little unwinding of an RNA substrate by NS3h, consistent with a recent report describing the lack of RNA unwinding activity of NS3h (FIG. 12B) (45).

[0223] Therefore, we measured unwinding rates using a DNA substrate. NS3h and the two mutant enzymes unwound the 45:30mer DNA at approximately 1 nM/min under steady state conditions (FIG. 12C). The conditions used in this assay are likely to favor a monomeric form of NS3h because it is highly unlikely that two molecules bind to the same substrate molecule in the presence of a large excess of DNA. This observation further confirms that NS3h KDA monomer is not functionally impaired. NS3h DDDD unwinds DNA almost as well as NS3h wild type under steady state conditions, despite its reduced binding affinity for nucleic acid. Steady state unwinding rates reflect a number of possible steps in the reaction, including association, dissociation, and DNA unwinding, so it is not possible to state that NS3h DDDD unwinding is the same as the wild type NS3h based solely on this experiment. However, the result with NS3h DDDD does indicate that it can unwind the substrate.

[0224] The uncertainties associated with measuring unwinding under steady state conditions can be overcome by measuring unwinding under single turnover conditions in the presence of excess enzyme (43, 49). Initial attempts to perform single turnover experiments were hampered by substantial ATP-independent unwinding with wild type NS3h (data not shown), consistent with previous reports (57). To compare the ATP-independent unwinding activity of the mutant and wild type forms of NS3h, excess enzyme was incubated with substrate in the absence of ATP, and the reaction was stopped by addition of excess poly dT to trap the enzyme. Interestingly, NS3h exhibited much greater ATP-independent unwinding than NS3h KDA or NS3h DDDD (FIG. 13). This result may reflect reduced protein-protein interactions in the case of NS3h KDA, because the nucleic acid binding affinity of this mutant is the same as the wild type NS3h (FIG. 10).

[0225] A different experimental protocol was required to measure the unwinding activity under single turnover conditions in the presence of excess enzyme due to the ATP-independent unwinding activity of NS3h. NS3h was rapidly mixed with substrate and incubated for a 10 s interval, followed by a second rapid mixing step in which ATP and Mg.sup.+2 were introduced (FIG. 14). The initial 10 s incubation time was too short for ATP-independent unwinding to occur. The Kintek Chemical Quench-Flow instrument is designed to readily perform such a `double-mixing` experiment which was recently used by Levin et al. to measure NS3h unwinding activity (49). Under these conditions, NS3h exhibited a lag phase very similar to that observed previously for a substrate of similar length (49). The lag phase represents multiple steps that are believed to occur prior to complete unwinding of the duplex (52, 53, 58). No unwinding was observed for NS3h DDDD under single turnover conditions (not shown). However, NS3h KDA unwound the substrate with a similar lag phase as the wild type enzyme, but significantly lower amplitude (FIG. 14). The amplitude for unwinding reflects the degree of processivity of the enzyme (49, 59); therefore NS3h KDA has lower processivity than NS3h.

DISCUSSION

[0226] We describe here the first x-ray crystal structure of NS3 helicase bound to a DNA molecule of sufficient length to accommodate binding of two molecules of enzyme. Our structure shows a nucleic acid binding site for each monomer that is consistent with a published crystal structure of a single monomer bound to a shorter DNA molecule (41). In each case, the DNA binds to a cleft between domains 1 and 2 on one side and domain 3 on the other. DNA binding is non-sequence specific, with the majority of protein-DNA contacts involving the phosphate backbone. The structure also reveals a protein-protein interface between two DNA-bound helicase monomers. The protein-protein contacts are mostly hydrophobic and involve domain 2 of one subunit (chain A) and domain 3 of the second subunit (chain B). The two nucleic acid binding sites of the dimer are therefore aligned in such a way as to force a significant bend in the bound DNA that appears to induce a base stacking motif in the oligonucleotide.

[0227] The structure and function HCV helicase have been investigated extensively. Important amino acid sequence motifs that play critical roles in biochemical function have been identified on both domains 1 and 2. The function of domain 3 has been less well characterized, presumably due to the facts that in superfamily II helicases, domain 3 is less conserved compared to domains 1 and 2 and that among the helicase superfamilies, there is no apparent homology in domain 3 (60). In the structure reported here, surface regions on domain 3 interact with domain 2 of the second molecule. Biological studies demonstrate that mutations in these surface regions interfere with viral replication in cell culture. This indicates that, besides its critical role in substrate binding, domain 3 also plays a role in mediating the formation of protein-protein complexes between NS3 monomers or between NS3 and other protein partners to ensure viral survival and growth.

[0228] NS3h exhibits relatively weak protein-protein interactions whereas full-length NS3 appears to interact with itself much more strongly (Raney and Chen, in preparation), which may account for some of the differences in activities observed between the two forms of the enzyme. For example, NS3h unwound only 17% of the 45:30mer whereas full-length NS3 unwound greater than 80% of the same substrate under the same conditions (unpublished data). Therefore, NS3h exhibits lower processivity in DNA unwinding than the full-length protein, which may reflect the relative strength of protein-protein interactions. Monomeric helicases are generally associated with low processivity; meaning that these enzymes can unwind only a few base pairs prior to dissociating from the DNA. Moderately processive helicases such as hexameric helicase DnaB (58) are able to unwinding hundreds of base pairs prior to dissociating from the DNA, although the replicative helicases are much more processive when associated with the replication complex. One highly processive helicase, RecBCD, has two helicase motors, one for each DNA strand, which leads to very high processivity (61, 62). The Rep helicase reportedly is unable to unwind DNA as a monomer, but readily melts the duplex as a dimer (63). The mutations at the interface of the putative dimer were designed to disrupt protein-protein interactions. These mutations would not be expected to disrupt the biochemical activities of monomeric NS3h. Indeed, NS3h KDA exhibits activities that mirror the wild-type enzyme in terms of nucleic acid binding, ATPase activity, and steady state DNA unwinding. The only biochemical activities of NS3h KDA that are reduced are processivity and ATP-independent unwinding, each of which would be expected to rely more heavily on protein-protein interactions. Thus, enzymatic activities of NS3h KDA are consistent with the biochemical relevance of the dimeric structure reported here. However, the reduction in amplitude for unwinding under single turnover conditions is less than two-fold, which would seem unlikely to be responsible for the dramatic reduction in HCV replicative capacity exhibited with the NS3h KDA mutations. Hence, the biochemical results are not sufficient to explain the biological results. The results with NS3h DDDD emphasize this point. NS3h DDDD has much lower affinity for nucleic acid than wild-type NS3h, resulting in essentially no product formation under single turnover unwinding conditions. However, the replicative capacity of the HCV replicon containing the DDDD mutation is greater than the HCV replicon containing KDA mutation. Thus, the biochemical activities of NS3h do not appear to reflect all of the biological activities of this enzyme. It remains a strong possibility that the protein surface implicated in formation of a dimeric helicase in vitro mediates additional interactions in vivo that are required for formation of a multi-protein viral replication complex.

[0229] By solving the x-ray crystal structure of two molecules of NS3 helicase domain bound to the same oligonucleotide, we have identified a region on the surface of the HCV NS3 helicase that is required for efficient viral replication. The amino residues in this region are highly conserved amongst isolates of HCV. The surface region appears to be capable of mediating protein-protein interactions, but does not appear to be essential in the nucleic acid binding or known enzymatic activities of NS3h. These data illustrate that NS3 has additional biochemical activities and/or protein-protein interactions in vivo that are not revealed by the known biochemical assays.

CONCLUSIONS

[0230] The hepatitis C virus non-structural protein 3 is a multi-functional enzyme with serine protease and DExD/H-box helicase domains. The helicase domain was crystallized in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two helicase molecules appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations introduced into domain 3 to disrupt the interface dramatically reduced replication capacity in a subgenomic replicon system. Purified mutant helicase exhibited lower processivity during DNA unwinding, consistent with the outcome predicted by the x-ray crystal structure. However, the overall biochemical activities of the mutant enzyme do not reflect the large reduction in HCV replication capacity seen in the biological experiment. Hence, the surface residues identified here, in addition to being essential for NS3-NS3 interactions that increase NS3 helicase activity, are probably required for a biological function of the helicase domain unrelated to known biochemical activities.

Example 4

A Peptide Inhibitor of NS3-NS3 Interactions Inhibits Hepatitis C Virus Replication

[0231] The data from the structural, biological, and biochemical data indicated the importance of domain 3 and in particular, the amino acid sequence from His541 through Lys551. This region of the protein adopts an alpha helical structure that appears to mediate protein-protein interactions (Example 3). We designed a peptide that mimicked this sequence in order to determine whether such a peptide could substitute for the alpha helix and thereby disrupt protein-protein interactions that are required for HCV replication. The peptide was 25 amino acids in length and contained the following amino acid sequence: HIDAHFLSQTK-GGG-YARAAARQARA (SEQ ID NO:2). The amino-terminal region of this peptide (HIDAHFLSQTK, SEQ ID NO:1) is identical to the sequence of residues 541-551 of the NS3 helicase (SEQ ID NO:3). This region is the `inhibitor` region of the peptide. The three glycines served as a linker between the inhibitor and a peptide sequence that has been shown to be able to improve the uptake of peptides in cells. The c-terminal domain (YARAAARQARA, SEQ ID NO:4) is referred to as the peptide transduction domain of the HCV inhibitor peptide. This sequence can be likely be substituted with other sequences.

[0232] The peptide was introduced into Huh-7 cells containing the HCV luciferase replicon. 48 hours after introduction of the peptide, luciferase activity was measured. The peptide strongly reduced the luciferase activity, indicating that HCV replication was strongly reduced (FIG. 15). These results indicate that it is possible to design small molecule inhibitors of protein-protein interactions that will reduce HCV replication. Such small molecules can be used for the treatment of HCV infection. Small molecules that are designed to mimic the activity of the peptide at sequence His 541 through Lys551 should act similarly to the observed activity of the HCV inhibitor peptide.

Example 5

An Adenoviral Vector for Genetic Therapy Using Mutant NS3 Expression in Vitro

[0233] A DNA encoding the D290A ATPase-deficient NS3 is cloned for expression in an adenovirus vector for gene therapy of HCV infection. Adenoviral vectors are a well characterized method of gene transfer in both cell culture and in vivo. Two advantages to the use of adenoviral vectors are (i) efficient transfer to multiple cell types and lines, and (ii) efficient transfer to cells that are not actively replicating.

Vector Production

[0234] The adenoviral vectors are produced using commercially available methods and materials, including the pAdEasy-1 vector system from Stratagene (La Jolla, Calif.) (64, 65). The D290A NS3 DNA is cloned behind the cauliflower mosaic virus (CMV) promoter in pShuttle-CMV (64) (SEQ ID NO:5) in E. coli. The CMV promoter is nucleotides 345-932 of SEQ ID NO:5. The resultant vector is linearized by PmeI and cotransformed into E. coli strain BJ5183 with the adenoviral backbone plasmid pAdEasy-1 (SEQ ID NO:7), which lacks the E1 and E3 genes, making it replication defective without those functions being provided in trans. The shuttle vector recombines into pAdEasy-1 by homologous recombination in vivo in E. coli. The recombinant vector carries a kanamycin resistance cassette. The pAdEasy-1 vector carries an ampicillin-resistance cassette that is lost in the recombination. E. coli colonies resistant to kanamycin are selected, followed by purification of plasmid. Recombinant adenoviral plasmids are screened by restriction digestion with PacI, resulting in fragments of 30 kb and approximately 3-4.5 kb. Non-recombinant plasmids have only the 30 kb fragment.

[0235] Once recombinant adenoviral plasmid clones are identified, they are digested with PacI and transfected into HEK293 cells. HEK293 cells are human embryonic kidney cells that have been transformed with sheared Ad5 DNA. They express the transforming genes of Ad5, including E1. The cells produce recombinant viral particles that can be used to transform other cells. Titer is determined by protocols of the product literature.

Cellular Assay

[0236] Similar to the assay of Example 1, Huh-7 cells harboring subgenomic HCV replicon are exposed to a concentration series of recombinant mutant-NS3-expressing adenoviral particles. Luciferase activity is measured after 48 hours, as described above. Green fluorescent protein (GFP) is encoded in the adenovirus vector and is used to determine the efficiency of transfection. To verify that the NS3 construct is being expressed, Huh-7 cells that do not contain HCV replicon will also be exposed to viral vectors. These cells are lysed at 48 hours post-transfection, and western blots are performed on the lysate.

[0237] Replication of the HCV replicon is monitored by the luciferase assay described in Example 1 to show that adenovirus expressing ATPase-deficient NS3 inhibits HCV replication.

REFERENCE LIST

[0238] 1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 359-362 [0239] 2. Blanchard, E., Brand, D., Trassard, S., Goudeau, A., and Roingeard, P. (2002) J. Virol. 76, 4073-4079 [0240] 3. Tan, S. L., Pause, A., Shi, Y., and Sonenberg, N. (2002) Nat. Rev. Drug Discov. 1, 867-881 [0241] 4. Shimotohno, K. (2000) Semin. Cancer Biol. 10, 233-240 [0242] 5. Terrault, N. A. (2002) Hepatology 36, S99-105 [0243] 6. Blight K J, Kolykhalov A A, Reed K E, Agapov E V, Rice C M (1998) Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets. Antivir Ther 3: 71-81 [0244] 7. Reed, K. E., Grakoui, A., and Rice, C. M. (1995) J. Virol. 69, 4127-4136 [0245] 8. Rosenberg, S. (2001) J. Mol. Biol. 313, 451-464 [0246] 9. Xu, Z., Choi, J., Yen, T. S., Lu, W., Strohecker, A., Govindarajan, S., Chien, D., Selby, M. J., and Ou, J. (2001) EMBO J. 20, 3840-3848 [0247] 10. Caruthers, J. M., and McKay, D. B. (2002) Current Opinion in Structural Biology 12, 123-133 [0248] 11. Kwong, A. D., Kim, J. L., and Lin, C. (2000) Curr. Top. Microbiol. Immunol. 242, 171-196 [0249] 12. Cho, Y. G., Yang, S. H., and Sung, Y. C. (1998) J. Virol. Methods 72, 109-115 [0250] 13. Porter, D. J. (1998) J. Biol. Chem. 273, 14247-14253 [0251] 14. Porter, D. J. (1998) J. Biol. Chem. 273, 7390-7396 [0252] 15. Khu, Y. L., Koh, E., Lim, S. P., Tan, Y. H., Brenner, S., Lim, S. G., Hong, W. J., and Goh, P. Y. (2001) J. Virol. 75, 205-214 [0253] 16. Levin, M. K. and Patel, S. S. (1999) The helicase from hepatitis C virus is active as an oligomer. J. Biol. Chem. 274, 31839-31846 [0254] 17. Bartenschlager, R. (2002) Virus Res. 82, 25-32 [0255] 18. Blight, K. J., McKeating, J. A., and Rice, C. M. (2002) J. Virol. 76, 13001-13014 [0256] 19. Krieger, N., Lohmann, V., and Bartenschlager, R. (2001) J. Virol. 75, 4614-4624 [0257] 20. Tackett, A. J., Wei, L., Cameron, C. E., and Raney, K. D. (2001) Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex. Nucleic Acids Res. 29, 565-572 [0258] 21. Morris, P. D., Tackett, A. J., Babb, K., Nanduri, B., Chick, C., Scott, J., and Raney, K. D. (2001) J. Biol. Chem. 276, 19691-19698 [0259] 22. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K. D., and McCarthy, J. E. (2002) Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 277, 45670-45679 [0260] 23. Raney, K. D. and Benkovic, S. J. (1995) J. Biol. Chem. 270, 22236-22242 [0261] 24. He, Y., Yan, W., Coito, C., Li, Y., Gale, M., Jr., and Katze, M. G. (2003) J. Gen. Virol. 84, 535-543 [0262] 25. He, Y., Yan, W., Coito, C., Li, Y., Gale, M., Jr., and Katze, M. G. (2003) J. Gen. Virol. 84, 535-543 [0263] 26. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., and Bartenschlager, R. (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110-113 [0264] 27. Goregaoker, S. P. and Culver, J. N. (2003) J. Virol. 77, 3549-3556 [0265] 28. Ng, P., Parks, R. J., and Graham, F. L. (2002) Preparation of helper-dependent adenoviral vectors, pp. 371-388 in Gene Therapy Protocols, J. R. Morgan ed., Humana Press, Totowa, N.J. [0266] 29. Sandig, V., et al. (2000) Proc. Nat'l Acad. Sci. USA 97, 1002-1007 [0267] 30. Cristiano, R. J. et al. (1993) Proc. Nat'l. Acad. Sci. USA 90, 11548-11552 [0268] 31. Wu, C. H., Walton, C. M., and Wu, G. Y. (2002) Targeted gene transfer to liver using protein-DNA complexes, pp. 15-23 in Gene Therapy Protocols, J. R. Morgan ed., Humana Press, Totowa, N.J. [0269] 32. Templeton, N. S., and Lasic, D. D., eds., (2000) Gene Therapy: Therapeutic Mechanisms and Strategies, Marcel Dekker, Inc., New York. [0270] 33. Zhang, G., et al. (2002) Meth. Enzymol. 346, 125 [0271] 34. Bianchi et al. (1996) Anal. Biochem. 237, 239-244 [0272] 35. Lin C, Thomson J A, Rice C M (1995) A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol 69: 4373-4380 [0273] 36. Qin W, Luo H, Nomura T, Hayashi N, Yamashita T, Murakami S (2002) Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase. J Biol Chem 277: 2132-2137 [0274] 37. Mottola G, Cardinali G, Ceccacci A, Trozzi C, Bartholomew L, Torrisi M R, Pedrazzini E, Bonatti S, Migliaccio G (2002) Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293: 31-43 [0275] 38. Shi S T, Lee K J, Aizaki H, Hwang S B, Lai M M (2003) Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77: 4160-4168 [0276] 39. Kim D W, Gwack Y, Han J H, Choe J (1995) C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215: 160-166 [0277] 40. Cho H S, Ha N C, Kang L W, Chung K M, Back S H, Jang S K, Oh B H (1998) Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem 273: 15045-15052 [0278] 41. Kim J L, Morgenstern K A, Griffith J P, Dwyer M D, Thomson J A, Murcko M A, Lin C, Caron PR (1998) Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6: 89-100 [0279] 42. Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkuhler C, De Francesco R (1998) Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. J Virol 72: 6758-6769 [0280] 43. Serebrov V, Pyle A M (2004) Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430: 476-480 [0281] 44. Locatelli G A, Spadari S, Maga G (2002) Hepatitis C virus NS3 ATPase/helicase: an ATP switch regulates the cooperativity among the different substrate binding sites. Biochemistry 41: 10332-10342 [0282] 45. Frick D N, Rypma R S, Lam A M, Gu B (2004) The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Biol Chem 279: 1269-1280 [0283] 46. Yao N, Reichert P, Taremi S S, Prosise W W, Weber P C (1999) Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure Fold Des 7: 1353-1363 [0284] 48. Levin M K, Patel S S (2002) Helicase from hepatitis C virus, energetics of DNA binding. J Biol Chem 277: 29377-29385 [0285] 49. Levin M K, Wang Y H, Patel S S (2004) The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J Biol Chem 279: 26005-26012 [0286] 49b. Navaza J (2001) Implementation of molecular replacement in AMoRe. Acta Crystallogr D Biol Crystallogr 57: 1367-1372 [0287] 50. Brunger A T, Adams P D, Clore G M, DeLano W L, Gros P, Grosse-Kunstleve R W, Jiang J S, Kuszewski J, Nilges M, Pannu N S, Read R J, Rice L M, Simonson T, Warren G L (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54 (Pt 5): 905-921 [0288] 51. McRee D E (1999) XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 125: 156-165 [0289] 52. Ali J A, Lohman T M (1997) Kinetic Measurement of the Step Size of DNA Unwinding by Escherichia coli UvrD Helicase. Science 275: 377-380 [0290] 53. Lucius A L, Maluf N K, Fischer C J, Lohman T M (2003) General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding. Biophys J 85: 2224-39 [0291] 54. Hooft R W, Vriend G, Sander C, Abola E E (1996) Errors in protein structures. Nature 381: 272-- [0292] 55. Parkinson G (1996) New Parameters for the Refinement of Nucleic Acid-Containing Structures. Acta Crystallographica D52: 57-64 [0293] 55b. Vriend G (1990) WHAT IF: a molecular modeling and drug design program. J. Mol Graph 8: 52-6, 29 [0294] 56. Blight K J, Kolykhalov A A, Rice C M (2000) Efficient initiation of HCV RNA replication in cell culture. Science 290: 1972-1974 [0295] 57. Porter D J, Preugschat F (2000) Strand-separating activity of hepatitis C virus helicase in the absence of ATP. Biochemistry 39: 5166-5173 [0296] 58. Galletto R, Jezewska M J, Bujalowski W (2004) Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method. J Mol Biol 343: [0297] 59. Pang P S, Jankowsky E, Planet P J, Pyle A M (2002) The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21: 1168-1176 [0298] 60. Singleton M R, Wigley D B (2002) Modularity and specialization in superfamily 1 and 2 helicases. J Bacteriol 184: 1819-1826 [0299] 61. Dillingham M S, Spies M, Kowalczykowski S C (2003) RecBCD enzyme is a bipolar DNA helicase. Nature 423: 893-897 [0300] 62. Singleton M R, Dillingham M S, Gaudier M, Kowalczykowski S C, Wigley D B (2004) Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432: 187-193 [0301] 63. Ha T, Rasnik I, Cheng W, Babcock H P, Gauss G H, Lohman T M, Chu S (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419: 638-41 [0302] 64. Hosfield T and Eldrige L, Generate adenovirus vectors in E. coli by homologous recombination with the AdEasy adenoviral vector system. Strategies 13: 100-102 [0303] 65. He T-C, Zho S, Da Costa L T, Yu J, Kinzler K W, and Vogelstein B (1998) A simplified system for generating recombinant adenovirus. Proc. Natl. Acad. Sci. USA 95: 2509-14 [0304] 66. Thoren P E, Persson D, Lincoln P, and Norden B (2005) Membrane destabilizing properties of cell-penetrating peptides. Biophys. Chem. 114: 169-179 [0305] 67. Thoren P E, Persson D, Isakson P, Goksor M, Onfelt A, and Norden B (2003) Uptake of analogs of pentratin, Tat(48-60) and oligoarginine in live cells. Biochem. Biophys. Res. Commun. 307: 100-107 [0306] 68. Goodford P J (1985) A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules. J. Med. Chem. 28: 849-857 [0307] 69. Miranker A and Karplus M (1991) Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method. Proteins: Structure. Function and Genetics 11: 29-34 [0308] 70. Goodsell D S and Olsen A J (1990) Automated Docking of Substrates to Proteins by Simulated Annealing. Proteins: Structure. Function and Genetics 8: [0309] 71. Kuntz I D et al. (1982) A Geometric Approach to Macromolecule-Ligand Interactions. J. Mol. Biol. 161: 269-288 [0310] 72. Rusinko A (1993) Chem. Des. Auto. News 8: 44-47 [0311] 73. Bartlett P A et al (1989) CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules, in Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78: 182-196 [0312] 74. Martin Y C (1992) 3D Database Searching in Drug Design. J. Med. Chem. 35: [0313] 75. Bohm H-J (1992) The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors. J. Comp. Aid. Molec. Design 6: 61-78 [0314] 76. Nishibata Y and Itai A (1991) Tetrahedron 47: 8985 [0315] 77. Cohen N C et al. (1990) Molecular Modeling Software and Methods for Medicinal Chemistry. J. Med. Chem. 33: 883-894 [0316] 78. Navia M A and Murcko M A (1992) The Use of Structural Information in Drug Design. Current Opinions in Structural Biology 2: 202-210 [0317] 79. Farmer P S (1980) Drug Design, Ariens, E. J., ed., Vol. 10, pp 119-143 (Academic Press, New York, 1980). [0318] 80. Verlinde C (1994) Structure 2: 577-587 [0319] 81. Kuntz I D (1992) Science 257: 1078-1082

[0320] All patents, patent-related documents, and references cited herein are hereby incorporated by reference.

Sequence CWU 1

1

8111PRTArtificialNS3 oligomerization inhibitor peptide 1His Ile Asp Ala His Phe Leu Ser Gln Thr Lys1 5 10225PRTArtificialNS3-oligomerization inhibitor peptide with cell entry peptide 2His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gly Gly Gly Tyr Ala1 5 10 15Arg Ala Ala Ala Arg Gln Ala Arg Ala20 253631PRTHepatitis C virus 3Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys1 5 10 15Ile Ile Thr Ser Leu Thr Gly Arg Asp Arg Asn Gln Val Glu Gly Glu20 25 30Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val35 40 45Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu50 55 60Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln65 70 75 80Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro85 90 95Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp100 105 110Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser115 120 125Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu130 135 140Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr145 150 155 160Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu165 170 175Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala180 185 190Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser195 200 205Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys210 215 220Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala225 230 235 240Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val245 250 255Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys260 265 270Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile275 280 285Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly290 295 300Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu305 310 315 320Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile325 330 335Glu Glu Val Ala Leu Ser Ser Thr Gly Glu Ile Pro Phe Tyr Gly Lys340 345 350Ala Ile Pro Ile Glu Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys355 360 365His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu370 375 380Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile385 390 395 400Pro Thr Ser Gly Asp Val Ile Val Val Ala Thr Asp Ala Leu Met Thr405 410 415Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val420 425 430Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr435 440 445Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg450 455 460Thr Gly Arg Gly Arg Met Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu465 470 475 480Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp485 490 495Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg500 505 510Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His515 520 525Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala530 535 540His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu545 550 555 560Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro565 570 575Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu580 585 590His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu595 600 605Val Thr Thr Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser610 615 620Ala Asp Leu Glu Val Val Thr625 630411PRTArtificialCell entry peptide 4Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala1 5 1057453DNAArtificialpShuttle-CMV 5catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa nnnntaatag taatcaatta 360cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 420gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 480ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 540ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 600atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 660cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 720acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 780acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 840actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 900gagctggttt agtgaaccgt cagatccgct agagatctgg taccgtcgac gcggccgctc 960gagcctaagc ttctagataa gatatccgat ccaccggatc tagataactg atcataatca 1020gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga 1080acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg 1140gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 1200ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cnnnntaagg gtgggaaaga 1260atatataagg tgggggtctt atgtagtttt gtatctgttt tgcagcagcc gccgccgcca 1320tgagcaccaa ctcgtttgat ggaagcattg tgagctcata tttgacaacg cgcatgcccc 1380catgggccgg ggtgcgtcag aatgtgatgg gctccagcat tgatggtcgc cccgtcctgc 1440ccgcaaactc tactaccttg acctacgaga ccgtgtctgg aacgccgttg gagactgcag 1500cctccgccgc cgcttcagcc gctgcagcca ccgcccgcgg gattgtgact gactttgctt 1560tcctgagccc gcttgcaagc agtgcagctt cccgttcatc cgcccgcgat gacaagttga 1620cggctctttt ggcacaattg gattctttga cccgggaact taatgtcgtt tctcagcagc 1680tgttggatct gcgccagcag gtttctgccc tgaaggcttc ctcccctccc aatgcggttt 1740aaaacataaa taaaaaacca gactctgttt ggatttggat caagcaagtg tcttgctgtc 1800tttatttagg ggttttgcgc gcgcggtagg cccgggacca gcggtctcgg tcgttgaggg 1860tcctgtgtat tttttccagg acgtggtaaa ggtgactctg gatgttcaga tacatgggca 1920taagcccgtc tctggggtgg aggtagcacc actgcagagc ttcatgctgc ggggtggtgt 1980tgtagatgat ccagtcgtag caggagcgct gggcgtggtg cctaaaaatg tctttcagta 2040gcaagctgat tgccaggggc aggcccttgg tgtaagtgtt tacaaagcgg ttaagctggg 2100atgggtgcat acgtggggat atgagatgca tcttggactg tatttttagg ttggctatgt 2160tcccagccat atccctccgg ggattcatgt tgtgcagaac caccagcaca gtgtatccgg 2220tgcacttggg aaatttgtca tgtagcttag aaggaaatgc gtggaagaac ttggagacgc 2280ccttgtgacc tccaagattt tccatgcatt cgtccataat gatggcaatg ggcccacggg 2340cggcggcctg ggcgaagata tttctgggat cactaacgtc atagttgtgt tccaggatga 2400gatcgtcata ggccattttt acaaagcgcg ggcggagggt gccagactgc ggtataatgg 2460ttccatccgg cccaggggcg tagttaccct cacagatttg catttcccac gctttgagtt 2520cagatggggg gatcatgtct acctgcgggg cgatgaagaa aacggtttcc ggggtagggg 2580agatcagctg ggaagaaagc aggttcctga gcagctgcga cttaccgcag ccggtgggcc 2640cgtaaatcac acctattacc gggtgcaact ggtagttaag agagctgcag ctgccgtcat 2700ccctgagcag gggggccact tcgttaagca tgtccctgac tcgcatgttt tccctgacca 2760aatccgccag aaggcgctcg ccgcccagcg atagcagttc ttgcaaggaa gcaaagtttt 2820tcaacggttt gagaccgtcc gccgtaggca tgcttttgag cgtttgacca agcagttcca 2880ggcggtccca cagctcggtc acctgctcta cggcatctcg atccagcata tctcctcgtt 2940tcgcgggttg gggcggcttt cgctgtacgg cagtagtcgg tgctcgtcca gacgggccag 3000ggtcatgtct ttccacgggc gcagggtcct cgtcagcgta gtctgggtca cggtgaaggg 3060gtgcgctccg ggctgcgcgc tggccagggt gcgcttgagg ctggtcctgc tggtgctgaa 3120gcgctgccgg tcttcgccct gcgcgtcggc caggtagcat ttgaccatgg tgtcatagtc 3180cagcccctcc gcggcgtggc ccttggcgcg cagcttgccc ttggaggagg cgccgcacga 3240ggggcagtgc agacttttga gggcgtagag cttgggcgcg agaaataccg attccgggga 3300gtaggcatcc gcgccgcagg ccccgcagac ggtctcgcat tccacgagcc aggtgagctc 3360tggccgttcg gggtcaaaaa ccaggtttcc cccatgcttt ttgatgcgtt tcttacctct 3420ggtttccatg agccggtgtc cacgctcggt gacgaaaagg ctgtccgtgt ccccgtatac 3480agacttgaga gggagtttaa acgaattcaa tagcttgttg catgggcggc gatataaaat 3540gcaaggtgct gctcaaaaaa tcaggcaaag cctcgcgcaa aaaagaaagc acatcgtagt 3600catgctcatg cagataaagg caggtaagct ccggaaccac cacagaaaaa gacaccattt 3660ttctctcaaa catgtctgcg ggtttctgca taaacacaaa ataaaataac aaaaaaacat 3720ttaaacatta gaagcctgtc ttacaacagg aaaaacaacc cttataagca taagacggac 3780tacggccatg ccggcgtgac cgtaaaaaaa ctggtcaccg tgattaaaaa gcaccaccga 3840cagctcctcg gtcatgtccg gagtcataat gtaagactcg gtaaacacat caggttgatt 3900catcggtcag tgctaaaaag cgaccgaaat agcccggggg aatacatacc cgcaggcgta 3960gagacaacat tacagccccc ataggaggta taacaaaatt aataggagag aaaaacacat 4020aaacacctga aaaaccctcc tgcctaggca aaatagcacc ctcccgctcc agaacaacat 4080acagcgcttc acagcggcag cctaacagtc agccttacca gtaaaaaaga aaacctatta 4140aaaaaacacc actcgacacg gcaccagctc aatcagtcac agtgtaaaaa agggccaagt 4200gcagagcgag tatatatagg actaaaaaat gacgtaacgg ttaaagtcca caaaaaacac 4260ccagaaaacc gcacgcgaac ctacgcccag aaacgaaagc caaaaaaccc acaacttcct 4320caaatcgtca cttccgtttt cccacgttac gtaacttccc attttaagaa aactacaatt 4380cccaacacat acaagttact ccgccctaaa acctacgtca cccgccccgt tcccacgccc 4440cgcgccacgt cacaaactcc accccctcat tatcatattg gcttcaatcc aaaataaggt 4500atattattga tgatgttaat taacatgcat ggatccatat gcggtgtgaa ataccgcaca 4560gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 4620tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4680tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4740ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4800agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4860accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4920ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 4980gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5040ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 5100gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5160taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 5220tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5280gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5340cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5400agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5460cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5520cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5580ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5640taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 5700tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5760ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 5820atagtttgcg caacgttgtt gccattgctg cagccatgag attatcaaaa aggatcttca 5880cctagatcct tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg 5940tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt 6000gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg 6060aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg 6120ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat 6180gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 6240tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 6300tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 6360ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 6420cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 6480aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 6540tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 6600aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 6660atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 6720cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 6780tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 6840accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 6900gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 6960tctatcgcct tcttgacgag ttcttctgaa ttttgttaaa atttttgtta aatcagctca 7020ttttttaacc aataggccga aatcggcaac atcccttata aatcaaaaga atagaccgcg 7080atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 7140aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 7200aaatcaagtt ttttgcggtc gaggtgccgt aaagctctaa atcggaaccc taaagggagc 7260ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 7320gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 7380acacccgcgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggatcgaatt 7440aattcttaat taa 745369605DNAHepatitis C virus 6gccagccccc gattgggggc gacactccac catagatcac tcccctgtga ggaactactg 60tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc 240gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac 360ctcaaagaaa aaccaaacgt aacaccaacc gccgcccaca ggacgtcaag ttcccgggcg 420gtggtcagat cgtcggtgga gtttacctgt tgccgcgcag gggccccagg ttgggtgtgc 480gcgcgactag gaagacttcc gagcggtcgc aacctcgtgg aaggcgacaa cctatcccca 540aggctcgcca gcccgagggt agggcctggg ctcagcccgg gtacccctgg cccctctatg 600gcaatgaggg cttggggtgg gcaggatggc tcctgtcacc ccgtggctct cggcctagtt 660ggggccccac ggacccccgg cgtaggtcgc gcaatttggg taaggtcatc gataccctca 720cgtgcggctt cgccgatctc atggggtaca ttccgctcgt cggcgccccc ctagggggcg 780ctgccagggc cctggcgcat ggcgtccggg ttctggagga cggcgtgaac tatgcaacag 840ggaatctgcc cggttgctcc ttttctatct tccttttggc tttgctgtcc tgtttgacca 900tcccagcttc cgcttatgaa gtgcgcaacg tatccggagt gtaccatgtc acgaacgact 960gctccaacgc aagcattgtg tatgaggcag cggacatgat catgcatacc cccgggtgcg 1020tgccctgcgt tcgggagaac aactcctccc gctgctgggt agcgctcact cccacgctcg 1080cggccaggaa cgctagcgtc cccactacga cgatacgacg ccatgtcgat ttgctcgttg 1140gggcggctgc tctctgctcc gctatgtacg tgggagatct ctgcggatct gttttcctcg 1200tcgcccagct gttcaccttc tcgcctcgcc ggcacgagac agtacaggac tgcaattgct 1260caatatatcc cggccacgtg acaggtcacc gtatggcttg ggatatgatg atgaactggt 1320cacctacagc agccctagtg gtatcgcagt tactccggat cccacaagct gtcgtggata 1380tggtggcggg ggcccattgg ggagtcctag cgggccttgc ctactattcc atggtgggga 1440actgggctaa ggttctgatt gtgatgctac tctttgccgg cgttgacggg ggaacctatg 1500tgacaggggg gacgatggcc aaaaacaccc tcgggattac gtccctcttt tcacccgggt 1560catcccagaa aatccagctt gtaaacacca acggcagctg gcacatcaac aggactgccc 1620tgaactgcaa tgactccctc aacactgggt tccttgctgc gctgttctac gtgcacaagt 1680tcaactcatc tggatgccca gagcgcatgg ccagctgcag ccccatcgac gcgttcgctc 1740aggggtgggg gcccatcact tacaatgagt cacacagctc ggaccagagg ccttattgtt 1800ggcactacgc accccggccg tgcggtatcg tacccgcggc gcaggtgtgt ggtccagtgt 1860actgcttcac cccaagccct gtcgtggtgg ggacgaccga ccggttcggc gtccctacgt 1920acagttgggg ggagaatgag acggacgtgc tgcttcttaa caacacgcgg ccgccgcaag 1980gcaactggtt tggctgtaca tggatgaata gcactgggtt caccaagacg tgcgggggcc 2040ccccgtgtaa catcgggggg atcggcaata aaaccttgac ctgccccacg gactgcttcc 2100ggaagcaccc cgaggccact tacaccaagt gtggttcggg gccttggttg acacccagat 2160gcttggtcca ctacccatac aggctttggc actacccctg cactgtcaac tttaccatct 2220tcaaggttag gatgtacgtg gggggagtgg agcacaggct cgaagccgca tgcaattgga 2280ctcgaggaga gcgttgtaac ctggaggaca gggacagatc agagcttagc ccgctgctgc 2340tgtctacaac ggagtggcag gtattgccct gttccttcac caccctaccg gctctgtcca 2400ctggtttgat ccatctccat cagaacgtcg tggacgtaca atacctgtac ggtatagggt 2460cggcggttgt ctcctttgca atcaaatggg agtatgtcct gttgctcttc cttcttctgg 2520cggacgcgcg cgtctgtgcc tgcttgtgga tgatgctgct gatagctcaa gctgaggccg 2580ccctagagaa cctggtggtc ctcaacgcgg catccgtggc cggggcgcat ggcattctct 2640ccttcctcgt gttcttctgt gctgcctggt acatcaaggg caggctggtc cctggggcgg 2700catatgccct ctacggcgta tggccgctac tcctgctcct gctggcgtta ccaccacgag 2760catacgccat ggaccgggag atggcagcat cgtgcggagg cgcggttttc gtaggtctga 2820tactcttgac cttgtcaccg cactataagc tgttcctcgc taggctcata tggtggttac 2880aatattttat caccagggcc gaggcacact tgcaagtgtg gatccccccc ctcaacgttc 2940gggggggccg cgatgccgtc atcctcctca cgtgcgcgat ccacccagag ctaatcttta 3000ccatcaccaa aatcttgctc gccatactcg gtccactcat ggtgctccag gctggtataa 3060ccaaagtgcc gtacttcgtg cgcgcacacg ggctcattcg tgcatgcatg ctggtgcgga 3120aggttgctgg gggtcattat gtccaaatgg ctctcatgaa gttggccgca ctgacaggta 3180cgtacgttta tgaccatctc accccactgc gggactgggc ccacgcgggc ctacgagacc 3240ttgcggtggc agttgagccc gtcgtcttct ctgatatgga gaccaaggtt atcacctggg 3300gggcagacac cgcggcgtgt ggggacatca tcttgggcct gcccgtctcc gcccgcaggg 3360ggagggagat acatctggga ccggcagaca gccttgaagg gcaggggtgg cgactcctcg 3420cgcctattac ggcctactcc caacagacgc gaggcctact tggctgcatc atcactagcc

3480tcacaggccg ggacaggaac caggtcgagg gggaggtcca agtggtctcc accgcaacac 3540aatctttcct ggcgacctgc gtcaatggcg tgtgttggac tgtctatcat ggtgccggct 3600caaagaccct tgccggccca aagggcccaa tcacccaaat gtacaccaat gtggaccagg 3660acctcgtcgg ctggcaagcg ccccccgggg cgcgttcctt gacaccatgc acctgcggca 3720gctcggacct ttacttggtc acgaggcatg ccgatgtcat tccggtgcgc cggcggggcg 3780acagcagggg gagcctactc tcccccaggc ccgtctccta cttgaagggc tcttcgggcg 3840gtccactgct ctgcccctcg gggcacgctg tgggcatctt tcgggctgcc gtgtgcaccc 3900gaggggttgc gaaggcggtg gactttgtac ccgtcgagtc tatggaaacc actatgcggt 3960ccccggtctt cacggacaac tcgtcccctc cggccgtacc gcagacattc caggtggccc 4020atctacacgc ccctactggt agcggcaaga gcactaaggt gccggctgcg tatgcagccc 4080aagggtataa ggtgcttgtc ctgaacccgt ccgtcgccgc caccctaggt ttcggggcgt 4140atatgtctaa ggcacatggt atcgacccta acatcagaac cggggtaagg accatcacca 4200cgggtgcccc catcacgtac tccacctatg gcaagtttct tgccgacggt ggttgctctg 4260ggggcgccta tgacatcata atatgtgatg agtgccactc aactgactcg accactatcc 4320tgggcatcgg cacagtcctg gaccaagcgg agacggctgg agcgcgactc gtcgtgctcg 4380ccaccgctac gcctccggga tcggtcaccg tgccacatcc aaacatcgag gaggtggctc 4440tgtccagcac tggagaaatc cccttttatg gcaaagccat ccccatcgag accatcaagg 4500gggggaggca cctcattttc tgccattcca agaagaaatg tgatgagctc gccgcgaagc 4560tgtccggcct cggactcaat gctgtagcat attaccgggg ccttgatgta tccgtcatac 4620caactagcgg agacgtcatt gtcgtagcaa cggacgctct aatgacgggc tttaccggcg 4680atttcgactc agtgatcgac tgcaatacat gtgtcaccca gacagtcgac ttcagcctgg 4740acccgacctt caccattgag acgacgaccg tgccacaaga cgcggtgtca cgctcgcagc 4800ggcgaggcag gactggtagg ggcaggatgg gcatttacag gtttgtgact ccaggagaac 4860ggccctcggg catgttcgat tcctcggttc tgtgcgagtg ctatgacgcg ggctgtgctt 4920ggtacgagct cacgcccgcc gagacctcag ttaggttgcg ggcttaccta aacacaccag 4980ggttgcccgt ctgccaggac catctggagt tctgggagag cgtctttaca ggcctcaccc 5040acatagacgc ccatttcttg tcccagacta agcaggcagg agacaacttc ccctacctgg 5100tagcatacca ggctacggtg tgcgccaggg ctcaggctcc acctccatcg tgggaccaaa 5160tgtggaagtg tctcatacgg ctaaagccta cgctgcacgg gccaacgccc ctgctgtata 5220ggctgggagc cgttcaaaac gaggttacta ccacacaccc cataaccaaa tacatcatgg 5280catgcatgtc ggctgacctg gaggtcgtca cgagcacctg ggtgctggta ggcggagtcc 5340tagcagctct ggccgcgtat tgcctgacaa caggcagcgt ggtcattgtg ggcaggatca 5400tcttgtccgg aaagccggcc atcattcccg acagggaagt cctttaccgg gagttcgatg 5460agatggaaga gtgcgcctca cacctccctt acatcgaaca gggaatgcag ctcgccgaac 5520aattcaaaca gaaggcaatc gggttgctgc aaacagccac caagcaagcg gaggctgctg 5580ctcccgtggt ggaatccaag tggcggaccc tcgaagcctt ctgggcgaag catatgtgga 5640atttcatcag cgggatacaa tatttagcag gcttgtccac tctgcctggc aaccccgcga 5700tagcatcact gatggcattc acagcctcta tcaccagccc gctcaccacc caacataccc 5760tcctgtttaa catcctgggg ggatgggtgg ccgcccaact tgctcctccc agcgctgctt 5820ctgctttcgt aggcgccggc atcgctggag cggctgttgg cagcataggc cttgggaagg 5880tgcttgtgga tattttggca ggttatggag caggggtggc aggcgcgctc gtggccttta 5940aggtcatgag cggcgagatg ccctccaccg aggacctggt taacctactc cctgctatcc 6000tctcccctgg cgccctagtc gtcggggtcg tgtgcgcagc gatactgcgt cggcacgtgg 6060gcccagggga gggggctgtg cagtggatga accggctgat agcgttcgct tcgcggggta 6120accacgtctc ccccacgcac tatgtgcctg agagcgacgc tgcagcacgt gtcactcaga 6180tcctctctag tcttaccatc actcagctgc tgaagaggct tcaccagtgg atcaacgagg 6240actgctccac gccatgctcc ggctcgtggc taagagatgt ttgggattgg atatgcacgg 6300tgttgactga tttcaagacc tggctccagt ccaagctcct gccgcgattg ccgggagtcc 6360ccttcttctc atgtcaacgt gggtacaagg gagtctggcg gggcgacggc atcatgcaaa 6420ccacctgccc atgtggagca cagatcaccg gacatgtgaa aaacggttcc atgaggatcg 6480tggggcctag gacctgtagt aacacgtggc atggaacatt ccccattaac gcgtacacca 6540cgggcccctg cacgccctcc ccggcgccaa attattctag ggcgctgtgg cgggtggctg 6600ctgaggagta cgtggaggtt acgcgggtgg gggatttcca ctacgtgacg ggcatgacca 6660ctgacaacgt aaagtgcccg tgtcaggttc cggcccccga attcttcaca gaagtggatg 6720gggtgcggtt gcacaggtac gctccagcgt gcaaacccct cctacgggag gaggtcacat 6780tcctggtcgg gctcaatcaa tacctggttg ggtcacagct cccatgcgag cccgaaccgg 6840acgtagcagt gctcacttcc atgctcaccg acccctccca cattacggcg gagacggcta 6900agcgtaggct ggccagggga tctcccccct ccttggccag ctcatcagct agccagctgt 6960ctgcgccttc cttgaaggca acatgcacta cccgtcatga ctccccggac gctgacctca 7020tcgaggccaa cctcctgtgg cggcaggaga tgggcgggaa catcacccgc gtggagtcag 7080aaaataaggt agtaattttg gactctttcg agccgctcca agcggaggag gatgagaggg 7140aagtatccgt tccggcggag atcctgcgga ggtccaggaa attccctcga gcgatgccca 7200tatgggcacg cccggattac aaccctccac tgttagagtc ctggaaggac ccggactacg 7260tccctccagt ggtacacggg tgtccattgc cgcctgccaa ggcccctccg ataccacctc 7320cacggaggaa gaggacggtt gtcctgtcag aatctaccgt gtcttctgcc ttggcggagc 7380tcgccacaaa gaccttcggc agctccgaat cgtcggccgt cgacagcggc acggcaacgg 7440cctctcctga ccagccctcc gacgacggcg acgcgggatc cgacgttgag tcgtactcct 7500ccatgccccc ccttgagggg gagccggggg atcccgatct cagcgacggg tcttggtcta 7560ccgtaagcga ggaggctagt gaggacgtcg tctgctgctc gatgtcctac acatggacag 7620gcgccctgat cacgccatgc gctgcggagg aaaccaagct gcccatcaat gcactgagca 7680actctttgct ccgtcaccac aacttggtct atgctacaac atctcgcagc gcaagcctgc 7740ggcagaagaa ggtcaccttt gacagactgc aggtcctgga cgaccactac cgggacgtgc 7800tcaaggagat gaaggcgaag gcgtccacag ttaaggctaa acttctatcc gtggaggaag 7860cctgtaagct gacgccccca cattcggcca gatctaaatt tggctatggg gcaaaggacg 7920tccggaacct atccagcaag gccgttaacc acatccgctc cgtgtggaag gacttgctgg 7980aagacactga gacaccaatt gacaccacca tcatggcaaa aaatgaggtt ttctgcgtcc 8040aaccagagaa ggggggccgc aagccagctc gccttatcgt attcccagat ttgggggttc 8100gtgtgtgcga gaaaatggcc ctttacgatg tggtctccac cctccctcag gccgtgatgg 8160gctcttcata cggattccaa tactctcctg gacagcgggt cgagttcctg gtgaatgcct 8220ggaaagcgaa gaaatgccct atgggcttcg catatgacac ccgctgtttt gactcaacgg 8280tcactgagaa tgacatccgt gttgaggagt caatctacca atgttgtgac ttggcccccg 8340aagccagaca ggccataagg tcgctcacag agcggcttta catcgggggc cccctgacta 8400attctaaagg gcagaactgc ggctatcgcc ggtgccgcgc gagcggtgta ctgacgacca 8460gctgcggtaa taccctcaca tgttacttga aggccgctgc ggcctgtcga gctgcgaagc 8520tccaggactg cacgatgctc gtatgcggag acgaccttgt cgttatctgt gaaagcgcgg 8580ggacccaaga ggacgaggcg agcctacggg ccttcacgga ggctatgact agatactctg 8640ccccccctgg ggacccgccc aaaccagaat acgacttgga gttgataaca tcatgctcct 8700ccaatgtgtc agtcgcgcac gatgcatctg gcaaaagggt gtactatctc acccgtgacc 8760ccaccacccc ccttgcgcgg gctgcgtggg agacagctag acacactcca gtcaattcct 8820ggctaggcaa catcatcatg tatgcgccca ccttgtgggc aaggatgatc ctgatgactc 8880atttcttctc catccttcta gctcaggaac aacttgaaaa agccctagat tgtcagatct 8940acggggcctg ttactccatt gagccacttg acctacctca gatcattcaa cgactccatg 9000gccttagcgc attttcactc catagttact ctccaggtga gatcaatagg gtggcttcat 9060gcctcaggaa acttggggta ccgcccttgc gagtctggag acatcgggcc agaagtgtcc 9120gcgctaggct actgtcccag ggggggaggg ctgccacttg tggcaagtac ctcttcaact 9180gggcagtaag gaccaagctc aaactcactc caatcccggc tgcgtcccag ttggatttat 9240ccagctggtt cgttgctggt tacagcgggg gagacatata tcacagcctg tctcgtgccc 9300gaccccgctg gttcatgtgg tgcctactcc tactttctgt aggggtaggc atctatctac 9360tccccaaccg atgaacgggg agctaaacac tccaggccaa taggccatcc tgtttttttc 9420cctttttttt tttctttttt tttttttttt tttttttttt ttttttttct cctttttttt 9480tcctcttttt ttccttttct ttcctttggt ggctccatct tagccctagt cacggctagc 9540tgtgaaaggt ccgtgagccg cttgactgca gagagtgctg atactggcct ctctgcagat 9600caagt 9605733450DNAArtificialpAdEasy vector 7ttaattaaca tgcatggatc ctcgtctcga cgatgccctt gagagccttc aacccagtca 60gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta 120tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct 180ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc 240tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta 300tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg acgcgaggct 360ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgc 420aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa ggatcgctcg 480cggctcttac cagcctaact tcgatcactg gaccgctgat cgtcacggcg atttatgccg 540cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta taccttgtct 600gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg 660gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag 720aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag 780cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt 840gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga 900atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc 960aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc 1020gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac 1080acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg 1140ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc 1200atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa 1260cagaaattcc cccttacacg gaggcatcaa gtgaccaaac aggaaaaaac cgcccttaac 1320atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac 1380gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc 1440agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 1500acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 1560gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg 1620tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt 1680gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 1740cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 1800aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 1860aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 1920tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 1980caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 2040cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 2100ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 2160gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 2220agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 2280gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 2340acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 2400gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 2460gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 2520cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 2580caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 2640gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 2700cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 2760cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 2820caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 2880gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 2940gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3000cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 3060catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 3120gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 3180ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 3240gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 3300cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 3360tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 3420gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 3480atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 3540ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 3600gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 3660nnngaattcg aatctagtat cgattcgaan nncttaaggg tgggaaagaa tatataaggt 3720gggggtctta tgtagttttg tatctgtttt gcagcagccg ccgccgccat gagcaccaac 3780tcgtttgatg gaagcattgt gagctcatat ttgacaacgc gcatgccccc atgggccggg 3840gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc ccgtcctgcc cgcaaactct 3900actaccttga cctacgagac cgtgtctgga acgccgttgg agactgcagc ctccgccgcc 3960gcttcagccg ctgcagccac cgcccgcggg attgtgactg actttgcttt cctgagcccg 4020cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg acaagttgac ggctcttttg 4080gcacaattgg attctttgac ccgggaactt aatgtcgttt ctcagcagct gttggatctg 4140cgccagcagg tttctgccct gaaggcttcc tcccctccca atgcggttta aaacataaat 4200aaaaaaccag actctgtttg gatttggatc aagcaagtgt cttgctgtct ttatttaggg 4260gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt cgttgagggt cctgtgtatt 4320ttttccagga cgtggtaaag gtgactctgg atgttcagat acatgggcat aagcccgtct 4380ctggggtgga ggtagcacca ctgcagagct tcatgctgcg gggtggtgtt gtagatgatc 4440cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt ctttcagtag caagctgatt 4500gccaggggca ggcccttggt gtaagtgttt acaaagcggt taagctggga tgggtgcata 4560cgtggggata tgagatgcat cttggactgt atttttaggt tggctatgtt cccagccata 4620tccctccggg gattcatgtt gtgcagaacc accagcacag tgtatccggt gcacttggga 4680aatttgtcat gtagcttaga aggaaatgcg tggaagaact tggagacgcc cttgtgacct 4740ccaagatttt ccatgcattc gtccataatg atggcaatgg gcccacgggc ggcggcctgg 4800gcgaagatat ttctgggatc actaacgtca tagttgtgtt ccaggatgag atcgtcatag 4860gccattttta caaagcgcgg gcggagggtg ccagactgcg gtataatggt tccatccggc 4920ccaggggcgt agttaccctc acagatttgc atttcccacg ctttgagttc agatgggggg 4980atcatgtcta cctgcggggc gatgaagaaa acggtttccg gggtagggga gatcagctgg 5040gaagaaagca ggttcctgag cagctgcgac ttaccgcagc cggtgggccc gtaaatcaca 5100cctattaccg ggtgcaactg gtagttaaga gagctgcagc tgccgtcatc cctgagcagg 5160ggggccactt cgttaagcat gtccctgact cgcatgtttt ccctgaccaa atccgccaga 5220aggcgctcgc cgcccagcga tagcagttct tgcaaggaag caaagttttt caacggtttg 5280agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa gcagttccag gcggtcccac 5340agctcggtca cctgctctac ggcatctcga tccagcatat ctcctcgttt cgcgggttgg 5400ggcggctttc gctgtacggc agtagtcggt gctcgtccag acgggccagg gtcatgtctt 5460tccacgggcg cagggtcctc gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg 5520gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt 5580cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt gtcatagtcc agcccctccg 5640cggcgtggcc cttggcgcgc agcttgccct tggaggaggc gccgcacgag gggcagtgca 5700gacttttgag ggcgtagagc ttgggcgcga gaaataccga ttccggggag taggcatccg 5760cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca ggtgagctct ggccgttcgg 5820ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt cttacctctg gtttccatga 5880gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc cccgtataca gacttgagag 5940gcctgtcctc gagcggtgtt ccgcggtcct cctcgtatag aaactcggac cactctgaga 6000caaaggctcg cgtccaggcc agcacgaagg aggctaagtg ggaggggtag cggtcgttgt 6060ccactagggg gtccactcgc tccagggtgt gaagacacat gtcgccctct tcggcatcaa 6120ggaaggtgat tggtttgtag gtgtaggcca cgtgaccggg tgttcctgaa ggggggctat 6180aaaagggggt gggggcgcgt tcgtcctcac tctcttccgc atcgctgtct gcgagggcca 6240gctgttgggg tgagtactcc ctctgaaaag cgggcatgac ttctgcgcta agattgtcag 6300tttccaaaaa cgaggaggat ttgatattca cctggcccgc ggtgatgcct ttgagggtgg 6360ccgcatccat ctggtcagaa aagacaatct ttttgttgtc aagcttggtg gcaaacgacc 6420cgtagagggc gttggacagc aacttggcga tggagcgcag ggtttggttt ttgtcgcgat 6480cggcgcgctc cttggccgcg atgtttagct gcacgtattc gcgcgcaacg caccgccatt 6540cgggaaagac ggtggtgcgc tcgtcgggca ccaggtgcac gcgccaaccg cggttgtgca 6600gggtgacaag gtcaacgctg gtggctacct ctccgcgtag gcgctcgttg gtccagcaga 6660ggcggccgcc cttgcgcgag cagaatggcg gtagggggtc tagctgcgtc tcgtccgggg 6720ggtctgcgtc cacggtaaag accccgggca gcaggcgcgc gtcgaagtag tctatcttgc 6780atccttgcaa gtctagcgcc tgctgccatg cgcgggcggc aagcgcgcgc tcgtatgggt 6840tgagtggggg accccatggc atggggtggg tgagcgcgga ggcgtacatg ccgcaaatgt 6900cgtaaacgta gaggggctct ctgagtattc caagatatgt agggtagcat cttccaccgc 6960ggatgctggc gcgcacgtaa tcgtatagtt cgtgcgaggg agcgaggagg tcgggaccga 7020ggttgctacg ggcgggctgc tctgctcgga agactatctg cctgaagatg gcatgtgagt 7080tggatgatat ggttggacgc tggaagacgt tgaagctggc gtctgtgaga cctaccgcgt 7140cacgcacgaa ggaggcgtag gagtcgcgca gcttgttgac cagctcggcg gtgacctgca 7200cgtctagggc gcagtagtcc agggtttcct tgatgatgtc atacttatcc tgtccctttt 7260ttttccacag ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 7320gaaacccgtc ggcctccgaa cggtaagagc ctagcatgta gaactggttg acggcctggt 7380aggcgcagca tcccttttct acgggtagcg cgtatgcctg cgcggccttc cggagcgagg 7440tgtgggtgag cgcaaaggtg tccctgacca tgactttgag gtactggtat ttgaagtcag 7500tgtcgtcgca tccgccctgc tcccagagca aaaagtccgt gcgctttttg gaacgcggat 7560ttggcagggc gaaggtgaca tcgttgaaga gtatctttcc cgcgcgaggc ataaagttgc 7620gtgtgatgcg gaagggtccc ggcacctcgg aacggttgtt aattacctgg gcggcgagca 7680cgatctcgtc aaagccgttg atgttgtggc ccacaatgta aagttccaag aagcgcggga 7740tgcccttgat ggaaggcaat tttttaagtt cctcgtaggt gagctcttca ggggagctga 7800gcccgtgctc tgaaagggcc cagtctgcaa gatgagggtt ggaagcgacg aatgagctcc 7860acaggtcacg ggccattagc atttgcaggt ggtcgcgaaa ggtcctaaac tggcgaccta 7920tggccatttt ttctggggtg atgcagtaga aggtaagcgg gtcttgttcc cagcggtccc 7980atccaaggtt cgcggctagg tctcgcgcgg cagtcactag aggctcatct ccgccgaact 8040tcatgaccag catgaagggc acgagctgct tcccaaaggc ccccatccaa gtataggtct 8100ctacatcgta ggtgacaaag agacgctcgg tgcgaggatg cgagccgatc gggaagaact 8160ggatctcccg ccaccaattg gaggagtggc tattgatgtg gtgaaagtag aagtccctgc 8220gacgggccga acactcgtgc tggcttttgt aaaaacgtgc gcagtactgg cagcggtgca 8280cgggctgtac atcctgcacg aggttgacct gacgaccgcg cacaaggaag cagagtggga 8340atttgagccc ctcgcctggc gggtttggct ggtggtcttc tacttcggct gcttgtcctt 8400gaccgtctgg ctgctcgagg ggagttacgg tggatcggac caccacgccg cgcgagccca 8460aagtccagat gtccgcgcgc ggcggtcgga gcttgatgac aacatcgcgc agatgggagc 8520tgtccatggt ctggagctcc cgcggcgtca ggtcaggcgg gagctcctgc aggtttacct 8580cgcatagacg ggtcagggcg cgggctagat ccaggtgata cctaatttcc aggggctggt 8640tggtggcggc gtcgatggct tgcaagaggc cgcatccccg cggcgcgact acggtaccgc 8700gcggcgggcg gtgggccgcg ggggtgtcct tggatgatgc atctaaaagc ggtgacgcgg 8760gcgagccccc ggaggtaggg ggggctccgg acccgccggg agagggggca ggggcacgtc 8820ggcgccgcgc gcgggcagga gctggtgctg

cgcgcgtagg ttgctggcga acgcgacgac 8880gcggcggttg atctcctgaa tctggcgcct ctgcgtgaag acgacgggcc cggtgagctt 8940gagcctgaaa gagagttcga cagaatcaat ttcggtgtcg ttgacggcgg cctggcgcaa 9000aatctcctgc acgtctcctg agttgtcttg ataggcgatc tcggccatga actgctcgat 9060ctcttcctcc tggagatctc cgcgtccggc tcgctccacg gtggcggcga ggtcgttgga 9120aatgcgggcc atgagctgcg agaaggcgtt gaggcctccc tcgttccaga cgcggctgta 9180gaccacgccc ccttcggcat cgcgggcgcg catgaccacc tgcgcgagat tgagctccac 9240gtgccgggcg aagacggcgt agtttcgcag gcgctgaaag aggtagttga gggtggtggc 9300ggtgtgttct gccacgaaga agtacataac ccagcgtcgc aacgtggatt cgttgatatc 9360ccccaaggcc tcaaggcgct ccatggcctc gtagaagtcc acggcgaagt tgaaaaactg 9420ggagttgcgc gccgacacgg ttaactcctc ctccagaaga cggatgagct cggcgacagt 9480gtcgcgcacc tcgcgctcaa aggctacagg ggcctcttct tcttcttcaa tctcctcttc 9540cataagggcc tccccttctt cttcttctgg cggcggtggg ggagggggga cacggcggcg 9600acgacggcgc accgggaggc ggtcgacaaa gcgctcgatc atctccccgc ggcgacggcg 9660catggtctcg gtgacggcgc ggccgttctc gcgggggcgc agttggaaga cgccgcccgt 9720catgtcccgg ttatgggttg gcggggggct gccatgcggc agggatacgg cgctaacgat 9780gcatctcaac aattgttgtg taggtactcc gccgccgagg gacctgagcg agtccgcatc 9840gaccggatcg gaaaacctct cgagaaaggc gtctaaccag tcacagtcgc aaggtaggct 9900gagcaccgtg gcgggcggca gcgggcggcg gtcggggttg tttctggcgg aggtgctgct 9960gatgatgtaa ttaaagtagg cggtcttgag acggcggatg gtcgacagaa gcaccatgtc 10020cttgggtccg gcctgctgaa tgcgcaggcg gtcggccatg ccccaggctt cgttttgaca 10080tcggcgcagg tctttgtagt agtcttgcat gagcctttct accggcactt cttcttctcc 10140ttcctcttgt cctgcatctc ttgcatctat cgctgcggcg gcggcggagt ttggccgtag 10200gtggcgccct cttcctccca tgcgtgtgac cccgaagccc ctcatcggct gaagcagggc 10260taggtcggcg acaacgcgct cggctaatat ggcctgctgc acctgcgtga gggtagactg 10320gaagtcatcc atgtccacaa agcggtggta tgcgcccgtg ttgatggtgt aagtgcagtt 10380ggccataacg gaccagttaa cggtctggtg acccggctgc gagagctcgg tgtacctgag 10440acgcgagtaa gccctcgagt caaatacgta gtcgttgcaa gtccgcacca ggtactggta 10500tcccaccaaa aagtgcggcg gcggctggcg gtagaggggc cagcgtaggg tggccggggc 10560tccgggggcg agatcttcca acataaggcg atgatatccg tagatgtacc tggacatcca 10620ggtgatgccg gcggcggtgg tggaggcgcg cggaaagtcg cggacgcggt tccagatgtt 10680gcgcagcggc aaaaagtgct ccatggtcgg gacgctctgg ccggtcaggc gcgcgcaatc 10740gttgacgctc taccgtgcaa aaggagagcc tgtaagcggg cactcttccg tggtctggtg 10800gataaattcg caagggtatc atggcggacg accggggttc gagccccgta tccggccgtc 10860cgccgtgatc catgcggtta ccgcccgcgt gtcgaaccca ggtgtgcgac gtcagacaac 10920gggggagtgc tccttttggc ttccttccag gcgcggcggc tgctgcgcta gcttttttgg 10980ccactggccg cgcgcagcgt aagcggttag gctggaaagc gaaagcatta agtggctcgc 11040tccctgtagc cggagggtta ttttccaagg gttgagtcgc gggacccccg gttcgagtct 11100cggaccggcc ggactgcggc gaacgggggt ttgcctcccc gtcatgcaag accccgcttg 11160caaattcctc cggaaacagg gacgagcccc ttttttgctt ttcccagatg catccggtgc 11220tgcggcagat gcgcccccct cctcagcagc ggcaagagca agagcagcgg cagacatgca 11280gggcaccctc ccctcctcct accgcgtcag gaggggcgac atccgcggtt gacgcggcag 11340cagatggtga ttacgaaccc ccgcggcgcc gggcccggca ctacctggac ttggaggagg 11400gcgagggcct ggcgcggcta ggagcgccct ctcctgagcg gtacccaagg gtgcagctga 11460agcgtgatac gcgtgaggcg tacgtgccgc ggcagaacct gtttcgcgac cgcgagggag 11520aggagcccga ggagatgcgg gatcgaaagt tccacgcagg gcgcgagctg cggcatggcc 11580tgaatcgcga gcggttgctg cgcgaggagg actttgagcc cgacgcgcga accgggatta 11640gtcccgcgcg cgcacacgtg gcggccgccg acctggtaac cgcatacgag cagacggtga 11700accaggagat taactttcaa aaaagcttta acaaccacgt gcgtacgctt gtggcgcgcg 11760aggaggtggc tataggactg atgcatctgt gggactttgt aagcgcgctg gagcaaaacc 11820caaatagcaa gccgctcatg gcgcagctgt tccttatagt gcagcacagc agggacaacg 11880aggcattcag ggatgcgctg ctaaacatag tagagcccga gggccgctgg ctgctcgatt 11940tgataaacat cctgcagagc atagtggtgc aggagcgcag cttgagcctg gctgacaagg 12000tggccgccat caactattcc atgcttagcc tgggcaagtt ttacgcccgc aagatatacc 12060atacccctta cgttcccata gacaaggagg taaagatcga ggggttctac atgcgcatgg 12120cgctgaaggt gcttaccttg agcgacgacc tgggcgttta tcgcaacgag cgcatccaca 12180aggccgtgag cgtgagccgg cggcgcgagc tcagcgaccg cgagctgatg cacagcctgc 12240aaagggccct ggctggcacg ggcagcggcg atagagaggc cgagtcctac tttgacgcgg 12300gcgctgacct gcgctgggcc ccaagccgac gcgccctgga ggcagctggg gccggacctg 12360ggctggcggt ggcacccgcg cgcgctggca acgtcggcgg cgtggaggaa tatgacgagg 12420acgatgagta cgagccagag gacggcgagt actaagcggt gatgtttctg atcagatgat 12480gcaagacgca acggacccgg cggtgcgggc ggcgctgcag agccagccgt ccggccttaa 12540ctccacggac gactggcgcc aggtcatgga ccgcatcatg tcgctgactg cgcgcaatcc 12600tgacgcgttc cggcagcagc cgcaggccaa ccggctctcc gcaattctgg aagcggtggt 12660cccggcgcgc gcaaacccca cgcacgagaa ggtgctggcg atcgtaaacg cgctggccga 12720aaacagggcc atccggcccg acgaggccgg cctggtctac gacgcgctgc ttcagcgcgt 12780ggctcgttac aacagcggca acgtgcagac caacctggac cggctggtgg gggatgtgcg 12840cgaggccgtg gcgcagcgtg agcgcgcgca gcagcagggc aacctgggct ccatggttgc 12900actaaacgcc ttcctgagta cacagcccgc caacgtgccg cggggacagg aggactacac 12960caactttgtg agcgcactgc ggctaatggt gactgagaca ccgcaaagtg aggtgtacca 13020gtctgggcca gactattttt tccagaccag tagacaaggc ctgcagaccg taaacctgag 13080ccaggctttc aaaaacttgc aggggctgtg gggggtgcgg gctcccacag gcgaccgcgc 13140gaccgtgtct agcttgctga cgcccaactc gcgcctgttg ctgctgctaa tagcgccctt 13200cacggacagt ggcagcgtgt cccgggacac atacctaggt cacttgctga cactgtaccg 13260cgaggccata ggtcaggcgc atgtggacga gcatactttc caggagatta caagtgtcag 13320ccgcgcgctg gggcaggagg acacgggcag cctggaggca accctaaact acctgctgac 13380caaccggcgg cagaagatcc cctcgttgca cagtttaaac agcgaggagg agcgcatttt 13440gcgctacgtg cagcagagcg tgagccttaa cctgatgcgc gacggggtaa cgcccagcgt 13500ggcgctggac atgaccgcgc gcaacatgga accgggcatg tatgcctcaa accggccgtt 13560tatcaaccgc ctaatggact acttgcatcg cgcggccgcc gtgaaccccg agtatttcac 13620caatgccatc ttgaacccgc actggctacc gccccctggt ttctacaccg ggggattcga 13680ggtgcccgag ggtaacgatg gattcctctg ggacgacata gacgacagcg tgttttcccc 13740gcaaccgcag accctgctag agttgcaaca gcgcgagcag gcagaggcgg cgctgcgaaa 13800ggaaagcttc cgcaggccaa gcagcttgtc cgatctaggc gctgcggccc cgcggtcaga 13860tgctagtagc ccatttccaa gcttgatagg gtctcttacc agcactcgca ccacccgccc 13920gcgcctgctg ggcgaggagg agtacctaaa caactcgctg ctgcagccgc agcgcgaaaa 13980aaacctgcct ccggcatttc ccaacaacgg gatagagagc ctagtggaca agatgagtag 14040atggaagacg tacgcgcagg agcacaggga cgtgccaggc ccgcgcccgc ccacccgtcg 14100tcaaaggcac gaccgtcagc ggggtctggt gtgggaggac gatgactcgg cagacgacag 14160cagcgtcctg gatttgggag ggagtggcaa cccgtttgcg caccttcgcc ccaggctggg 14220gagaatgttt taaaaaaaaa aaagcatgat gcaaaataaa aaactcacca aggccatggc 14280accgagcgtt ggttttcttg tattcccctt agtatgcggc gcgcggcgat gtatgaggaa 14340ggtcctcctc cctcctacga gagtgtggtg agcgcggcgc cagtggcggc ggcgctgggt 14400tctcccttcg atgctcccct ggacccgccg tttgtgcctc cgcggtacct gcggcctacc 14460ggggggagaa acagcatccg ttactctgag ttggcacccc tattcgacac cacccgtgtg 14520tacctggtgg acaacaagtc aacggatgtg gcatccctga actaccagaa cgaccacagc 14580aactttctga ccacggtcat tcaaaacaat gactacagcc cgggggaggc aagcacacag 14640accatcaatc ttgacgaccg gtcgcactgg ggcggcgacc tgaaaaccat cctgcatacc 14700aacatgccaa atgtgaacga gttcatgttt accaataagt ttaaggcgcg ggtgatggtg 14760tcgcgcttgc ctactaagga caatcaggtg gagctgaaat acgagtgggt ggagttcacg 14820ctgcccgagg gcaactactc cgagaccatg accatagacc ttatgaacaa cgcgatcgtg 14880gagcactact tgaaagtggg cagacagaac ggggttctgg aaagcgacat cggggtaaag 14940tttgacaccc gcaacttcag actggggttt gaccccgtca ctggtcttgt catgcctggg 15000gtatatacaa acgaagcctt ccatccagac atcattttgc tgccaggatg cggggtggac 15060ttcacccaca gccgcctgag caacttgttg ggcatccgca agcggcaacc cttccaggag 15120ggctttagga tcacctacga tgatctggag ggtggtaaca ttcccgcact gttggatgtg 15180gacgcctacc aggcgagctt gaaagatgac accgaacagg gcgggggtgg cgcaggcggc 15240agcaacagca gtggcagcgg cgcggaagag aactccaacg cggcagccgc ggcaatgcag 15300ccggtggagg acatgaacga tcatgccatt cgcggcgaca cctttgccac acgggctgag 15360gagaagcgcg ctgaggccga agcagcggcc gaagctgccg cccccgctgc gcaacccgag 15420gtcgagaagc ctcagaagaa accggtgatc aaacccctga cagaggacag caagaaacgc 15480agttacaacc taataagcaa tgacagcacc ttcacccagt accgcagctg gtaccttgca 15540tacaactacg gcgaccctca gaccggaatc cgctcatgga ccctgctttg cactcctgac 15600gtaacctgcg gctcggagca ggtctactgg tcgttgccag acatgatgca agaccccgtg 15660accttccgct ccacgcgcca gatcagcaac tttccggtgg tgggcgccga gctgttgccc 15720gtgcactcca agagcttcta caacgaccag gccgtctact cccaactcat ccgccagttt 15780acctctctga cccacgtgtt caatcgcttt cccgagaacc agattttggc gcgcccgcca 15840gcccccacca tcaccaccgt cagtgaaaac gttcctgctc tcacagatca cgggacgcta 15900ccgctgcgca acagcatcgg aggagtccag cgagtgacca ttactgacgc cagacgccgc 15960acctgcccct acgtttacaa ggccctgggc atagtctcgc cgcgcgtcct atcgagccgc 16020actttttgag caagcatgtc catccttata tcgcccagca ataacacagg ctggggcctg 16080cgcttcccaa gcaagatgtt tggcggggcc aagaagcgct ccgaccaaca cccagtgcgc 16140gtgcgcgggc actaccgcgc gccctggggc gcgcacaaac gcggccgcac tgggcgcacc 16200accgtcgatg acgccatcga cgcggtggtg gaggaggcgc gcaactacac gcccacgccg 16260ccaccagtgt ccacagtgga cgcggccatt cagaccgtgg tgcgcggagc ccggcgctat 16320gctaaaatga agagacggcg gaggcgcgta gcacgtcgcc accgccgccg acccggcact 16380gccgcccaac gcgcggcggc ggccctgctt aaccgcgcac gtcgcaccgg ccgacgggcg 16440gccatgcggg ccgctcgaag gctggccgcg ggtattgtca ctgtgccccc caggtccagg 16500cgacgagcgg ccgccgcagc agccgcggcc attagtgcta tgactcaggg tcgcaggggc 16560aacgtgtatt gggtgcgcga ctcggttagc ggcctgcgcg tgcccgtgcg cacccgcccc 16620ccgcgcaact agattgcaag aaaaaactac ttagactcgt actgttgtat gtatccagcg 16680gcggcggcgc gcaacgaagc tatgtccaag cgcaaaatca aagaagagat gctccaggtc 16740atcgcgccgg agatctatgg ccccccgaag aaggaagagc aggattacaa gccccgaaag 16800ctaaagcggg tcaaaaagaa aaagaaagat gatgatgatg aacttgacga cgaggtggaa 16860ctgctgcacg ctaccgcgcc caggcgacgg gtacagtgga aaggtcgacg cgtaaaacgt 16920gttttgcgac ccggcaccac cgtagtcttt acgcccggtg agcgctccac ccgcacctac 16980aagcgcgtgt atgatgaggt gtacggcgac gaggacctgc ttgagcaggc caacgagcgc 17040ctcggggagt ttgcctacgg aaagcggcat aaggacatgc tggcgttgcc gctggacgag 17100ggcaacccaa cacctagcct aaagcccgta acactgcagc aggtgctgcc cgcgcttgca 17160ccgtccgaag aaaagcgcgg cctaaagcgc gagtctggtg acttggcacc caccgtgcag 17220ctgatggtac ccaagcgcca gcgactggaa gatgtcttgg aaaaaatgac cgtggaacct 17280gggctggagc ccgaggtccg cgtgcggcca atcaagcagg tggcgccggg actgggcgtg 17340cagaccgtgg acgttcagat acccactacc agtagcacca gtattgccac cgccacagag 17400ggcatggaga cacaaacgtc cccggttgcc tcagcggtgg cggatgccgc ggtgcaggcg 17460gtcgctgcgg ccgcgtccaa gacctctacg gaggtgcaaa cggacccgtg gatgtttcgc 17520gtttcagccc cccggcgccc gcgcggttcg aggaagtacg gcgccgccag cgcgctactg 17580cccgaatatg ccctacatcc ttccattgcg cctacccccg gctatcgtgg ctacacctac 17640cgccccagaa gacgagcaac tacccgacgc cgaaccacca ctggaacccg ccgccgccgt 17700cgccgtcgcc agcccgtgct ggccccgatt tccgtgcgca gggtggctcg cgaaggaggc 17760aggaccctgg tgctgccaac agcgcgctac caccccagca tcgtttaaaa gccggtcttt 17820gtggttcttg cagatatggc cctcacctgc cgcctccgtt tcccggtgcc gggattccga 17880ggaagaatgc accgtaggag gggcatggcc ggccacggcc tgacgggcgg catgcgtcgt 17940gcgcaccacc ggcggcggcg cgcgtcgcac cgtcgcatgc gcggcggtat cctgcccctc 18000cttattccac tgatcgccgc ggcgattggc gccgtgcccg gaattgcatc cgtggccttg 18060caggcgcaga gacactgatt aaaaacaagt tgcatgtgga aaaatcaaaa taaaaagtct 18120ggactctcac gctcgcttgg tcctgtaact attttgtaga atggaagaca tcaactttgc 18180gtctctggcc ccgcgacacg gctcgcgccc gttcatggga aactggcaag atatcggcac 18240cagcaatatg agcggtggcg ccttcagctg gggctcgctg tggagcggca ttaaaaattt 18300cggttccacc gttaagaact atggcagcaa ggcctggaac agcagcacag gccagatgct 18360gagggataag ttgaaagagc aaaatttcca acaaaaggtg gtagatggcc tggcctctgg 18420cattagcggg gtggtggacc tggccaacca ggcagtgcaa aataagatta acagtaagct 18480tgatccccgc cctcccgtag aggagcctcc accggccgtg gagacagtgt ctccagaggg 18540gcgtggcgaa aagcgtccgc gccccgacag ggaagaaact ctggtgacgc aaatagacga 18600gcctccctcg tacgaggagg cactaaagca aggcctgccc accacccgtc ccatcgcgcc 18660catggctacc ggagtgctgg gccagcacac acccgtaacg ctggacctgc ctccccccgc 18720cgacacccag cagaaacctg tgctgccagg cccgaccgcc gttgttgtaa cccgtcctag 18780ccgcgcgtcc ctgcgccgcg ccgccagcgg tccgcgatcg ttgcggcccg tagccagtgg 18840caactggcaa agcacactga acagcatcgt gggtctgggg gtgcaatccc tgaagcgccg 18900acgatgcttc tgaatagcta acgtgtcgta tgtgtgtcat gtatgcgtcc atgtcgccgc 18960cagaggagct gctgagccgc cgcgcgcccg ctttccaaga tggctacccc ttcgatgatg 19020ccgcagtggt cttacatgca catctcgggc caggacgcct cggagtacct gagccccggg 19080ctggtgcagt ttgcccgcgc caccgagacg tacttcagcc tgaataacaa gtttagaaac 19140cccacggtgg cgcctacgca cgacgtgacc acagaccggt cccagcgttt gacgctgcgg 19200ttcatccctg tggaccgtga ggatactgcg tactcgtaca aggcgcggtt caccctagct 19260gtgggtgata accgtgtgct ggacatggct tccacgtact ttgacatccg cggcgtgctg 19320gacaggggcc ctacttttaa gccctactct ggcactgcct acaacgccct ggctcccaag 19380ggtgccccaa atccttgcga atgggatgaa gctgctactg ctcttgaaat aaacctagaa 19440gaagaggacg atgacaacga agacgaagta gacgagcaag ctgagcagca aaaaactcac 19500gtatttgggc aggcgcctta ttctggtata aatattacaa aggagggtat tcaaataggt 19560gtcgaaggtc aaacacctaa atatgccgat aaaacatttc aacctgaacc tcaaatagga 19620gaatctcagt ggtacgaaac tgaaattaat catgcagctg ggagagtcct taaaaagact 19680accccaatga aaccatgtta cggttcatat gcaaaaccca caaatgaaaa tggagggcaa 19740ggcattcttg taaagcaaca aaatggaaag ctagaaagtc aagtggaaat gcaatttttc 19800tcaactactg aggcgaccgc aggcaatggt gataacttga ctcctaaagt ggtattgtac 19860agtgaagatg tagatataga aaccccagac actcatattt cttacatgcc cactattaag 19920gaaggtaact cacgagaact aatgggccaa caatctatgc ccaacaggcc taattacatt 19980gcttttaggg acaattttat tggtctaatg tattacaaca gcacgggtaa tatgggtgtt 20040ctggcgggcc aagcatcgca gttgaatgct gttgtagatt tgcaagacag aaacacagag 20100ctttcatacc agcttttgct tgattccatt ggtgatagaa ccaggtactt ttctatgtgg 20160aatcaggctg ttgacagcta tgatccagat gttagaatta ttgaaaatca tggaactgaa 20220gatgaacttc caaattactg ctttccactg ggaggtgtga ttaatacaga gactcttacc 20280aaggtaaaac ctaaaacagg tcaggaaaat ggatgggaaa aagatgctac agaattttca 20340gataaaaatg aaataagagt tggaaataat tttgccatgg aaatcaatct aaatgccaac 20400ctgtggagaa atttcctgta ctccaacata gcgctgtatt tgcccgacaa gctaaagtac 20460agtccttcca acgtaaaaat ttctgataac ccaaacacct acgactacat gaacaagcga 20520gtggtggctc ccgggttagt ggactgctac attaaccttg gagcacgctg gtcccttgac 20580tatatggaca acgtcaaccc atttaaccac caccgcaatg ctggcctgcg ctaccgctca 20640atgttgctgg gcaatggtcg ctatgtgccc ttccacatcc aggtgcctca gaagttcttt 20700gccattaaaa acctccttct cctgccgggc tcatacacct acgagtggaa cttcaggaag 20760gatgttaaca tggttctgca gagctcccta ggaaatgacc taagggttga cggagccagc 20820attaagtttg atagcatttg cctttacgcc accttcttcc ccatggccca caacaccgcc 20880tccacgcttg aggccatgct tagaaacgac accaacgacc agtcctttaa cgactatctc 20940tccgccgcca acatgctcta ccctataccc gccaacgcta ccaacgtgcc catatccatc 21000ccctcccgca actgggcggc tttccgcggc tgggccttca cgcgccttaa gactaaggaa 21060accccatcac tgggctcggg ctacgaccct tattacacct actctggctc tataccctac 21120ctagatggaa ccttttacct caaccacacc tttaagaagg tggccattac ctttgactct 21180tctgtcagct ggcctggcaa tgaccgcctg cttaccccca acgagtttga aattaagcgc 21240tcagttgacg gggagggtta caacgttgcc cagtgtaaca tgaccaaaga ctggttcctg 21300gtacaaatgc tagctaacta caacattggc taccagggct tctatatccc agagagctac 21360aaggaccgca tgtactcctt ctttagaaac ttccagccca tgagccgtca ggtggtggat 21420gatactaaat acaaggacta ccaacaggtg ggcatcctac accaacacaa caactctgga 21480tttgttggct accttgcccc caccatgcgc gaaggacagg cctaccctgc taacttcccc 21540tatccgctta taggcaagac cgcagttgac agcattaccc agaaaaagtt tctttgcgat 21600cgcacccttt ggcgcatccc attctccagt aactttatgt ccatgggcgc actcacagac 21660ctgggccaaa accttctcta cgccaactcc gcccacgcgc tagacatgac ttttgaggtg 21720gatcccatgg acgagcccac ccttctttat gttttgtttg aagtctttga cgtggtccgt 21780gtgcaccggc cgcaccgcgg cgtcatcgaa accgtgtacc tgcgcacgcc cttctcggcc 21840ggcaacgcca caacataaag aagcaagcaa catcaacaac agctgccgcc atgggctcca 21900gtgagcagga actgaaagcc attgtcaaag atcttggttg tgggccatat tttttgggca 21960cctatgacaa gcgctttcca ggctttgttt ctccacacaa gctcgcctgc gccatagtca 22020atacggccgg tcgcgagact gggggcgtac actggatggc ctttgcctgg aacccgcact 22080caaaaacatg ctacctcttt gagccctttg gcttttctga ccagcgactc aagcaggttt 22140accagtttga gtacgagtca ctcctgcgcc gtagcgccat tgcttcttcc cccgaccgct 22200gtataacgct ggaaaagtcc acccaaagcg tacaggggcc caactcggcc gcctgtggac 22260tattctgctg catgtttctc cacgcctttg ccaactggcc ccaaactccc atggatcaca 22320accccaccat gaaccttatt accggggtac ccaactccat gctcaacagt ccccaggtac 22380agcccaccct gcgtcgcaac caggaacagc tctacagctt cctggagcgc cactcgccct 22440acttccgcag ccacagtgcg cagattagga gcgccacttc tttttgtcac ttgaaaaaca 22500tgtaaaaata atgtactaga gacactttca ataaaggcaa atgcttttat ttgtacactc 22560tcgggtgatt atttaccccc acccttgccg tctgcgccgt ttaaaaatca aaggggttct 22620gccgcgcatc gctatgcgcc actggcaggg acacgttgcg atactggtgt ttagtgctcc 22680acttaaactc aggcacaacc atccgcggca gctcggtgaa gttttcactc cacaggctgc 22740gcaccatcac caacgcgttt agcaggtcgg gcgccgatat cttgaagtcg cagttggggc 22800ctccgccctg cgcgcgcgag ttgcgataca cagggttgca gcactggaac actatcagcg 22860ccgggtggtg cacgctggcc agcacgctct tgtcggagat cagatccgcg tccaggtcct 22920ccgcgttgct cagggcgaac ggagtcaact ttggtagctg ccttcccaaa aagggcgcgt 22980gcccaggctt tgagttgcac tcgcaccgta gtggcatcaa aaggtgaccg tgcccggtct 23040gggcgttagg atacagcgcc tgcataaaag ccttgatctg cttaaaagcc acctgagcct 23100ttgcgccttc agagaagaac atgccgcaag acttgccgga aaactgattg gccggacagg 23160ccgcgtcgtg cacgcagcac cttgcgtcgg tgttggagat ctgcaccaca tttcggcccc 23220accggttctt cacgatcttg gccttgctag actgctcctt cagcgcgcgc tgcccgtttt 23280cgctcgtcac atccatttca atcacgtgct ccttatttat cataatgctt ccgtgtagac 23340acttaagctc gccttcgatc tcagcgcagc ggtgcagcca caacgcgcag cccgtgggct 23400cgtgatgctt gtaggtcacc tctgcaaacg actgcaggta cgcctgcagg aatcgcccca 23460tcatcgtcac aaaggtcttg ttgctggtga aggtcagctg caacccgcgg tgctcctcgt 23520tcagccaggt cttgcatacg gccgccagag cttccacttg gtcaggcagt agtttgaagt 23580tcgcctttag atcgttatcc acgtggtact tgtccatcag cgcgcgcgca gcctccatgc 23640ccttctccca cgcagacacg atcggcacac tcagcgggtt catcaccgta atttcacttt 23700ccgcttcgct gggctcttcc tcttcctctt gcgtccgcat accacgcgcc actgggtcgt 23760cttcattcag ccgccgcact gtgcgcttac ctcctttgcc atgcttgatt agcaccggtg 23820ggttgctgaa acccaccatt tgtagcgcca catcttctct ttcttcctcg ctgtccacga 23880ttacctctgg tgatggcggg cgctcgggct

tgggagaagg gcgcttcttt ttcttcttgg 23940gcgcaatggc caaatccgcc gccgaggtcg atggccgcgg gctgggtgtg cgcggcacca 24000gcgcgtcttg tgatgagtct tcctcgtcct cggactcgat acgccgcctc atccgctttt 24060ttgggggcgc ccggggaggc ggcggcgacg gggacgggga cgacacgtcc tccatggttg 24120ggggacgtcg cgccgcaccg cgtccgcgct cgggggtggt ttcgcgctgc tcctcttccc 24180gactggccat ttccttctcc tataggcaga aaaagatcat ggagtcagtc gagaagaagg 24240acagcctaac cgccccctct gagttcgcca ccaccgcctc caccgatgcc gccaacgcgc 24300ctaccacctt ccccgtcgag gcacccccgc ttgaggagga ggaagtgatt atcgagcagg 24360acccaggttt tgtaagcgaa gacgacgagg accgctcagt accaacagag gataaaaagc 24420aagaccagga caacgcagag gcaaacgagg aacaagtcgg gcggggggac gaaaggcatg 24480gcgactacct agatgtggga gacgacgtgc tgttgaagca tctgcagcgc cagtgcgcca 24540ttatctgcga cgcgttgcaa gagcgcagcg atgtgcccct cgccatagcg gatgtcagcc 24600ttgcctacga acgccaccta ttctcaccgc gcgtaccccc caaacgccaa gaaaacggca 24660catgcgagcc caacccgcgc ctcaacttct accccgtatt tgccgtgcca gaggtgcttg 24720ccacctatca catctttttc caaaactgca agatacccct atcctgccgt gccaaccgca 24780gccgagcgga caagcagctg gccttgcggc agggcgctgt catacctgat atcgcctcgc 24840tcaacgaagt gccaaaaatc tttgagggtc ttggacgcga cgagaagcgc gcggcaaacg 24900ctctgcaaca ggaaaacagc gaaaatgaaa gtcactctgg agtgttggtg gaactcgagg 24960gtgacaacgc gcgcctagcc gtactaaaac gcagcatcga ggtcacccac tttgcctacc 25020cggcacttaa cctacccccc aaggtcatga gcacagtcat gagtgagctg atcgtgcgcc 25080gtgcgcagcc cctggagagg gatgcaaatt tgcaagaaca aacagaggag ggcctacccg 25140cagttggcga cgagcagcta gcgcgctggc ttcaaacgcg cgagcctgcc gacttggagg 25200agcgacgcaa actaatgatg gccgcagtgc tcgttaccgt ggagcttgag tgcatgcagc 25260ggttctttgc tgacccggag atgcagcgca agctagagga aacattgcac tacacctttc 25320gacagggcta cgtacgccag gcctgcaaga tctccaacgt ggagctctgc aacctggtct 25380cctaccttgg aattttgcac gaaaaccgcc ttgggcaaaa cgtgcttcat tccacgctca 25440agggcgaggc gcgccgcgac tacgtccgcg actgcgttta cttatttcta tgctacacct 25500ggcagacggc catgggcgtt tggcagcagt gcttggagga gtgcaacctc aaggagctgc 25560agaaactgct aaagcaaaac ttgaaggacc tatggacggc cttcaacgag cgctccgtgg 25620ccgcgcacct ggcggacatc attttccccg aacgcctgct taaaaccctg caacagggtc 25680tgccagactt caccagtcaa agcatgttgc agaactttag gaactttatc ctagagcgct 25740caggaatctt gcccgccacc tgctgtgcac ttcctagcga ctttgtgccc attaagtacc 25800gcgaatgccc tccgccgctt tggggccact gctaccttct gcagctagcc aactaccttg 25860cctaccactc tgacataatg gaagacgtga gcggtgacgg tctactggag tgtcactgtc 25920gctgcaacct atgcaccccg caccgctccc tggtttgcaa ttcgcagctg cttaacgaaa 25980gtcaaattat cggtaccttt gagctgcagg gtccctcgcc tgacgaaaag tccgcggctc 26040cggggttgaa actcactccg gggctgtgga cgtcggctta ccttcgcaaa tttgtacctg 26100aggactacca cgcccacgag attaggttct acgaagacca atcccgcccg ccaaatgcgg 26160agcttaccgc ctgcgtcatt acccagggcc acattcttgg ccaattgcaa gccatcaaca 26220aagcccgcca agagtttctg ctacgaaagg gacggggggt ttacttggac ccccagtccg 26280gcgaggagct caacccaatc cccccgccgc cgcagcccta tcagcagcag ccgcgggccc 26340ttgcttccca ggatggcacc caaaaagaag ctgcagctgc cgccgccacc cacggacgag 26400gaggaatact gggacagtca ggcagaggag gttttggacg aggaggagga ggacatgatg 26460gaagactggg agagcctaga cgaggaagct tccgaggtcg aagaggtgtc agacgaaaca 26520ccgtcaccct cggtcgcatt cccctcgccg gcgccccaga aatcggcaac cggttccagc 26580atggctacaa cctccgctcc tcaggcgccg ccggcactgc ccgttcgccg acccaaccgt 26640agatgggaca ccactggaac cagggccggt aagtccaagc agccgccgcc gttagcccaa 26700gagcaacaac agcgccaagg ctaccgctca tggcgcgggc acaagaacgc catagttgct 26760tgcttgcaag actgtggggg caacatctcc ttcgcccgcc gctttcttct ctaccatcac 26820ggcgtggcct tcccccgtaa catcctgcat tactaccgtc atctctacag cccatactgc 26880accggcggca gcggcagcgg cagcaacagc agcggccaca cagaagcaaa ggcgaccgga 26940tagcaagact ctgacaaagc ccaagaaatc cacagcggcg gcagcagcag gaggaggagc 27000gctgcgtctg gcgcccaacg aacccgtatc gacccgcgag cttagaaaca ggatttttcc 27060cactctgtat gctatatttc aacagagcag gggccaagaa caagagctga aaataaaaaa 27120caggtctctg cgatccctca cccgcagctg cctgtatcac aaaagcgaag atcagcttcg 27180gcgcacgctg gaagacgcgg aggctctctt cagtaaatac tgcgcgctga ctcttaagga 27240ctagtttcgc gccctttctc aaatttaagc gcgaaaacta cgtcatctcc agcggccaca 27300cccggcgcca gcacctgtcg tcagcgccat tatgagcaag gaaattccca cgccctacat 27360gtggagttac cagccacaaa tgggacttgc ggctggagct gcccaagact actcaacccg 27420aataaactac atgagcgcgg gaccccacat gatatcccgg gtcaacggaa tccgcgccca 27480ccgaaaccga attctcttgg aacaggcggc tattaccacc acacctcgta ataaccttaa 27540tccccgtagt tggcccgctg ccctggtgta ccaggaaagt cccgctccca ccactgtggt 27600acttcccaga gacgcccagg ccgaagttca gatgactaac tcaggggcgc agcttgcggg 27660cggctttcgt cacagggtgc ggtcgcccgg gcagggtata actcacctga caatcagagg 27720gcgaggtatt cagctcaacg acgagtcggt gagctcctcg cttggtctcc gtccggacgg 27780gacatttcag atcggcggcg ccggccgtcc ttcattcacg cctcgtcagg caatcctaac 27840tctgcagacc tcgtcctctg agccgcgctc tggaggcatt ggaactctgc aatttattga 27900ggagtttgtg ccatcggtct actttaaccc cttctcggga cctcccggcc actatccgga 27960tcaatttatt cctaactttg acgcggtaaa ggactcggcg gacggctacg actgaatgtt 28020aagtggagag gcagagcaac tgcgcctgaa acacctggtc cactgtcgcc gccacaagtg 28080ctttgcccgc gactccggtg agttttgcta ctttgaattg cccgaggatc atatcgaggg 28140cccggcgcac ggcgtccggc ttaccgccca gggagagctt gcccgtagcc tgattcggga 28200gtttacccag cgccccctgc tagttgagcg ggacagggga ccctgtgttc tcactgtgat 28260ttgcaactgt cctaaccttg gattacatca agatcctcta gttataacta gagtacccgg 28320ggatcttatt ccctttaact aataaaaaaa aataataaag catcacttac ttaaaatcag 28380ttagcaaatt tctgtccagt ttattcagca gcacctcctt gccctcctcc cagctctggt 28440attgcagctt cctcctggct gcaaactttc tccacaatct aaatggaatg tcagtttcct 28500cctgttcctg tccatccgca cccactatct tcatgttgtt gcagatgaag cgcgcaagac 28560cgtctgaaga taccttcaac cccgtgtatc catatgacac ggaaaccggt cctccaactg 28620tgccttttct tactcctccc tttgtatccc ccaatgggtt tcaagagagt ccccctgggg 28680tactctcttt gcgcctatcc gaacctctag ttacctccaa tggcatgctt gcgctcaaaa 28740tgggcaacgg cctctctctg gacgaggccg gcaaccttac ctcccaaaat gtaaccactg 28800tgagcccacc tctcaaaaaa accaagtcaa acataaacct ggaaatatct gcacccctca 28860cagttacctc agaagcccta actgtggctg ccgccgcacc tctaatggtc gcgggcaaca 28920cactcaccat gcaatcacag gccccgctaa ccgtgcacga ctccaaactt agcattgcca 28980cccaaggacc cctcacagtg tcagaaggaa agctagccct gcaaacatca ggccccctca 29040ccaccaccga tagcagtacc cttactatca ctgcctcacc ccctctaact actgccactg 29100gtagcttggg cattgacttg aaagagccca tttatacaca aaatggaaaa ctaggactaa 29160agtacggggc tcctttgcat gtaacagacg acctaaacac tttgaccgta gcaactggtc 29220caggtgtgac tattaataat acttccttgc aaactaaagt tactggagcc ttgggttttg 29280attcacaagg caatatgcaa cttaatgtag caggaggact aaggattgat tctcaaaaca 29340gacgccttat acttgatgtt agttatccgt ttgatgctca aaaccaacta aatctaagac 29400taggacaggg ccctcttttt ataaactcag cccacaactt ggatattaac tacaacaaag 29460gcctttactt gtttacagct tcaaacaatt ccaaaaagct tgaggttaac ctaagcactg 29520ccaaggggtt gatgtttgac gctacagcca tagccattaa tgcaggagat gggcttgaat 29580ttggttcacc taatgcacca aacacaaatc ccctcaaaac aaaaattggc catggcctag 29640aatttgattc aaacaaggct atggttccta aactaggaac tggccttagt tttgacagca 29700caggtgccat tacagtagga aacaaaaata atgataagct aactttgtgg accacaccag 29760ctccatctcc taactgtaga ctaaatgcag agaaagatgc taaactcact ttggtcttaa 29820caaaatgtgg cagtcaaata cttgctacag tttcagtttt ggctgttaaa ggcagtttgg 29880ctccaatatc tggaacagtt caaagtgctc atcttattat aagatttgac gaaaatggag 29940tgctactaaa caattccttc ctggacccag aatattggaa ctttagaaat ggagatctta 30000ctgaaggcac agcctataca aacgctgttg gatttatgcc taacctatca gcttatccaa 30060aatctcacgg taaaactgcc aaaagtaaca ttgtcagtca agtttactta aacggagaca 30120aaactaaacc tgtaacacta accattacac taaacggtac acaggaaaca ggagacacaa 30180ctccaagtgc atactctatg tcattttcat gggactggtc tggccacaac tacattaatg 30240aaatatttgc cacatcctct tacacttttt catacattgc ccaagaataa agaatcgttt 30300gtgttatgtt tcaacgtgtt tatttttcaa ttgcagaaaa tttcaagtca tttttcattc 30360agtagtatag ccccaccacc acatagctta tacagatcac cgtaccttaa tcaaactcac 30420agaaccctag tattcaacct gccacctccc tcccaacaca cagagtacac agtcctttct 30480ccccggctgg ccttaaaaag catcatatca tgggtaacag acatattctt aggtgttata 30540ttccacacgg tttcctgtcg agccaaacgc tcatcaagtg atattaataa actccccggg 30600cagctcactt aagttcatgt cgctgtccag ctgctgagcc acaggctgct gtccaacttg 30660cggttgctta acgggcggcg aaggagaagt ccacgcctac atggggggag agtcataatc 30720gtgcatcagg atagggcggt ggtgctgcag cagcgcgcga ataaactgct gccgccgccg 30780ctccgtcctg caggaataca acatggcagt ggtctcctca gcgatgattc gcaccgcccg 30840cagcataagg cgcttgtcct ccgggcacag cagcgcaccc tgatctcact taaatcagca 30900cagtaactgc agcacagcac cacaatattg ttcaaaatcc cacagtgcaa ggcgctgtat 30960ccaaagctca tggcggggac cacagaaccc acgtggccat cataccacaa gcgcaggtag 31020attaagtggc gacccctcat aaacacgctg gacataaaca ttacctcttt tggcatgttg 31080taattcacca cctcccggta ccatataaac ctctgattaa acatggcgcc atccaccacc 31140atcctaaacc agctggccaa aacctgcccc gccgggntat acactgcagg gaaccgggac 31200ttggacaatg acaagtggga gagcccagga ctcgtaacca tggatcatca tgctcgtcat 31260gatatcaatg ttggcacaac acaggcacac gtgcatacac ttcctcagga ttacaagctc 31320ctcccgcgtt agaaccatat cccagggaac aacccattcc tgaatcagcg taaatcccac 31380actgcaggga agacctcgca cgtaactcac gttgtgcatt gtcaaagtgt tacattcggg 31440cagcagcgga tgatcctcca gtatggtagc gcgggtttct gtctcaaaag gaggtagacg 31500atccctactg tacggagtgc gccgagacaa ccgagatcgt gttggtcgta gtgtcatgcc 31560aaatggaacg ccggacgtag tcatatttcc tgaagcaaaa ccaggtgcgg gcgtgacaaa 31620cagatctgcg tctccggtct cgccgcttag atcgctctgt gtagtagttg tagtatatcc 31680actctctcaa agcatccagg cgccccctgg cttcgggttc tatgtaaact ccttcatgcg 31740ccgctgccct gataacatcc accaccgcag aataagccac acccagccaa cctacacatt 31800cgttctgcga gtcacacacg ggaggagcgg gaagagctgg aagaaccatg tttttttttt 31860tattccaaaa gattatccaa aacctcaaaa tgaagatcta ttaagtgaac gcgctcccct 31920ccggtggcgt ggtcaaactc tacagccaaa gaacagataa tggcatttgt aagatgttgc 31980acaatggctt ccaaaaggca aacggccctc acgtccaagt ggacgtaaag gctaaaccct 32040tcagggtgaa tctcctctat aaacattcca gcaccttcaa ccatgcccaa ataattctca 32100tctcgccacc ttctcaatat atctctaagc aaatcccgaa tatttaagtc cgggccattg 32160taaaaaattt ggctccagag cgccctccac cttcagcctc aagcagcgaa tcatgattgc 32220aaaaattcag gttcctcaca gacctgtata agattcaaaa gcggaacatt aacaaaaata 32280ccgcgatccc gtaggtccct tcgcagggcc agctgaacat aatcgtgcag gtctgcacgg 32340accagcgcgg ccacttcccc gccaggaacc atgacaaaag aacccacact gattatgaca 32400cgcatactcg gagctatgct aaccagcgta gccccgatgt aagcttgttg catgggcggc 32460gatataaaat gcaaggtgct gctcaaaaaa tcaggcaaag cctcgcgcaa aaaagaaagc 32520acatcgtagt catgctcatg cagataaagg caggtaagct ccggaaccac cacagaaaaa 32580gacaccattt ttctctcaaa catgtctgcg ggtttctgca taaacacaaa ataaaataac 32640aaaaaaacat ttaaacatta gaagcctgtc ttacaacagg aaaaacaacc cttataagca 32700taagacggac tacggccatg ccggcgtgac cgtaaaaaaa ctggtcaccg tgattaaaaa 32760gcaccaccga cagctcctcg gtcagtccgg agtcataatg taagactcgg taaacacatc 32820aggttgattc acatcggtca gtgttaaaaa gcgaccgaaa tagccngggg gaatacaata 32880cccgcaggcg tagagacaac attacagccc ccataggagg tataacaaaa ttaataggag 32940agaaaaacac ataaacacct gaaaaaccct cctgcctagg caaaatagca ccctcccgct 33000ccagaacaac atacagcgct tccacagcgg cagccataac agtcagcctt accagtaaaa 33060aagaaaacct attaaaaaaa caccactcga cacggcacca gctcaatcag tcacagtgta 33120aaaaagggcc aagtgcagag cgagtatata taggactaaa aaatgacggt aacggttaaa 33180gtccacaaaa aacacccaga aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 33240aacccacaac ttcctcaaat cgtcacttcc gttttcccac gttacgtcac ttcccatttt 33300aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc 33360cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc 33420aatccaaaat aaggtatatt attgatgatg 3345083010PRTHepatitis C virus 8Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro50 55 60Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr180 185 190Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser195 200 205Asn Ala Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr245 250 255Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Leu Cys260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ala275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Thr Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ser Gln325 330 335Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp355 360 365Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly370 375 380Thr Tyr Val Thr Gly Gly Thr Met Ala Lys Asn Thr Leu Gly Ile Thr385 390 395 400Ser Leu Phe Ser Pro Gly Ser Ser Gln Lys Ile Gln Leu Val Asn Thr405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser420 425 430Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Val His Lys Phe Asn435 440 445Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Ser Pro Ile Asp Ala450 455 460Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Asn Glu Ser His Ser Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile485 490 495Val Pro Ala Ala Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Ser515 520 525Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro530 535 540Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ile Gly Asn565 570 575Lys Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala580 585 590Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu595 600 605Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe610 615 620Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu625 630 635 640Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu Asp645 650 655Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp660 665 670Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly675 680 685Leu Ile His Leu His Gln Asn Val Val Asp Val Gln Tyr Leu Tyr Gly690 695 700Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Val Leu705 710 715 720Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp725 730 735Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val740 745 750Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe755 760 765Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro770 775 780Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu785 790 795 800Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala805 810 815Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ile Leu Leu Thr Leu Ser820 825 830Pro His Tyr Lys Leu Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr835 840 845Phe Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro Pro Leu850 855 860Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Thr Cys Ala Ile865 870 875 880His Pro Glu Leu Ile Phe Thr Ile Thr Lys Ile Leu Leu Ala Ile Leu885 890 895Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Lys Val Pro Tyr Phe900 905 910Val Arg Ala His Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val915 920 925Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu Ala Ala Leu930 935 940Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp Trp Ala945 950 955 960His Ala Gly Leu Arg Asp Leu

Ala Val Ala Val Glu Pro Val Val Phe965 970 975Ser Asp Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala980 985 990Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Arg995 1000 1005Glu Ile His Leu Gly Pro Ala Asp Ser Leu Glu Gly Gln Gly Trp1010 1015 1020Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly1025 1030 1035Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Arg Asn1040 1045 1050Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser1055 1060 1065Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His1070 1075 1080Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr1085 1090 1095Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala1100 1105 1110Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser1115 1120 1125Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg1130 1135 1140Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val1145 1150 1155Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser1160 1165 1170Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly1175 1180 1185Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr1190 1195 1200Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala1205 1210 1215Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly1220 1225 1230Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly1235 1240 1245Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly1250 1255 1260Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile1265 1270 1275Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr1280 1285 1290Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly1295 1300 1305Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser1310 1315 1320Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr1325 1330 1335Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly1340 1345 1350Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser1355 1360 1365Ser Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu1370 1375 1380Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys1385 1390 1395Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Leu Asn1400 1405 1410Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr1415 1420 1425Ser Gly Asp Val Ile Val Val Ala Thr Asp Ala Leu Met Thr Gly1430 1435 1440Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val1445 1450 1455Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu1460 1465 1470Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg1475 1480 1485Gly Arg Thr Gly Arg Gly Arg Met Gly Ile Tyr Arg Phe Val Thr1490 1495 1500Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys1505 1510 1515Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala1520 1525 1530Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu1535 1540 1545Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr1550 1555 1560Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln1565 1570 1575Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val1580 1585 1590Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp1595 1600 1605Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro1610 1615 1620Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Thr Thr1625 1630 1635His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu1640 1645 1650Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala1655 1660 1665Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val1670 1675 1680Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg1685 1690 1695Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ala Ser1700 1705 1710His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe1715 1720 1725Lys Gln Lys Ala Ile Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala1730 1735 1740Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Thr Leu Glu1745 1750 1755Ala Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln1760 1765 1770Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala1775 1780 1785Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr1790 1795 1800Gln His Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala1805 1810 1815Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly1820 1825 1830Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu1835 1840 1845Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu1850 1855 1860Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp1865 1870 1875Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val1880 1885 1890Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro1895 1900 1905Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala1910 1915 1920Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser1925 1930 1935Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu Thr Ile1940 1945 1950Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys1955 1960 1965Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp1970 1975 1980Ile Cys Thr Val Leu Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys1985 1990 1995Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg2000 2005 2010Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Gln Thr Thr2015 2020 2025Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser2030 2035 2040Met Arg Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly2045 2050 2055Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser2060 2065 2070Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu2075 2080 2085Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr2090 2095 2100Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala2105 2110 2115Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr2120 2125 2130Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Glu Val Thr Phe Leu2135 2140 2145Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu2150 2155 2160Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro2165 2170 2175Ser His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly2180 2185 2190Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala2195 2200 2205Pro Ser Leu Lys Ala Thr Cys Thr Thr Arg His Asp Ser Pro Asp2210 2215 2220Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly2225 2230 2235Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu2240 2245 2250Asp Ser Phe Glu Pro Leu Gln Ala Glu Glu Asp Glu Arg Glu Val2255 2260 2265Ser Val Pro Ala Glu Ile Leu Arg Arg Ser Arg Lys Phe Pro Arg2270 2275 2280Ala Met Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu2285 2290 2295Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly2300 2305 2310Cys Pro Leu Pro Pro Ala Lys Ala Pro Pro Ile Pro Pro Pro Arg2315 2320 2325Arg Lys Arg Thr Val Val Leu Ser Glu Ser Thr Val Ser Ser Ala2330 2335 2340Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Glu Ser Ser2345 2350 2355Ala Val Asp Ser Gly Thr Ala Thr Ala Ser Pro Asp Gln Pro Ser2360 2365 2370Asp Asp Gly Asp Ala Gly Ser Asp Val Glu Ser Tyr Ser Ser Met2375 2380 2385Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly2390 2395 2400Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys2405 2410 2415Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys2420 2425 2430Ala Ala Glu Glu Thr Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser2435 2440 2445Leu Leu Arg His His Asn Leu Val Tyr Ala Thr Thr Ser Arg Ser2450 2455 2460Ala Ser Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val2465 2470 2475Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys2480 2485 2490Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys2495 2500 2505Lys Leu Thr Pro Pro His Ser Ala Arg Ser Lys Phe Gly Tyr Gly2510 2515 2520Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala Val Asn His Ile2525 2530 2535Arg Ser Val Trp Lys Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile2540 2545 2550Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro2555 2560 2565Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp2570 2575 2580Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val2585 2590 2595Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe Gln2600 2605 2610Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala Trp Lys2615 2620 2625Ala Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys Phe2630 2635 2640Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile2645 2650 2655Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg2660 2665 2670Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser2675 2680 2685Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val2690 2695 2700Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala2705 2710 2715Ala Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu2720 2725 2730Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr2735 2740 2745Gln Glu Asp Glu Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr2750 2755 2760Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Lys Pro Glu Tyr Asp2765 2770 2775Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His2780 2785 2790Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr2795 2800 2805Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro2810 2815 2820Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu2825 2830 2835Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu2840 2845 2850Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly2855 2860 2865Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln2870 2875 2880Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro2885 2890 2895Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val2900 2905 2910Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala2915 2920 2925Arg Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr2930 2935 2940Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile2945 2950 2955Pro Ala Ala Ser Gln Leu Asp Leu Ser Ser Trp Phe Val Ala Gly2960 2965 2970Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro2975 2980 2985Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly2990 2995 3000Ile Tyr Leu Leu Pro Asn Arg3005 3010

Claims

1-9. (canceled)

10. A compound of molecular weight 10,000 or less, wherein the compound interacts with NS3 to inhibit NS3 oligomerization and wherein the compound inhibits hepatitis C virus (HCV) replication.

11. The compound of claim 10 wherein the compound comprises an inhibitory peptide comprising 4 or more contiguous residues of SEQ ID NO:1.

12. The compound of claim 11 wherein the inhibitory peptide comprises 6 or more contiguous residues of SEQ ID NO:1.

13. The compound of claim 12 wherein the inhibitory peptide comprises 8 or more contiguous residues of SEQ ID NO:1.

14. The compound of claim 13 wherein the inhibitory peptide comprises SEQ ID NO:1.

15. The compound of claim 14 wherein the inhibitory peptide comprises SEQ ID NO:2.

16. The compound of claim 11 wherein the compound further comprises a cell-entry vehicle coupled to the inhibitor peptide.

17. The compound of claim 10 wherein the structure of the compound fits a molecular interface of NS3 such that a free energy calculation predicts the compound is expected to bind to the molecular interface of NS3.

18. The compound of claim 10 wherein the surface of NS3 which the compound fits includes at least one amino acid residue selected from residues 541-553, 584-591, 435-453, 477-488, and 524-536 of SEQ ID NO:3.

19-31. (canceled)

32. The compound of claim 10 wherein the compound comprises an inhibitory peptide comprising at least 4 contiguous residues of reverse D sequence of SEQ ID NO:1.