U.S. patent application number 10/734801 was filed with the patent office on 2004-07-01 for in vitro system for replication of rna-dependent rna polymerase (rdrp) viruses.
Invention is credited to Jeffries, Matthew W., King, Robert W., Pasquinelli, Claudio.
Application Number | 20040126388 10/734801 |
Document ID | / |
Family ID | 23010445 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126388 |
Kind Code |
A1 |
King, Robert W. ; et
al. |
July 1, 2004 |
In vitro system for replication of RNA-dependent RNA polymerase
(RDRP) viruses
Abstract
An in vitro method to conduct genomic replication of the viral
genomes of viruses that utilize RNA-dependent RNA polymerase for
replication (RDRP viruses), such as HCV. The method employs a
construct comprising the 3' and 5' untranslated regions (UTRs) of
the viral genome which are operably linked on the 5' and 3' ends of
a reporter sequence, in antisense orientation, such that when viral
replication is occurring within the cell which produces RDRP, the
reporter protein will be made. The method of the invention provides
an efficient means for measuring genomic replication in RDRP
viruses, and also for the rapid screening of compounds for their
ability to inhibit genomic replication of RDRP viruses, including
the Hepatitis C virus (HCV).
Inventors: |
King, Robert W.; (West
Chester, PA) ; Jeffries, Matthew W.; (Wilmington,
DE) ; Pasquinelli, Claudio; (Media, PA) |
Correspondence
Address: |
STEPHEN B. DAVIS
BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
23010445 |
Appl. No.: |
10/734801 |
Filed: |
December 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10734801 |
Dec 12, 2003 |
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10066130 |
Jan 31, 2002 |
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6699657 |
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60265437 |
Jan 31, 2001 |
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Current U.S.
Class: |
424/189.1 ;
435/5 |
Current CPC
Class: |
C12N 15/85 20130101;
C12Q 1/70 20130101; C12N 2770/24211 20130101; Y02A 50/30 20180101;
C12N 2770/24011 20130101; C12N 2840/203 20130101 |
Class at
Publication: |
424/189.1 ;
435/005 |
International
Class: |
C12Q 001/70; A61K
039/29 |
Claims
What is claimed is:
1. An in vitro method for measuring the genomic replication of a
virus that is dependent for replication upon RNA-dependent RNA
polymerase (an RDRP virus) comprising the steps of: a) culturing
virally-compatible eukaryotic cells which have been transfected
with the cDNA of the genomic sequence of said RDRP virus; b)
transfecting said cultured cells with a construct comprising the
cDNA, in antisense orientation, of a reporter gene sequence wherein
said reporter gene cDNA sequence is operably linked on its 5' end
with the cDNA of the untranslated region (UTR), in antisense
orientation, of the native 3' end of said RDRP virus and is
operably linked on its 3' end with the cDNA of the UTR, in
antisense orientation, of the native 5' end of said RDRP virus; c)
culturing said cells for a sufficient period of time under
conditions which are permissive for replication of said RDRP virus;
and d) analyzing the cells for the presence of the protein encoded
by the reporter gene sequence, whereby detection of said protein
provides a means to measure the genomic replication of said RDRP
virus.
2. The method of claim 1, wherein the RDRP virus is a member of the
Flaviviridae family.
3. The method of claim 2, wherein the RDRP virus is HCV.
4. The method of claim 1, wherein the virally-compatible eukaryotic
cell is human.
5. The method of claim 4, wherein the virally-compatible eukaryotic
cell is a human liver or kidney cell.
6. The method of claim 3, wherein the virally-compatible eukaryotic
cells of step (a) are from the human cell line 293 FL#9.
7. The method of claim 1 wherein at step (b), transfection of
cultured cells is performed using the method selected from:
electroporation, liposomal transfer, CaPO.sub.4 shock, and
DEAE-dextran shock.
8. The method of claim 1, wherein at step (b), the construct
further comprises the cDNA, in the sense orientation, of a delta
ribozyme operably linked to the 3' end of the 5' UTR sequence.
9. The method of claim 1, wherein the reporter gene is selected
from: luciferase, secreted alkaline phosphatase,
beta-galactosidase, Hepatitis B virus surface antigen, herpes
simplex virus thymidine kinase, genticin-resistance,
zeocin-resistance, hygromycin-resistance, and
puromycin-resistance.
10. The method of claim 9, wherein the reporter gene is
luciferase.
11. The method of claim 3, wherein at step (b) the construct
further comprises the cDNA, in sense orientation, of the hepatitis
delta ribozyme operably linked to the end of the 5' UTR
sequence.
12. The method of claim 3 wherein the construct at step (b)
comprises SEQ ID NO: 18.
13. The method of claim 6 wherein the construct at step (b)
comprises SEQ ID NO: 18.
14. A construct comprising the cDNA in antisense orientation of a
reporter gene sequence wherein said reporter gene cDNA sequence is
operably linked on its 5' end with the cDNA of the untranslated
region (UTR), in antisense orientation, of the native 3' end of an
RDRP virus and is operably linked on its 3' end with the cDNA of
the UTR, in antisense orientation, of the native 5' end of the RDRP
virus.
15. The construct of claim 14, wherein the RDRP virus is HCV.
16. The construct of claim 14, wherein the construct further
comprises the cDNA, in sense orientation, of the hepatitis delta
ribozyme operably linked to the 3' end of the 5' UTR sequence.
17. The construct of claim 16, wherein the RDRP virus is HCV, and
the reporter gene is selected from: luciferase, secreted alkaline
phosphatase, beta-galactosidase, hepatitis B virus surface antigen,
herpes simplex virus thymidine kinase, genticin-resistance,
zeocin-resistance, hygromycin-resistance, and
puromycin-resistance.
18. A construct comprising SEQ ID NO:17.
19. A cell containing the construct of claim 14.
20. A cell containing the construct of claim 16.
21. A 293B4.alpha. cell.
22. An in vitro method for identifying compounds or conditions
which inhibit the genomic replication of a virus that is dependent
for replication on RNA-dependent RNA polymerase (an RDRP virus)
comprising the steps of: a) culturing virally-compatible eukaryotic
cells which have been transfected with the cDNA of all or a portion
of the genomic sequence of said RDRP virus; b) transfecting said
cultured cells with a construct comprising the cDNA, in antisense
orientation, of a reporter gene sequence wherein said reporter gene
cDNA sequence is operably linked on its 5' end with the cDNA of the
untranslated region (UTR), in antisense orientation, from the
native 3' end of said RDRP virus and is operably linked on its 3'
end with the UTR, in antisense orientation, from the native 5' end
of said RDRP virus; c) exposing said cultured cells to a compound
or condition suspected of being capable of inhibiting the genomic
replication of said RDRP virus; d) culturing said cells for a
sufficient period of time under conditions which are permissive for
genomic replication of said RDRP virus; and e) analyzing the cells
for the presence of the protein encoded by the reporter gene
sequence, whereby a decrease in the level of said protein encoded
by the reporter gene sequence indicates that said compound or
condition is capable of inhibiting the genomic replication of said
RDRP virus.
23. The method of claim 22, wherein the RDRP virus is a member of
the Flaviviridae family.
24. The method of claim 23, wherein the RDRP virus is HCV.
25. The method of claim 22, wherein the virally-compatible
eukaryotic cell is human.
26. The method of claim 25, wherein the virally compatible
eukaryotic cell is a liver or kidney cell.
27. The method of claim 22, wherein the virally-compatible
eukaryotic cells of step (a) are from the human cell line 293
FL#9.
28. The method of claim 22 wherein at step (b), transfection of
cultured cells is performed using the method selected from:
electroporation, liposomal transfer, CaPO.sub.4 shock, and
DEAE-dextran shock.
29. The method of claim 22, wherein at step (b), the construct
further comprises the cDNA, in the sense orientation, of a delta
ribozyme sequence operably linked to the 3' end of the 5' UTR
sequence.
30. The method of claim 22, wherein the reporter gene is selected
from: luciferase, secreted alkaline phosphatase,
beta-galactosidase, hepatitis B virus surface antigen, herpes
simplex virus thymidine kinase, genticin-resistance,
zeocin-resistance, hygromycin-resistance, and
puromycin-resistance.
31. The method of claim 30, wherein the reporter gene is
luciferase.
32. The method of claim 24, wherein at step (b) the construct
further comprises the cDNA, in sense orientation, of the hepatitis
delta ribozyme operably linked to the end of the 5' UTR
sequence.
33. The method of claim 22, wherein said compound or condition is
selected from the group consisting of: small molecular weight
synthetic chemicals, organic compounds that are derived from living
or once living organisms, synthetic chemical compounds based on
organic compounds derived from living or once living organisms,
sound, light and temperature.
34. The method of claim 33 wherein said compound is a small
molecular weight synthetic chemical.
35. The method of claim 32, wherein at step (a) said virally
compatible cells have been transfected with the cDNA of all of the
genomic sequence of HCV.
36. The method of claim 24, wherein at a step (a) said portion of
the genomic sequence of said RDRP virus consists essentially of the
genomic sequences encoding from the NS2 to the NS5b region.
37. The method of claim 24, wherein at a step (a) said portion of
the genomic sequence of said RDRP virus consists essentially of the
genomic sequence encoding the NS5b portion.
38. The method of claim 35 wherein the cultured cells at step (e)
are 293B4(cells.
39. The method of claim 24 wherein the construct of step (b)
comprises SEQ ID NO: 18.
40. A method of selectively affecting a cell infected with a virus
that is dependent for genomic replication upon RNA-dependent RNA
polymerase (an RDRP virus) comprising the steps of: a) transfecting
said infected cell with a construct comprising the cDNA, in
antisense orientation, of a gene sequence which encodes a protein
that is capable of affecting said cell, wherein said cDNA sequence
is operably linked on its 5' end with the cDNA of the untranslated
region (UTR), in antisense orientation, of the native 3' end of
said RDRP virus and is operably linked on its 3' end with the cDNA
of the UTR, in antisense orientation, of the native 5' end of said
RDRP virus; and b) allowing a sufficient period of time for genomic
replication of said RDRP virus, whereby upon genomic replication of
said RDRP virus, RNA-dependent RNA polymerase produced by said
replicating RDRP virus will cause expression of the construct of
step (a), whereby the cell is affected.
41. The method of claim 40, wherein the RDRP virus is HCV.
42. The method of claim 40, wherein at step (a) the construct
further comprises the cDNA of a delta ribozyme sequence, in sense
orientation, operably linked to said 3' end of the cDNA of the UTR,
in antisense orientation of the native 5' end of said RDRP virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/265,437, filed Jan. 31, 2001, the contents of
which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention is directed toward the pharmaceutical and
molecular biology arts, more particularly this invention is an in
vitro system for the replication of the viral genomes of viruses
that depend upon the enzyme RNA-dependent RNA polymerase (RDRP) for
replication. The method of the invention provides an efficient
means for measuring genomic replication in RDRP viruses, and also
for the rapid screening of compounds for their ability to inhibit
genomic replication of RDRP viruses, including the Hepatitis C
virus (HCV).
BACKGROUND OF THE INVENTION
[0003] It is known that viral genomes can be made of DNA or RNA and
can be double-stranded or single-stranded. Typically, viral genomes
encode viral coat proteins that serve to package the genome after
replication, and also nonstructural proteins that facilitate
enzymatic replication of the viral genome in conjunction with
cellular enzymes. In the case of some viruses having a
single-stranded RNA genome, one of the nonstructural proteins
encoded by the viral genome is RNA-dependent RNA polymerase (RDRP),
which is needed by the virus to replicate its genomic sequence. The
viral enzyme RNA-dependent RNA polymerase is also called RNA
replicase.
[0004] The viral family Flaviviridae is one such type of virus
which is dependent upon its own RNA-dependent RNA polymerase in
order to replicate. Flaviviridae is a family of viruses having a
single-stranded RNA genome in the (+)orientation. The term
"(+)orientation" is a convention used to designate single-stranded
nucleic acid molecules which exist in the coding or sense
orientation when read from the 5' to 3' direction. The Flaviviridae
family comprises the flaviviruses, the animal pathogenic
pestiviruses, the recently characterized GB viruses (GBV-A, GBV-B
and GBV-C/hepatitis G), and most importantly from a human disease
perspective, the genus Hepacivirus or Hepatitis C virus (HCV). The
RNA genome of these viruses typically includes a single long open
reading frame encoding a polyprotein that is proteolyically cleaved
into a set of distinct structural and nonstructural protein
products. Translation of the open reading frame of the genome is
directed via a 5' untranslated region (UTR) which functions as an
internal ribosomal entry site (IRES). The 3' end of the genome in
these viruses comprises a highly conserved UTR region of variable
length which is thought to be essential for replication.
[0005] The most well-known member of the Flaviviridae family of
viruses is the Hepatitis C virus ("HCV"), which is a parenterally
transmitted, hepatotropic virus that in primates causes acute and
chronic hepatitis, as well as hepatocellular carcinoma.
Approximately 2% of the world's human population is thought to be
afflicted with HCV infections. No vaccine for HCV is currently
available, and present treatment is generally limited to interferon
monotherapy, or the combination of alpha-interferon with the
nucleoside analog ribavirin. (1)(2)(3)(4)(5).
[0006] HCV is a positive-stranded RNA virus having a genome 9.6 kb
long comprised of a single, uninterrupted open reading frame
encoding a polyprotein of about 3000-3011 amino acids. The HCV
polyprotein is a precursor to the individual HCV proteins necessary
for replication, packaging and infectivity. The structural region
of the polyprotein precursor (including the C, E1, E2 and p7
proteins) is processed by host cell signal peptidases. The
nonstructural region of the precursor (including the NS2, NS3,
NS4A, NS4B, NS5A and NS5B proteins) is processed between NS2 and
NS3 by NS2-3 protease, while processing in the NS3-NS5B region of
the polyprotein is accomplished by NS3 protease activity. (6) (7)
(8) (9) (10).
[0007] The mode of replication of the HCV virus is still
speculative and current understanding is based upon analogy with
other of the flavi- and pestiviruses. It is believed that HCV
replication begins by viral penetration of the host cell and
liberation of the viral genomic (+)single-stranded RNA from the
virus particle into the cytoplasm of the cell. The viral RNA is
translated by cellular enzymes, and the encoded viral polyprotein
is processed into several distinct functional viral proteins
including RNA-dependent RNA polymerase protein (RDRP). RDRP then
proceeds to synthesize (-)stranded RNA intermediates (from template
viral genomes) which in turn serve as templates for synthesis of
new (+)stranded RNA molecules. These (+)stranded viral RNA
molecules can then be used for further viral polyprotein
expression, for synthesis of new (-)stranded RNA molecules, or for
packaging into progeny virions which can then be released from the
infected cell to spread the HCV infection. (1).
[0008] Presently, there are no efficient systems for in vitro
monitoring of the replication of the RDRP viruses of the
Flaviviridae family. As a result, there is a lack of means for
studying the mechanism of replication of these (+)stranded RNA
viruses, or for determining the ability of a compound or condition
to inhibit such replication. While cell-based systems for HCV
replication have been described (11), these systems rely on
protocols and endpoints that are not easily formatted into
platforms for screening large numbers of compounds for anti-viral
activity (12). The present invention provides a solution to these
problems by providing a system for the efficient in vitro
manipulation and monitoring of the replication of RDRP viruses. The
system of the invention can be assembled so as to provide a
convenient platform for screening inhibitors to RDRP viral
replication. The method of the invention also provides a means to
design therapies for the in vivo treatment of cells that are
infected with RDRP viruses.
SUMMARY OF THE INVENTION
[0009] The present invention provides an efficient in vitro method
for measuring the replication of the genome of viruses that are
dependent upon RNA-dependent RNA polymerase for replication (these
types of viruses are herein referred to as "RDRP viruses"). The
method comprises the steps of culturing virally-compatible
eukaryotic cells, which have been transfected with the cDNA of the
genome of the RDRP virus, and transfecting these cultured cells
with a construct of the invention, which construct comprises the
cDNA, in antisense orientation, of a reporter gene sequence. The
reporter gene cDNA sequence of the construct is operably linked on
its 5' end with the cDNA of the untranslated region (hereinafter
"UTR") in antisense orientation of the native 3' end of said RDRP
virus, and is operably linked on its 3' end with the cDNA of the
UTR in antisense orientation of the native 5' end of said RDRP
virus. Thus, the construct will be comprised of the cDNA, in
antisense orientation, of a reporter gene flanked by the 3' and 5'
UTRs of the native RDRP viral genome. Transfected cells containing
the construct of the invention are cultured for a sufficient period
of time under conditions which are permissive for replication of
said RDRP virus, and the cells are analyzed for the presence of the
protein encoded by the reporter gene. If the cDNA of the RDRP viral
genome has been replicated and processed within the cultured cell,
viral RDRP enzyme will have been synthesized, thereby enabling
polymerization of the construct and synthesis of the protein
encoded by the reporter gene. Thus, detection of the reporter
protein in the cells provides a means to monitor and measure the
genomic replication of said RDRP virus.
[0010] In another aspect, the invention provides an efficient in
vitro method for identifying compounds or conditions which inhibit
the genomic replication of viruses that are dependent for
replication on RNA-dependent RNA polymerase (an RDRP virus). The
method comprises the steps of culturing virally-compatible
eukaryotic cells, which have been transfected with the cDNA of all
or a portion of the genomic sequence of the RDRP virus, and
transfecting these cultured cells with a construct of the
invention, which comprises the cDNA in antisense orientation of a
reporter gene sequence. The reporter gene cDNA sequence is operably
linked on its 5' end with the cDNA of the untranslated region
(UTR), in antisense orientation, from the native 3' end of said
RDRP virus, and is operably linked on its 3' end with the UTR, in
antisense orientation, from the native 5' end of the RDRP virus.
The cultured cells are exposed to a compound or condition suspected
of being capable of inhibiting the genomic replication of the RDRP
virus, and thereafter or concurrently the cells are cultured for a
period of time under conditions which are permissive for genomic
replication of the RDRP virus. The cells are analyzed for the
presence of the protein encoded by the reporter gene sequence,
whereby a decrease in the level of the reporter protein indicates
that the suspected compound or condition is capable of inhibiting
genomic replication of the RDRP virus.
[0011] The present invention also provides a method of selectively
affecting a cell which is infected with a virus that is dependent
for genomic replication upon RNA-dependent RNA polymerase (an RDRP
virus). The method comprises transfecting tissues, or cells which
are infected with an RDRP virus, with a construct of the invention
comprising the cDNA in antisense orientation of a gene or sequence
which encodes a protein that is capable of affecting the cell,
wherein the cDNA sequence encoding said protein is operably linked
on its 5' end with the cDNA of the untranslated region (UTR), in
antisense orientation, of the native 3' end of said RDRP virus and
is operably linked on its 3' end with the cDNA of the UTR, in
antisense orientation, of the native 5' end of said RDRP virus.
Sufficient time for genomic replication of said RDRP virus is
allowed. Thus, upon genomic replication of the RDRP virus,
RNA-dependent RNA polymerase (RDRP) is produced which will cause
polymerization of the construct thereby allowing synthesis within
infected cells of the affecting protein. In this manner, only cells
that are infected with the RDRP virus will be affected, thereby
affording a mechanism to selectively affect RDRP virally infected
cells within a mixed population of infected and normal cells. In a
preferred aspect of this embodiment of the invention, the effect
achieved is to selectively harm or kill cells which are infected
with the RDRP virus by inserting into the construct the cDNA of a
sequence encoding a protein which is harmful or fatal to the
cell.
[0012] In all aspects of the present method of the invention, a
preferred embodiment includes wherein the RDRP virus is selected
from the viral family Flaviviridae. It is most preferred that the
RDRP virus is HCV.
[0013] A further preferred embodiment in all aspects of the method
of the invention includes wherein the construct of the invention
further comprises the cDNA of a delta ribozyme sequence, in sense
orientation, operably linked to the 3' end of the construct
adjacent to the 3' end. When the RDRP virus is HCV, the cDNA of
hepatitis delta ribozyme, in sense orientation, is operably linked
to the 3' end of the cDNA of the 5' UTR of the native HCV viral
genome.
[0014] In another aspect, the invention provides a construct
comprising the cDNA, in antisense orientation, of a reporter gene
sequence wherein said reporter gene cDNA sequence is operably
linked on its 5' end with the cDNA of the UTR, in antisense
orientation, of the native 3' end of an RDRP virus and is operably
linked on its 3' end with the cDNA of the UTR, in antisense
orientation, of the native 5' end of the RDRP virus. Alternatively,
in another aspect of the invention, instead of the antisense cDNA
of a reporter gene sequence, a construct may comprise the antisense
cDNA of an "affecting gene" wherein said gene encodes a protein
which is capable of affecting the cell, preferably harming or
killing the cell. In these aspects of the invention it is preferred
that the RDRP virus is HCV.
[0015] The constructs of the invention further comprise an operably
linked constitutive or inducible promoter.
[0016] It is also preferred that the constructs of the invention
further comprise the cDNA, in sense orientation, of the hepatitis
delta ribozyme operably linked to the 3' end of the cDNA, in
antisense orientation, of the 5' UTR of the native viral
genome.
[0017] And in another aspect, the invention provides a eukaryotic
cell which has been transfected with a construct of the invention,
preferably a primate cell, most preferably, a human cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Schematic for production of RDRP-dependent
luciferase activity in the 293B4.alpha. cell line.
[0019] FIG. 2. Cloning strategy for the construction of pMJ050.
[0020] FIG. 3. Nucleotide sequence of pMJ050, presented from left
to right in 5' to 3' orientation, FIG. 3A. showing the nucleotides
comprising the SV40 promoter and the HCV 3'UTR (in antisense
orientation); FIG. 3B. showing the luciferase coding region (in
antisense orientation); the HCV 5' UTR sequence (in antisense
orientation); and the hepatitis delta virus ribozyme sequence (in
sense orientation); and FIG. 3C. showing the plasmid backbone
sequence.
[0021] FIG. 4. Production of luciferase in 293FL#9 cells stably
transfected with pMJ050.
[0022] FIG. 5. Production of luciferase, HCV core, HCV serine
protease, and HCV RDRP in the 293B4.alpha. cell line.
[0023] FIG. 6. Production of luciferase sense and antisense RNA in
the 293B4.alpha. cell line.
[0024] FIG. 7. Schematic representation of the mechanism of the
invention in a B4alpha human kidney cell which has been transfected
with the genome of HCV, using luciferase as the reporter gene in a
construct of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] A convenient in vitro system that models viral replication
for several members of the important viral family Flaviviridae has
not been described to date. This lack of an in vitro system has
significantly hindered research in this field directed towards
development of antiviral agents for the treatment of viral
infection, particularly for HCV infection. Described herein is an
in vitro system that can be formatted to allow detection of cells
in which RDRP genomic replication is occurring. The method employs
a construct that expresses a detectable reporter protein in
response to RDRP viral genomic replication. The method of the
invention can be manipulated to screen for compounds or conditions
having the ability to inhibit RDRP viral genomic replication. The
method also provides a mechanism in which RDRP virally-infected
cells can be selectively affected.
[0026] Various definitions and abbreviations are provided
throughout this document. Most words, unless otherwise defined,
have the meaning that would be attributed to those words by one
skilled in the art of the invention.
[0027] The following abbreviations are used throughout this
application: HCV: Hepatitis C virus; DNA: deoxyribonucleic acid;
RNA: ribonucleic acid; UTR: untranslated region; hdvribo: hepatitis
delta virus ribozyme; PCR: polymerase chain reaction; RDRP:
RNA-dependent RNA polymerase enzyme; IRES: internal ribosome entry
site; RT: reverse transcriptase; and RT-PCR: reverse transcription
polymerase chain reaction.
[0028] As used herein the term "in vitro" means occurring outside
of a living organism; in contrast to the term "in vivo", which
means occurring within a living organism. In vitro can describe
processes and conditions occurring within cultured cells, or
occurring within cellular lysate systems that contain the cellular
components necessary to perform the process in question.
[0029] Applicants contemplate that the in vi tro methods of the
invention relating to measuring RDRP genomic replication and
identifying inhibitors of such replication can be conducted in cell
culture systems, or alternatively, in the cellular lysate systems
of virally compatible eukaryotic cells.
[0030] Within the context of this invention, the term
"virally-compatible cells" refers to eukaryotic cells that contain
the necessary cellular proteins required by the RDRP virus to
complete replication of the virus genome. Virally-compatible cells
include, but are not limited to, cells in which the viral particle
is able to complete its entire replication cycle i.e., the virus is
able to reproduce and generate other infectious viral particles.
Also included are cells that may not be able to sustain the entire
viral replication cycle, but which are able to sustain the
replication of the viral RNA genome. Examples of preferred
virally-compatible cells include mammalian, especially human, liver
and kidney cells, and B and T cells.
[0031] "Virally-compatible cells which have been transfected with
the cDNA of the genomic sequence of an RDRP virus" refers to
virally-compatible cells into which have been stably incorporated a
functional genomic sequence of the virus under study. When the
method is conducted in order to study and measure replication of
the viral genome, it will be preferable to incorporate most or all
of the native viral genomic sequence, in order to most effectively
mimic and study native replication. When the method is conducted in
order to identify inhibitors of viral replication, it is possible
to incorporate into the cellular genome all of the genome, or
alternatively only those selective portions of the viral genome
which encode proteins to be studied, so long as the selected
portion of the viral genome includes the sequence that encodes the
RNA-dependent RNA polymerase, which is known as the NS5B portion of
the HCV genome. Methods for stably transfecting all or selective
portions of the viral genome into suitable cell lines are known by
those skilled in the art. For example, such methods are reported in
"Continuous Human Cell Lines Inducibly Expressing Hepatitus C Virus
Structural and Nonstructural Proteins," Darius Marpour, Petra Kary,
Charles M. Rice and Huber E. Blum (1998) Hepatology 28:192201.
"Transfection of a Differentiated Human Hepatoma Cell Line (Huh7)
with In Vitro-Transcribed Hepatitis C Virus (HCV) RNA and
Establishment of a Long-Term Culture Persistently Infected with
HCV," Young J. Yoo et al, J. of Virology, Vol 69, No.1, January
1995, p. 32-38. Genomic sequences for the flaviviruses are
generally available in the scientific literature, for example, see
www.ncbi.nlm.nih.gov/genbank for the Genbank library of sequences
that includes viral and the flavivirus gene sequences.
[0032] Applicants contemplate that the in vitro methods of the
invention relating to measuring genomic replication of RDRP cells
and identifying inhibitors of such replication can be conducted not
only in cell culture systems of virally-compatible cells, but also
in cell lysate systems of those cells. For example, cells from
HCV-infected cell culture or tissues removed from an HCV-positive
individual can be used to create a cell lysate that can serve as a
source of the HCV replicative proteins. This lysate can be prepared
by lysing the infected culture or tissue cells by methods well
known in these art, for example, by chemical or physical means, and
clarifying the cell lysate of macromolecule cellular debris by
centrifugation. Reporter RNA produced by in vitro transcription of
the reporter constructs of the invention can be added to the cell
lysate described above and the lysate with added reporter RNA can
be incubated under proper conditions permissive for genomic
replication. Such conditions (e.g., temperature, pH, salt
concentrations, etc.) are known or can be readily determined
experimentally by those skilled in the art for the particular
system selected for the assay. Lysates are then assayed to see if
the reporter protein has been produced, thereby indicating that
viral genomic replication has occurred within the lysate system.
Such lysate systems are amenable to rapid, high throughput
screening for inhibitors of RDRP viral replication.
[0033] The term "replication" as used within the disclosure herein
regarding viruses relates to the replication of the genome of the
virus, rather than whole virus replication which results in an
infectious particle.
[0034] The term "transfecting" as used herein refers to the process
of inserting heterologous DNA into a eukaryotic cell by chemical,
physical or other means that include but are not limited to
liposomal transfer, in which liposomal micelles containing the
heterologous DNA transfer the DNA into the cell by fusion with the
cell membrane; CaPO.sub.4 or DEAE-dextran shock, in which these
chemical moieties physically disrupt the cell membrane allowing
macromolecules to pass from the outside to the inside of the cell;
and electroporation, in which electrical shock is used to disrupt
the cell membrane allowing macromolecules to pass from the outside
to the inside of the cell. Such methods are well known in these
arts. Newly emerging nucleic acid delivery systems include the
adenoviral and adeno-associated viral systems, which are being
developed and used to deliver heterologous DNA sequences into human
tissues for the purposes of gene therapy. Also, as used herein, the
term "transfected" includes both stably transfected cells, in which
the transfected DNA recombines with the host cell DNA such that it
becomes a permanent part of the genome of the host cell, and also
transiently transfected cells, in which the transfected DNA remains
independent of the host cell DNA and is either destroyed by host
cell mechanisms which act to defend the cell from "infection" with
heterologous DNA or is diluted out by the replication of the host
cell.
[0035] Moreover, the RNA which is used as the template for
replication can be delivered to the cell by methods including but
not limited to virus infection, transfection of in vitro
transcribed RNA, and transcription of DNA that is stably or
transiently transfected into the host cell. RNA transcription from
stably or transiently transfected heterologous DNA can occur either
constitutively or inducibly.
[0036] Within the context of the invention, those skilled in this
art will understand that transcription of transfected DNA will be
driven by an operably linked promoter system. A "promoter" is a
regulatory nucleic acid sequence that is capable of controlling the
expression of a coding sequence or functional RNA. In general, a
coding sequence will be located 3' to the promoter sequence.
Promoters may be derived in their entirety from a native gene, or
be comprised of different elements derived from different promoters
found in nature. It is understood that various well known promoters
are suitable to direct expression of any number of different coding
sequences depending on cell and tissue type, in response to
different stimuli, or at different stages of cellular or tissue
development. Furthermore, the promoter sequence, which is part of
the transfected DNA of the invention, will determine if expression
of the transfected DNA will be constitutive or inducible. Examples
of constitutive promoters include but are not limited to the
cytomegalovirus immediate-early promoter, the SV40 viral promoter,
human immunodeficiency virus long terminal repeat promoter, and the
chicken beta-actin promoter. Examples of inducible promoters
include but are not limited to the tetracycline-responsive
promoter, the ecdysone-inducible promoter, and the
mifepristone-inducible promoter.
[0037] The term "operably linked" refers to the association of two
or more nucleic acid sequences on a single nucleic acid fragment so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences such as promoters in the sense or antisense
orientation.
[0038] The term "RNA-dependent RNA polymerase virus" or "RDRP
virus" means a virus which is dependent upon a functional
RNA-dependent RNA polymerase for the replication of its nucleic
acid genome and the production of infectious virus. In particular
relating to the present invention, we include viruses that are
members of the Flaviviridae family. It has been shown that the
replication of all of the members of this virus family are
dependent upon the virus's RNA-dependent RNA polymerase for
replication of the virus genome. The RDRP performs several
essential steps in the replication of RNA including the interaction
of the RDRP with the 3' and 5' untranslated regions (UTR) of the
genome, initiation of the synthesis of the new RNA strand, and
continued elongation of the growing progeny RNA. The UTR's for each
of the different members of this family are unique to that
particular virus, and have been identified, sequenced and placed in
the public domain (i.e., Genebank data base), and are thus readily
obtainable. In a preferred aspect of this invention the sequence
for the ribozyme from the hepatitis delta virus is placed at the 3'
end of the constructs of the invention. This sequence, when
transcribed into RNA, has catalytic function and will cleave itself
from the 3' end of the RNA transcript. When using HCV, for example,
this cleavage event results in a proper 3' end for the HCV 3' UTR
in the antisense RNA transcripts and a proper 5' end in the HCV
5'UTR in the sense RNA transcripts of this invention. The catalysis
of the hepatitis delta virus ribozyme is regulated by sequences
contained within the ribozyme itself. This being so, the sequence
for the hepatitis delta ribozyme included in the description of
this construct can be used with other RDRP virus systems (besides
HCV) and accordingly in most or all of the antisense reporter
constructs of the invention.
[0039] Within the context of the present invention, the term
"expression" refers to the transcription of DNA resulting in the
production of sense or antisense RNA, and may also refer to
translation of mRNA into a polypeptide, such as the reporter
protein.
[0040] The term "reporter gene" or "reporter gene sequence" refers
to any gene encoding a protein which can be expressed and
conveniently detected in a eukaryotic cell including chemically,
spectrophotometrically, immunologically, calorimetrically,
radioactively or through a receptor-mediated cascade system.
Examples of reporters include but are not limited to luciferase,
secreted alkaline phosphatase, beta-galactosidase, hepatitis B
virus surface antigen, herpes simplex virus thymidine kinase,
genticin-resistance, zeocin-resistance, hygromycin-resistance, and
puromycin-resistance.
[0041] A reporter gene sequence affects cells or lysates within the
method of the invention by producing an effect that can be
detected. Applicants contemplate that other gene sequences could be
used in place of a reporter sequence when the goal of the
researcher is to selectively produce another specific effect within
cells wherein viral genomic replication is occurring. Thus, if a
gene or genes resulting in a deleterious effect are selected for
insertion into the construct in place of (or in addition to) the
reporter sequence, the method of the invention offers a mechanism
to design therapeutic methods for the selective treatment of cells
infected with a flavivirus, wherein the construct containing an
affecting gene or genes is delivered in vivo to cells known to be
infected with a replicating flavivirus.
[0042] The term "construct" when referring to the "construct of the
invention" as used herein refers to a DNA sequence which comprises
a coding sequence for a reporter gene or an affecting gene, as the
terms are used herein, plus regulatory sequences related to
expression of that coding sequence including particularly promoters
that facilitate transcription of the coding sequence and also
including any 5' and/or 3' transcribed but untranslated sequences
that are associated with the coding sequence and may be required,
plus the 3' and 5' untranslated regions (UTRs) of the RDRP virus
under study. These sequences may be in the sense or antisense
orientation. The total construct sequence is created using standard
molecular biology techniques. The construct of the invention may
also include an operably linked ribozyme sequence.
[0043] A representative construct of the invention is provided in
FIG. 1, and includes (from 5' to 3') an SV40 promoter sequence, in
sense orientation, operably linked to the HCV 3'UTR, in antisense
orientation, linked to a coding sequence for luciferase protein, in
antisense orientation, linked to the HCV 5'UTR, in antisense
orientation, linked to the coding sequence for the hepatitis delta
ribozyme, in sense orientation, wherein said construct is delivered
to the cell via the plasmid entitled pMJO50.
[0044] In one aspect of the invention, a method is provided for
screening inhibitors of viral replication. Compounds and conditions
that are potentially capable of inhibiting viral replication
include but are not limited to small molecular weight synthetic
chemicals, organic compounds that are derived from living or once
living organisms, synthetic chemical compounds based on organic
compounds derived from living or once living organisms, as well as
various conditions including different frequencies of sound, and
various wavelengths of light and temperature. By example, compounds
may include small molecules, peptides, proteins, sugars,
nucleotides or nucleic acids, and may be natural or synthetic.
[0045] The method of screening compounds for inhibitors of viral
replication includes any protocol which utilizes cells or cell
lysate containing all or a portion of a viral genome sufficient to
express a functional RNA-dependent RNA polymerase, to which cell or
lysate, is added the appropriate reporter construct of the
invention. The viral genome and reporter construct system are
placed in the presence of a potential inhibitor(s) of viral
replication, under conditions amenable to replication of that viral
genome. Thus, compounds or conditions capable of inhibiting
replication of the viral genome, and/or capable of inhibiting the
functionality of the expressed RDRP enzyme, can be identified via
inhibition of expression of the reporter sequence.
[0046] The methods of the invention are functional when enough of
the viral genome is present in the system to result in production
of functional RNA-dependent RNA polymerase. Thus, the coding
sequence of the viral genome that is used in the cultured cells or
cell lysate can contain the coding sequence of the RNA-dependent
RNA polymerase as part of the entire viral genome, or
alternatively, it can contain subgenomic fragments of the viral
genome, encoding, for example in HCV, the NS2 to NS5b region, or
from the NS3 to NS5b region. In light of this aspect, the methods
of the invention permit screening for inhibitors which have the
ability to inhibit not only the NS5b RDRP, but also the NS2
protease, NS3 protease, and NS3 helicase as well, individually or
collectively. Specifically, the ability to screen compounds for the
potential to inhibit many different targets allows for the testing
of different combinations of inhibitors targeted at one or more of
the essential enzyme functions establishing whether interaction
between the compounds favorably or deleteriously effects the
ability of the compounds to inhibit the replication of the RNA.
EXAMPLES
[0047] The following examples demonstrate the method of the
invention, but should not be viewed as limiting of the scope of the
invention. Based upon the present disclosure many possible
variations of the method of the invention will become apparent to
those skilled in these arts.
[0048] In Examples 1 through 3 examines HCV viral genomic
replication in human kidney cells, using firefly luciferase as a
reporter gene within the construct of the invention. Example 3
demonstrates use of the method to confirm known inhibitors of HCV
replication using the method of the invention. Example 4
demonstrates a semi high-throughput screening assay for inhibitors
or HCV genomic replication.
Example 1
[0049] It is known that Flaviviridae viral replication takes place
through a step catalyzed by the viral RNA-dependent RNA polymerase
(RDRP), an enzyme not normally found in eukaryotic cells. A
substrate for HCV RDRP was selected that consists of an antisense
sequence of the firefly luciferase gene, a common reporter gene
used in cell biology. To make this sequence appear "HCV-like" it
was flanked with the 5' and 3' untranslated regions (UTR) of the
native HCV viral genome in the same orientation as they are found
in the (-)strand of the HCV replicative intermediates. Using the
convention employed herein, the orientation existing in the
(-)strand of the RNA genome will be referred to as the antisense
orientation when read from the 5' to 3' direction. To demonstrate a
preferred embodiment of the method, the hepatitis delta ribozyme
hdvribo was attached to the 3' end of the HCV 5' UTR sequence, such
that when the hdvribo processes the RNA, the sequence integrity of
the 5' UTR would be maintained (the strategy for the reporter is
shown in FIG. 1). This was done because it is known to those
skilled in the art that the 5' UTR also acts as an internal
ribosomal entry site (IRES) (13), and it was desirable to keep the
5' UTR sequence as true as possible to that found in the native
virus.
[0050] This construct was stably transfected into a 293 cell line
(human embryonic kidney cells) and designated 293 FL#9 (this cell
line had been previously transfected to contain a full length cDNA
copy of the native HCV genotype lb genome). The cell line
containing the construct of the invention, 293B4.alpha., was
demonstrated to produce active, detectable luciferase as a result
of genomic replication of the HCV viral genome.
Preparation of the pMJ050 Construct
[0051] pMJ050 was prepared in three steps. First, the antisense
sequences of the 3' untranslated region of the HCV genome (3'UTR)
and the firefly luciferase gene were joined together; second, the
antisense sequence of the HCV 5' untranslated region (5'UTR) and
the sense sequence of the hdvribo were joined together; and finally
these two constructs were joined together resulting in a sequence
which consisted of the antisense sequences of the "3'UTR-firefly
luciferase-5'UTR-hdvribo (in the sense orientation)", respectively
as read from 5' to 3' (FIG. 2).
Construction of the 3'UTR and Luciferase Sequence
[0052] The antisense sequence of the HCV 3'UTR was PCR amplified
from plasmid p90 (supplied by Dr. Charles Rice, Washington
University at St. Louis) using PCR primers, 3'UTR5'(new) and
3'UTRHO (for the nucleotide sequences of all oligos, see Table 1).
The antisense sequence of the firefly luciferase gene was PCR
amplified from plasmid pGL3 (Promega Corporation, Madison Wis.)
using PCR primers LUCHO and LUCIF3'. To join these two PCR products
together, overlapping PCR was performed in which equimolar amounts
of the two PCR products were mixed with oligos, 3'UTR5' and
LUCIF3', and the DNA amplified by PCR.
1TABLE 1 Nucleotide Sequences of Oligos Used to Create and Sequence
pMJ050 OLIGO NAME/ SEQ ID Nos: OLIGO SEQUENCE (Read 5' TO 3')
3'UTR5'new GCG TTT AAG CTT ACA TGA TCT GCA GAG SEQ ID NO: 1 AGG
3'UTRHO GGC GGA AAG ATC GCC GTG TAA AGG TTG SEQ ID NO: 2 GGG TAA
ACA CTC CGG 5'UTR5' CTG TGG ACG TCG GTT GGT GTT ACG TTT SEQ ID NO:
3 GGT TTT TCT TTG AGG TTT AGG 5'UTRHO GGC TGG GAC CAT GCC GGC CGC
CAG CCC SEQ ID NO: 4 CCT GAT GGG GGC LUCHO CCG GAG TGT TTA CCC CAA
CCT TTA CAC SEQ ID NO: 5 GGC GAT CTT TCC GCC LUCIF3' TTG GTA GAC
GTC CAA TGG AAG ACG CCA SEQ ID NO: 6 AAA TAA AGA AAG G HEPHO GCC
CCC ATC AGG GGG CTG GCG GCC GGC SEQ ID NO: 7 ATG GTC CCA GCC
RIBOHD3' CTC AAG CTC TAG AGA GAT TTG TGG GTC SEQ ID NO: 8 CC LUCACA
(+) GAA GAC GCC AAA AAC ATA AAG AAG GGC SEQ ID NO: 9 CCG GCG CCA
LUCACA (-) TGG CGC CGG GCC CTT CTT TAT GTT TTT SEQ ID NO: 10 GGC
GTC TTC UTRRNA (+) CCT CTT AGG CCA TTT CCT GTT TTT TTT SEQ ID NO:
11 TTT UTRRNA (-) AAA AAA AAA AAC AGG AAA TGG CCT AAG SEQ ID NO: 12
AGG LUCFOR CCG AGT GTA GTA AAC ATT CC SEQ ID NO: 13 LUCREV CTC GCA
TGC CAG AGA TCC SEQ ID NO: 14 LITFOR GAT CTT CGA ATG CAT CGC GCG C
SEQ ID NO: 15 LITREV GGC CTT GAC TAG AGG GTA CC SEQ ID NO: 16
[0053] The product of the overlapping PCR was digested with the
restriction enzymes, Hind III and Aat II, and ligated into
pLitmus28 (New England Biolabs, Beverley, Mass.) which had been
linearized with Hind III and Aat II. The plasmid from the ligation
reaction, pLitmus283'UTR luciferase was transformed into chemically
competent E. coli DH5.alpha. cells. E. coli that had become
transformed with this plasmid were selected by the ability to grow
on solid nutrient agar containing ampicillin. Plasmid DNA was
isolated from ampicillin-resistant bacterial cells and the sequence
was verified by restriction enzyme analysis using BsrG I, Hind III
and Aat II, and sequence analysis using sequencing oligos LUCFOR,
LUCREV, LITFOR, and LITREV.
Construction of the Antisense 5'UTR and Sense hdvribo Sequence
[0054] The sequence of the hepatitis delta virus ribozyme (hdvribo)
was PCR amplified from plasmid pFullLengthVec (provided by Dr.
William Mason, Fox Chase Cancer Center, Philadelphia, Pa.) using
PCR primers HEPHO and RIBOHD3'. The 5'UTR of the HCV genome was PCR
amplified from plasmid pSignalIRES (provided by Robert Kovelman,
Signal Pharmaceuticals, San Diego, Calif.) and joined to the
hdvribo sequence by overlapping PCR using PCR primers 5'UTR5',
5'UTRHO, and RIBOHD3'.
[0055] The DNA product from the overlapping PCR was digested with
the restriction enzymes, Xba I and Aat II, and ligated into
pLitmus28 that had been linearized by digestion with Xba I and Aat
II. The recombinant plasmid, pLitmus285'UTRAribo was transformed
into chemically competent E. coli DH5 .alpha. cells. E. coli that
had been transformed with this plasmid were selected by the ability
to grow on solid nutrient agar containing ampicillin. Plasmid DNA
was isolated from ampicillin-resistant bacterial cells and the
sequence was verified by restriction enzyme analysis with Xba I and
Aat II, and sequence analysis using primers 5'UTR5' and
RIBOHD3'.
Construction of pMJ050
[0056] The inserts in plasmids, pLitmus283'UTR luciferase and
pLitmus285'UTR hdvribo were joined together by digesting both
plasmids with restriction enzymes Hind III and Aat II. Equimolar
amounts of DNA were mixed and ligated together. The DNA resulting
from the ligation reaction was transformed into chemically
competent E. coli DH5.alpha. cells. E. coli that had become
transformed were selected by the ability to grow on solid nutrient
agar containing ampicillin. Plasmid DNA was isolated from
ampicillin-resistant bacterial cells. Insertion of the reporter
gene was verified by restriction enzyme analysis using Hind III and
Xba I.
[0057] For the reporter gene to be transcribed in an eukaryotic
cell, the reporter gene from pLitmus28reporter had to be placed
into a plasmid that contained an eukaryotic promoter. To accomplish
this, the reporter gene was removed from pLitmus28reporter by
restriction digestion with Spe I and Xba I and ligated into the
plasmid pZeoSV that had been previously linearized by restriction
digest with Hind III and Spe I. The DNA resulting from the ligation
reaction was transformed into chemically competent E. coli
DH5.alpha.. E. coli that had become transformed were selected by
the ability to grow on solid nutrient agar containing zeocin.
Plasmid DNA was isolated from zeocin-resistant bacterial cells and
the sequence of the recombinant plasmid was verified by restriction
enzyme analysis using Hind III, TthIII I and Kpn I, and sequence
analysis with oligos 3'UTR5'new, 3'UTRHO, 5'UTR5', 5'UTRHO, LUCHO,
LUCIF3', HEPHO, RIBOHD3', LUCFOR, and LUCREV. The plasmid
containing the correct sequence construct was designated pMJ050
(FIG. 3).
Example 2
Creation of the 293B4.alpha. Cell Line
[0058] (a) Transfection and Selection of Zeocin-Resistant 293FL#9
Cells.
[0059] To create a cell line the would express the antisense
luciferase construct as RNA in the environment of the HCV proteins,
pMJ050 was transfected into 293FL#9 cells by electroporation (3-10
.mu.g of plasmid into 5.times.10.sup.6 cells; one pulse at
960.degree. F. and 0.2 kV in a BioRad electroporator).
Transfectants were grown in the presence of 250 .mu.g/ml each of
G418 and zeocin for several weeks to select for cells that had
stably integrated pMJ050 into their genome. Forty-eight
zeocin-resistant stable transfectants were randomly selected and
expanded further.
[0060] (b) Luciferase Assay
[0061] The 48 stable transfectants were tested for the ability to
express active luciferase using the commercially available
Luciferase Assay System (Promega Corp., Madison, Wis.) as directed
by the manufacturer. Briefly, the 1.times.10.sup.6 cells from each
of the 48 clonal cell lines was lysed with 100 .mu.l of Lysis
Buffer. The lysates were clarified by centrifugation and stored at
-80.degree. C. Twenty microliters of cell lysate was assayed for
luciferase activity by the addition of 100 .mu.l of luciferin
substrate and quantification on a luminometer.
[0062] Twelve of 48 resultant clones expressed various amounts of
luciferase activity. These twelve were grown for several more weeks
in the presence of zeocin and G418 and then retested for luciferase
activity (FIG. 4). The cell line, 293B4.alpha. consistently and
reproducibly had the highest level of luciferase activity and was
chosen for further study.
Characterization of the 293B4.alpha. Cell Line
[0063] (a) Protein Production
[0064] Western blot analysis was used to determine if the
293B4.alpha. cell line was producing luciferase, HCV core, HCV
serine protease (encoded by the HCV NS3 gene), and HCV RDRP
(encoded by the HCV NS5b). Western blot analysis showed that all 4
proteins were produced in the 293B4.alpha. cell line (FIG. 5).
[0065] (b) Luciferase RNA Production
[0066] Theoretically, the only way for luciferase protein to be
produced in 293B4.alpha. cells is if there is an RDRP present in
the cells to transcribe the antisense luciferase RNA into the sense
orientation. RT-PCR was used to determine (1) if antisense
luciferase RNA transcription, driven by the SV40 promoter, was
taking place, and (2) if the antisense RNA was being transcribed
into sense luciferase RNA. Oligos LUCFOR and LUCREV were used in
the RT-PCR to determine both of these.
[0067] Total cytoplasmic RNA was isolated from 5.times.10.sup.6
293B4.alpha. cells using the RNAgents RNA Isolation kit as directed
by the manufacturers (Promega Corp., Madison, Wis.). An aliquot,
which was equivalent to {fraction (1/50)} of the RNA isolated, was
used in each RT-PCR. To determine the presence of the antisense and
sense strands of RNA, the RT portion of the reaction was run in the
presence of only one of the oligos (i.e. LUCREV to detect the
antisense strand and LUCFOR to detect the sense strand). The
temperature of the reaction was increased to 95.degree. C. for 5
minutes to heat inactivate the RT enzyme and then the other oligo
was added and PCR proceeded as normal.
[0068] The cytoplasm of the 293B4.alpha. cells contained both
species of luciferase RNA, whereas 293FL#9 cells did not contain
either species (FIG. 6). Likewise, if the RT step was eliminated
from the RT-PCR or if the RNA samples were treated with RNase prior
to the RT-PCR, no products were produced indicating that the
product detected in the RT-PCR of the RNA of 293B4.alpha. cells was
from RNA and not DNA contamination. Moreover, treating the RNA
samples with DNase prior to RT-PCR had no effect on the quantity of
product produced in the RT-PCR.
Example 3
Inhibition of Luciferase Activity by Inhibitors of Luciferase, HCV
Serine Protease and IRES-Mediated Translation
[0069] Four chemical compounds, two known to inhibit the HCV serine
protease, one known to inhibit IRES-mediated translation of the HCV
RNA, and one known to inhibit firefly luciferase in the
293B4.alpha. cell line, were tested for the ability to reduce the
level of firefly luciferase in the 293B4.alpha. cell line.
Thirty-five millimeter plates were seeded with 5.times.10.sup.5
cells/plate and incubated at 37.degree. C. overnight. Media
containing various concentrations of the four chemical compounds
were added to the cells. Forty-eight hours after the addition of
compound, the cells were lysed with lysis buffer as described
above. Luciferase activity was quantified using the Luciferase
Assay System (Promega Corp., Madison, Wis.) as directed by the
manufacturer. All four compounds had the ability to inhibit
luciferase activity (Table 2).
2TABLE 2 Inhibition of luciferase activity in the 293B4.alpha. cell
line Compound Inhibitor Class Inhibition.sup.1 Cmpd A HCV Protease
++ Cmpd B HCV Protease ++ Cmpd C HCV RDRP + Cmpd D Luciferase +++
Vehicle (0.3% DMSO) N/A -- .sup.1Key to activity: +++: greater than
75% inhibition; ++: between 75% and 50% inhibition; +: between 49%
and 25% inhibition; and --: less than 25% inhibition.
Example 4
Semi High-Throughput Assay for Inhibitors of HCV Replication Using
the 293B4.alpha. Cell Line
[0070] The assay begins by plating 3000 293B4.alpha. cells/well in
96-well plates and incubating the cells at 37.degree. C. overnight
to allow for attachment of the cells to the bottom of the well.
Sixteen to twenty-four hours after plating the cells, various
concentrations of compound are added to the wells. Thirty-six to
forty-eight hours after the addition of compound, media are removed
from the cells and the cells are washed once with cold PBS. The
cells are lysed in 25 .mu.l of lysis buffer and the plates are
stored at -80.degree. C. The lysates are thawed at room temperature
and 20 .mu.l of lysate and 100 .mu.l of luciferin substrate are
placed into the well of an opaque microtiter plate. Luciferase
activity is quantified with a luminometer. The potency of the
individual compounds is calculated by linear regression. 293FL#9
cells were electroporated with pMJ050 and were selected in G418 and
zeocin. Forty-eight clones were randomly selected and tested for
the ability to produce luciferase. The luciferase activity in the
twelve clones that were able to produce luciferase, was quantified
and is shown in FIG. 4. The 293B4.alpha. cell line was selected for
further study.
[0071] 293B4.alpha. cells were lysed in lysis buffer and the
proteins in the lysates were separated by size on a 4-12%
poly-acrylamide gel. The proteins were transferred to
nitrocellulose by electrophoresis. Luciferase, HCV core, HCV serine
protease, and HCV RDRP were detected by antibodies specific for the
individual proteins.
[0072] Total cytoplasmic RNA was isolated for 293B4.alpha. cells
using the RNAgents RNA Isolation kit as directed by the
manufacturer (Promega Corp., Madison, Wis.). RT-PCR and 2% of each
RNA sample was used to produce DNA from either the sense or
antisense luciferase RNA. C=sense orientation of the luciferase
gene (coding); A=antisense orientation of the luciferase gene
(non-coding); and A/C=single tube RT-PCR, does not differentiate
between the coding and non-coding species of RNA. Plasmid DNA
containing the luciferase gene was used as a positive control for
the RT-PCR. (FIG. 6)
[0073] All references cited within this disclosure are hereby
incorporated by reference in their entirety.
REFERENCES
[0074] 1. Bartenschlager and Lohman (2000) J. Gen. Virol. 81,
1631-1648.
[0075] 2. Kolykhalov et al. (1997) Science 277, 570-574.
[0076] 3. Neuman et al. (1998) Science, 282, 103-107.
[0077] 4. Tong et al. (1995) Lancet 345, 1058-1059.
[0078] 5. Saito et al. (1990) Proc. Natl. Acad. Sci. USA 87,
6547-6549.
[0079] 6. Bartenschlager et al. (1994) J. Virol. 68, 5045-5055.
[0080] 7. Eckart et al. (1993) BBRC 192, 399-406.
[0081] 8. Grakoui et al. (1993) J. Virol. 67, 2832-2843.
[0082] 9. Lin et al. (1994a) J. Virol. 68, 5063-5073.
[0083] 10. Lin et al. (1994b) J. Virol. 68, 8147-8157.
[0084] 11. Lohmann V., Korner F., Koch J. O., Herian U., Theilmann
L. and Bartenschlager R. (1999) Replication of subgenomic hepatitis
C virus RNAs in a hepatoma cell line. Science. 285:110-113.
[0085] 12. Ohishi M., Sakisaka S., Harada H., Koga H., Taniguchi
E., Kawaguchi T., Sasatomi K., Sata M., Kurohiji T. and Tanikawa K.
(1999) Detection of hepatitis-C virus and hepatitis-C virus
replication in hepatocellular carcinoma by in situ hybridization.
Scandinavian J. Gastroenterology. 34:432-438.
[0086] 13. Rijnbrand R. C. A. and Lemon S. M. (2000) Internal
ribosomal entry site-mediated translation in hepatitis C virus
replication. In, Current Topics in Microbiology and Immunology,
Eds. Hagedorn, C. H. and Rice, C. M. pp. 85-116. Springer-Verlag
Berlin.
Sequence CWU 1
1
20 1 30 DNA Artificial Sequence oligonucleotide 1 gcgtttaagc
ttacatgatc tgcagagagg 30 2 42 DNA Artificial Sequence
oligonucleotide 2 ggcggaaaga tcgccgtgta aaggttgggg taaacactcc gg 42
3 48 DNA Artificial Sequence oligonucleotide 3 ctgtggacgt
cggttggtgt tacgtttggt ttttctttga ggtttagg 48 4 39 DNA Artificial
Sequence oligonucleotide 4 ggctgggacc atgccggccg ccagccccct
gatgggggc 39 5 42 DNA Artificial Sequence oligonucleotide 5
ccggagtgtt taccccaacc tttacacggc gatctttccg cc 42 6 40 DNA
Artificial Sequence oligonucleotide 6 ttggtagacg tccaatggaa
gacgccaaaa taaagaaagg 40 7 39 DNA Artificial Sequence
oligonucleotide 7 gcccccatca gggggctggc ggccggcatg gtcccagcc 39 8
29 DNA Artificial Sequence oligonucleotide 8 ctcaagctct agagagattt
gtgggtccc 29 9 36 DNA Artificial Sequence oligonucleotide 9
gaagacgcca aaaacataaa gaagggcccg gcgcca 36 10 36 DNA Artificial
Sequence oligonucleotide 10 tggcgccggg cccttcttta tgtttttggc gtcttc
36 11 30 DNA Artificial Sequence oligonucleotide 11 cctcttaggc
catttcctgt tttttttttt 30 12 30 DNA Artificial Sequence
oligonucleotide 12 aaaaaaaaaa acaggaaatg gcctaagagg 30 13 20 DNA
Artificial Sequence oligonucleotide 13 ccgagtgtag taaacattcc 20 14
18 DNA Artificial Sequence oligonucleotide 14 ctcgcatgcc agagatcc
18 15 22 DNA Artificial Sequence oligonucleotide 15 gatcttcgaa
tgcatcgcgc gc 22 16 20 DNA Artificial Sequence oligonucleotide 16
ggccttgact agagggtacc 20 17 5860 DNA viral 17 ggatccgctg tggaatgtgt
gtcagttagg gtgtggaaag tccccaggct ccccagcagg 60 cagaagtatg
caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 120
ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
180 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
ctccgcccca 240 tggctgacta atttttttta tttatgcaga ggccgaggcc
gcctcggcct ctgagctatt 300 ccagaagtag tgaggaggct tttttggagg
cctaggcttt tgcaaaaagc ttacatgatc 360 tgcagagagg ccagtatcag
cactctctgc agtcatgcgg ctcacggacc tttcacagct 420 agccgtgact
agggctaaga tggagccacc attaaagaag gaaggaaaag aaaggaaaaa 480
agaaggaaag aaaaaaaaaa aaaaaaaaaa ggaaaaaaaa aaaaaaaaag aaaaaaaaaa
540 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaacaggaaa
tggcctaaga 600 ggccggagtg tttaccccaa cctttaaacg gcgatctttc
cgcccttctt ggcctttatg 660 aggatctctc tgatttttct tgcgtcgagt
tttccggtaa gacctttcgg tacttcgtcc 720 acaaacacaa ctcctccgcg
caactttttc gcggttgtta cttgactggc gacgtaatcc 780 acgatctctt
tttccgtcat cgtctttccg tgctccaaaa caacaacggc ggcgggaagt 840
tcaccggcgt catcgtcggg aagacctgcg acacctgcgt cgaagatgtt ggggtgttgg
900 agcaagatgg attccaattc agcgggagcc acctgatagc ctttgtactt
aatcagagac 960 ttcaggcggt caacgatgaa gaagtgttcg tcttcgtccc
agtaagctat gtctccagaa 1020 tgtagccatc catccttgtc aatcaaggcg
ttggtcgctt ccggattgtt tacataaccg 1080 gacataatca taggacctct
cacacacagt tcgcctcttt gattaacgcc cagcgttttc 1140 ccggtatcca
gatccacaac cttcgcttca aaaaatggaa caactttacc gaccgcgccc 1200
ggtttatcat ccccctcggg tgtaatcaga atagctgatg tagtctcagt gagcccatat
1260 ccttgcctga tacctggcag atggaacctc ttggcaaccg cttccccgac
ttccttagag 1320 aggggagcgc caccagaagc aatttcgtgt aaattagata
aatcgtattt gtcaatcaga 1380 gtgcttttgg cgaagaagga gaatagggtt
ggcaccagca gcgcactttg aatcttgtaa 1440 tcctgaaggc tcctcagaaa
cagctcttct tcaaatctat acattaagac gactcgaaat 1500 ccacatatca
aatatccgag tgtagtaaac attccaaaac cgtgatggaa tggaacaaca 1560
cttaaaatcg cagtatccgg aatgatttga ttgccaaaaa taggatctct ggcatgcgag
1620 aatctcacgc aggcagttct atgaggcaga gcgacacctt taggcagacc
agtagatcca 1680 gaggagttca tgatcagtgc aattgtcttg tccctatcga
aggactctgg cacaaaatcg 1740 tattcattaa aaccgggagg tagatgagat
gtgacgaacg tgtacatcga ctgaaatccc 1800 tggtaatccg ttttagaatc
catgataata attttttgga tgattgggag ctttttttgc 1860 acgttcaaaa
ttttttgcaa cccctttttg gaaacgaaca ccacggtagg ctgcgaaatg 1920
cccatactgt tgagcaattc acgttcatta taaatgtcgt tcgcgggcgc aactgcaact
1980 ccgataaata acgcgcccaa caccggcata aagaattgaa gagagttttc
actgcatacg 2040 acgattctgt gatttgtatt cagcccatat cgtttcatag
cttctgccaa ccgaacggac 2100 atttcgaagt actcagcgta agtgatgtcc
acctcgatat gtgcatctgt aaaagcaatt 2160 gttccaggaa ccagggcgta
tctcttcata gccttatgca gttgctctcc agcggttcca 2220 tcttccagcg
gatagaatgg cgccgggcct ttctttatgt ttttggcgtc ttccatggga 2280
cgtcggttgg tgttacgttt ggtttttctt tgaggtttag gattcgtgct catgatgcac
2340 ggtctacgag acctcccggg gcactcgcaa gcaccctatc aggcagtacc
acaaggcctt 2400 tcgcgaccca acactactcg gctagcagtc ttgcgggggc
acgcccaaat ctccaggcat 2460 tgagcggggt tatccaagaa aggacccggt
cgtcctggca attccggtgt actcaccggt 2520 tccgcagacc actatggctc
tcccgggagg gggggtcctg gaggctgcac gacactcata 2580 ctaacgccat
ggctagacgc tttctgcgtg aagacagtag ttcctcacag gggagtgatt 2640
catggtggag tgtcgccccc atcagggggc tggcggccgg catggtccca gcctcctcgc
2700 tggcgccggc tgggcaacat tccgagggga ccgtcccctc ggtaatggcg
aatgggaccc 2760 acaaatctct ctagatacct aggtgagctc tcggtacctc
gagaattcga acgcgtgatc 2820 agctgttcta tagtgtcacc taaatagctt
cgaggtcgac ctcgaaactt gtttattgca 2880 gcttataatg gttacaaata
aagcaatagc atcacaaatt tcacaaataa agcatttttt 2940 tcactgcatt
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 3000
cctcggagat ctgggcccat gcggccgcgg atcgatgctc actcaaaggc ggtaatacgg
3060 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
ccagcaaaag 3120 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
ataggctccg cccccctgac 3180 gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa acccgacagg actataaaga 3240 taccaggcgt ttccccctgg
aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3300 accggatacc
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 3360
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
3420 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
caacccggta 3480 agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag agcgaggtat 3540 gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac tagaaggaca 3600 gtatttggta tctgcgctct
gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3660 tgatccggca
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3720
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
3780 cagtggaacg aaaactcacg ttaagggatt ttggtcatga cattaaccta
taaaaatagg 3840 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
acggtgaaaa cctctgacac 3900 atgcagctcc cggagacggt cacagcttgt
ctgtaagcgg atgccgggag cagacaagcc 3960 cgtcagggcg cgtcagcggg
tgttggcggg tgtcggggct ggcttaacta tgcggcatca 4020 gagcagattg
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 4080
agaaaatacc gcatcaggcg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
4140 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
ctttcgcttt 4200 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
caagctctaa atcgggggct 4260 ccctttaggg ttccgattta gagctttacg
gcacctcgac cgcaaaaaac ttgatttggg 4320 tgatggttca cgtagtgggc
catcgccctg atagacggtt tttcgccctt tgacgttgga 4380 gtccacgttc
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 4440
ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
4500 gctgatttaa caaatattta acgcgaattt taacaaaata ttaacgttta
caatttccat 4560 tcgccattca ggctgcaact agatctagag tccgttacat
aacttacggt aaatggcccg 4620 cctggctgac cgcccaacga cccccgccca
ttgacgtcaa taatgacgta tgttcccata 4680 gtaacgccaa tagggacttt
ccattgacgt caatgggtgg agtatttacg gtaaactgcc 4740 cacttggcag
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 4800
ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
4860 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
gcagtacatc 4920 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
gtctccaccc cattgacgtc 4980 aatgggagtt tgttttggca ccaaaatcaa
cgggactttc caaaatgtcg taacaactcc 5040 gccccattga cgcaaatggg
cggtaggcgt gtacggtggg aggtctatat aagcagagct 5100 cgtttagtga
accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 5160
agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac ggacctgcag
5220 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
gactcactat 5280 aggagggcca ccatggccaa gttgaccagt gccgttccgg
tgctcaccgc gcgcgacgtc 5340 gccggagcgg tcgagttctg gaccgaccgg
ctcgggttct cccgggactt cgtggaggac 5400 gacttcgccg gtgtggtccg
ggacgacgtg accctgttca tcagcgcggt ccaggaccag 5460 gtggtgccgg
acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 5520
gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag
5580 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
caactgcgtg 5640 cacttcgtgg ccgaggagca ggactgaccg acgccgacca
acaccgccgg tccgacggcg 5700 gcccacgggt cccagggggg tcgacctcga
aacttgttta ttgcagctta taatggttac 5760 aaataaagca atagcatcac
aaatttcaca aataaagcat ttttttcact gcattctagt 5820 tgtggtttgt
ccaaactcat caatgtatct tatcatgtct 5860 18 2771 DNA viral 18
ggatccgctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
60 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
agtccccagg 120 ctccccagca ggcagaagta tgcaaagcat gcatctcaat
tagtcagcaa ccatagtccc 180 gcccctaact ccgcccatcc cgcccctaac
tccgcccagt tccgcccatt ctccgcccca 240 tggctgacta atttttttta
tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 300 ccagaagtag
tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttacatgatc 360
tgcagagagg ccagtatcag cactctctgc agtcatgcgg ctcacggacc tttcacagct
420 agccgtgact agggctaaga tggagccacc attaaagaag gaaggaaaag
aaaggaaaaa 480 agaaggaaag aaaaaaaaaa aaaaaaaaaa ggaaaaaaaa
aaaaaaaaag aaaaaaaaaa 540 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaacaggaaa tggcctaaga 600 ggccggagtg tttaccccaa
cctttaaacg gcgatctttc cgcccttctt ggcctttatg 660 aggatctctc
tgatttttct tgcgtcgagt tttccggtaa gacctttcgg tacttcgtcc 720
acaaacacaa ctcctccgcg caactttttc gcggttgtta cttgactggc gacgtaatcc
780 acgatctctt tttccgtcat cgtctttccg tgctccaaaa caacaacggc
ggcgggaagt 840 tcaccggcgt catcgtcggg aagacctgcg acacctgcgt
cgaagatgtt ggggtgttgg 900 agcaagatgg attccaattc agcgggagcc
acctgatagc ctttgtactt aatcagagac 960 ttcaggcggt caacgatgaa
gaagtgttcg tcttcgtccc agtaagctat gtctccagaa 1020 tgtagccatc
catccttgtc aatcaaggcg ttggtcgctt ccggattgtt tacataaccg 1080
gacataatca taggacctct cacacacagt tcgcctcttt gattaacgcc cagcgttttc
1140 ccggtatcca gatccacaac cttcgcttca aaaaatggaa caactttacc
gaccgcgccc 1200 ggtttatcat ccccctcggg tgtaatcaga atagctgatg
tagtctcagt gagcccatat 1260 ccttgcctga tacctggcag atggaacctc
ttggcaaccg cttccccgac ttccttagag 1320 aggggagcgc caccagaagc
aatttcgtgt aaattagata aatcgtattt gtcaatcaga 1380 gtgcttttgg
cgaagaagga gaatagggtt ggcaccagca gcgcactttg aatcttgtaa 1440
tcctgaaggc tcctcagaaa cagctcttct tcaaatctat acattaagac gactcgaaat
1500 ccacatatca aatatccgag tgtagtaaac attccaaaac cgtgatggaa
tggaacaaca 1560 cttaaaatcg cagtatccgg aatgatttga ttgccaaaaa
taggatctct ggcatgcgag 1620 aatctcacgc aggcagttct atgaggcaga
gcgacacctt taggcagacc agtagatcca 1680 gaggagttca tgatcagtgc
aattgtcttg tccctatcga aggactctgg cacaaaatcg 1740 tattcattaa
aaccgggagg tagatgagat gtgacgaacg tgtacatcga ctgaaatccc 1800
tggtaatccg ttttagaatc catgataata attttttgga tgattgggag ctttttttgc
1860 acgttcaaaa ttttttgcaa cccctttttg gaaacgaaca ccacggtagg
ctgcgaaatg 1920 cccatactgt tgagcaattc acgttcatta taaatgtcgt
tcgcgggcgc aactgcaact 1980 ccgataaata acgcgcccaa caccggcata
aagaattgaa gagagttttc actgcatacg 2040 acgattctgt gatttgtatt
cagcccatat cgtttcatag cttctgccaa ccgaacggac 2100 atttcgaagt
actcagcgta agtgatgtcc acctcgatat gtgcatctgt aaaagcaatt 2160
gttccaggaa ccagggcgta tctcttcata gccttatgca gttgctctcc agcggttcca
2220 tcttccagcg gatagaatgg cgccgggcct ttctttatgt ttttggcgtc
ttccatggga 2280 cgtcggttgg tgttacgttt ggtttttctt tgaggtttag
gattcgtgct catgatgcac 2340 ggtctacgag acctcccggg gcactcgcaa
gcaccctatc aggcagtacc acaaggcctt 2400 tcgcgaccca acactactcg
gctagcagtc ttgcgggggc acgcccaaat ctccaggcat 2460 tgagcggggt
tatccaagaa aggacccggt cgtcctggca attccggtgt actcaccggt 2520
tccgcagacc actatggctc tcccgggagg gggggtcctg gaggctgcac gacactcata
2580 ctaacgccat ggctagacgc tttctgcgtg aagacagtag ttcctcacag
gggagtgatt 2640 catggtggag tgtcgccccc atcagggggc tggcggccgg
catggtccca gcctcctcgc 2700 tggcgccggc tgggcaacat tccgagggga
ccgtcccctc ggtaatggcg aatgggaccc 2760 acaaatctct c 2771 19 2674 DNA
viral 19 ggatccgctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
ccccagcagg 60 cagaagtatg caaagcatgc atctcaatta gtcagcaacc
aggtgtggaa agtccccagg 120 ctccccagca ggcagaagta tgcaaagcat
gcatctcaat tagtcagcaa ccatagtccc 180 gcccctaact ccgcccatcc
cgcccctaac tccgcccagt tccgcccatt ctccgcccca 240 tggctgacta
atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 300
ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttacatgatc
360 tgcagagagg ccagtatcag cactctctgc agtcatgcgg ctcacggacc
tttcacagct 420 agccgtgact agggctaaga tggagccacc attaaagaag
gaaggaaaag aaaggaaaaa 480 agaaggaaag aaaaaaaaaa aaaaaaaaaa
ggaaaaaaaa aaaaaaaaag aaaaaaaaaa 540 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaacaggaaa tggcctaaga 600 ggccggagtg
tttaccccaa cctttaaacg gcgatctttc cgcccttctt ggcctttatg 660
aggatctctc tgatttttct tgcgtcgagt tttccggtaa gacctttcgg tacttcgtcc
720 acaaacacaa ctcctccgcg caactttttc gcggttgtta cttgactggc
gacgtaatcc 780 acgatctctt tttccgtcat cgtctttccg tgctccaaaa
caacaacggc ggcgggaagt 840 tcaccggcgt catcgtcggg aagacctgcg
acacctgcgt cgaagatgtt ggggtgttgg 900 agcaagatgg attccaattc
agcgggagcc acctgatagc ctttgtactt aatcagagac 960 ttcaggcggt
caacgatgaa gaagtgttcg tcttcgtccc agtaagctat gtctccagaa 1020
tgtagccatc catccttgtc aatcaaggcg ttggtcgctt ccggattgtt tacataaccg
1080 gacataatca taggacctct cacacacagt tcgcctcttt gattaacgcc
cagcgttttc 1140 ccggtatcca gatccacaac cttcgcttca aaaaatggaa
caactttacc gaccgcgccc 1200 ggtttatcat ccccctcggg tgtaatcaga
atagctgatg tagtctcagt gagcccatat 1260 ccttgcctga tacctggcag
atggaacctc ttggcaaccg cttccccgac ttccttagag 1320 aggggagcgc
caccagaagc aatttcgtgt aaattagata aatcgtattt gtcaatcaga 1380
gtgcttttgg cgaagaagga gaatagggtt ggcaccagca gcgcactttg aatcttgtaa
1440 tcctgaaggc tcctcagaaa cagctcttct tcaaatctat acattaagac
gactcgaaat 1500 ccacatatca aatatccgag tgtagtaaac attccaaaac
cgtgatggaa tggaacaaca 1560 cttaaaatcg cagtatccgg aatgatttga
ttgccaaaaa taggatctct ggcatgcgag 1620 aatctcacgc aggcagttct
atgaggcaga gcgacacctt taggcagacc agtagatcca 1680 gaggagttca
tgatcagtgc aattgtcttg tccctatcga aggactctgg cacaaaatcg 1740
tattcattaa aaccgggagg tagatgagat gtgacgaacg tgtacatcga ctgaaatccc
1800 tggtaatccg ttttagaatc catgataata attttttgga tgattgggag
ctttttttgc 1860 acgttcaaaa ttttttgcaa cccctttttg gaaacgaaca
ccacggtagg ctgcgaaatg 1920 cccatactgt tgagcaattc acgttcatta
taaatgtcgt tcgcgggcgc aactgcaact 1980 ccgataaata acgcgcccaa
caccggcata aagaattgaa gagagttttc actgcatacg 2040 acgattctgt
gatttgtatt cagcccatat cgtttcatag cttctgccaa ccgaacggac 2100
atttcgaagt actcagcgta agtgatgtcc acctcgatat gtgcatctgt aaaagcaatt
2160 gttccaggaa ccagggcgta tctcttcata gccttatgca gttgctctcc
agcggttcca 2220 tcttccagcg gatagaatgg cgccgggcct ttctttatgt
ttttggcgtc ttccatggga 2280 cgtcggttgg tgttacgttt ggtttttctt
tgaggtttag gattcgtgct catgatgcac 2340 ggtctacgag acctcccggg
gcactcgcaa gcaccctatc aggcagtacc acaaggcctt 2400 tcgcgaccca
acactactcg gctagcagtc ttgcgggggc acgcccaaat ctccaggcat 2460
tgagcggggt tatccaagaa aggacccggt cgtcctggca attccggtgt actcaccggt
2520 tccgcagacc actatggctc tcccgggagg gggggtcctg gaggctgcac
gacactcata 2580 ctaacgccat ggctagacgc tttctgcgtg aagacagtag
ttcctcacag gggagtgatt 2640 catggtggag tgtcgccccc atcagggggc tggc
2674 20 2327 DNA viral 20 agcttacatg atctgcagag aggccagtat
cagcactctc tgcagtcatg cggctcacgg 60 acctttcaca gctagccgtg
actagggcta agatggagcc accattaaag aaggaaggaa 120 aagaaaggaa
aaaagaagga aagaaaaaaa aaaaaaaaaa aaaggaaaaa aaaaaaaaaa 180
aagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacagg
240 aaatggccta agaggccgga gtgtttaccc caacctttaa acggcgatct
ttccgccctt 300 cttggccttt atgaggatct ctctgatttt tcttgcgtcg
agttttccgg taagaccttt 360 cggtacttcg tccacaaaca caactcctcc
gcgcaacttt ttcgcggttg ttacttgact 420 ggcgacgtaa tccacgatct
ctttttccgt catcgtcttt ccgtgctcca aaacaacaac 480 ggcggcggga
agttcaccgg cgtcatcgtc gggaagacct gcgacacctg cgtcgaagat 540
gttggggtgt tggagcaaga tggattccaa ttcagcggga gccacctgat agcctttgta
600 cttaatcaga gacttcaggc ggtcaacgat gaagaagtgt tcgtcttcgt
cccagtaagc 660 tatgtctcca gaatgtagcc atccatcctt gtcaatcaag
gcgttggtcg cttccggatt 720 gtttacataa ccggacataa tcataggacc
tctcacacac agttcgcctc tttgattaac 780 gcccagcgtt ttcccggtat
ccagatccac aaccttcgct tcaaaaaatg gaacaacttt 840 accgaccgcg
cccggtttat catccccctc gggtgtaatc agaatagctg atgtagtctc 900
agtgagccca tatccttgcc tgatacctgg cagatggaac ctcttggcaa ccgcttcccc
960 gacttcctta gagaggggag cgccaccaga agcaatttcg tgtaaattag
ataaatcgta 1020 tttgtcaatc agagtgcttt tggcgaagaa ggagaatagg
gttggcacca gcagcgcact 1080 ttgaatcttg taatcctgaa ggctcctcag
aaacagctct tcttcaaatc tatacattaa 1140 gacgactcga aatccacata
tcaaatatcc gagtgtagta aacattccaa aaccgtgatg 1200 gaatggaaca
acacttaaaa tcgcagtatc cggaatgatt tgattgccaa aaataggatc 1260
tctggcatgc gagaatctca cgcaggcagt tctatgaggc agagcgacac ctttaggcag
1320 accagtagat ccagaggagt tcatgatcag tgcaattgtc ttgtccctat
cgaaggactc 1380 tggcacaaaa tcgtattcat taaaaccggg aggtagatga
gatgtgacga acgtgtacat 1440 cgactgaaat ccctggtaat ccgttttaga
atccatgata ataatttttt ggatgattgg 1500 gagctttttt tgcacgttca
aaattttttg caaccccttt ttggaaacga acaccacggt 1560 aggctgcgaa
atgcccatac tgttgagcaa ttcacgttca ttataaatgt cgttcgcggg 1620
cgcaactgca actccgataa ataacgcgcc caacaccggc ataaagaatt gaagagagtt
1680 ttcactgcat acgacgattc tgtgatttgt attcagccca tatcgtttca
tagcttctgc 1740 caaccgaacg gacatttcga agtactcagc gtaagtgatg
tccacctcga tatgtgcatc 1800 tgtaaaagca attgttccag gaaccagggc
gtatctcttc atagccttat gcagttgctc 1860 tccagcggtt ccatcttcca
gcggatagaa tggcgccggg cctttcttta tgtttttggc 1920 gtcttccatg
ggacgtcggt tggtgttacg tttggttttt
ctttgaggtt taggattcgt 1980 gctcatgatg cacggtctac gagacctccc
ggggcactcg caagcaccct atcaggcagt 2040 accacaaggc ctttcgcgac
ccaacactac tcggctagca gtcttgcggg ggcacgccca 2100 aatctccagg
cattgagcgg ggttatccaa gaaaggaccc ggtcgtcctg gcaattccgg 2160
tgtactcacc ggttccgcag accactatgg ctctcccggg agggggggtc ctggaggctg
2220 cacgacactc atactaacgc catggctaga cgctttctgc gtgaagacag
tagttcctca 2280 caggggagtg attcatggtg gagtgtcgcc cccatcaggg ggctggc
2327
* * * * *
References