U.S. patent application number 10/656441 was filed with the patent office on 2004-07-29 for multimerization of hiv-1 vif protein as a therapeutic target.
Invention is credited to Pomerantz, Roger J., Yang, Bin, Zhang, Hui.
Application Number | 20040146522 10/656441 |
Document ID | / |
Family ID | 23080751 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146522 |
Kind Code |
A1 |
Zhang, Hui ; et al. |
July 29, 2004 |
Multimerization of HIV-1 Vif protein as a therapeutic target
Abstract
One approach to treating individuals infected with HIV-1 is to
administer to such individuals compounds that directly interfere
with and intervene in the machinery by which HIV-1 replicates
itself within human cells. Although the specific role of HIV-1
viral protein Vif in the viral life cycle is not known, the vif
gene is essential for the pathogenic replication of lentiviruses in
vivo. The present invention relates to a method for treating an
individual exposed to or infected with HIV-1. Individuals
identified as being exposed to or infected by HIV-1 are
administered a therapeutically effective amount of one or more
compounds that inhibit or prevent replication of said HIV-1 by
interfering with the replicative or other essential functions of
HIV-1 viral protein Vif, by interactively blocking the
multimerization domain of Vif, thereby preventing multimerization
of Vif protein, which is important for Vif function in the
lentivirus life cycle. In preferred embodiments, the compound or
compounds that interactively block the multimerization domain of
Vif are Vif antagonists. Pharmaceutical compositions comprising
these compounds are also disclosed.
Inventors: |
Zhang, Hui; (US) ;
Pomerantz, Roger J.; (US) ; Yang, Bin;
(US) |
Correspondence
Address: |
DRINKER BIDDLE & REATH
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Family ID: |
23080751 |
Appl. No.: |
10/656441 |
Filed: |
September 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10656441 |
Sep 5, 2003 |
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10118575 |
Apr 8, 2002 |
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6653443 |
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60282270 |
Apr 6, 2001 |
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Current U.S.
Class: |
424/188.1 ;
530/350 |
Current CPC
Class: |
A61P 31/18 20180101;
A61K 38/00 20130101; C12N 2740/16322 20130101; C07K 14/005
20130101; A61P 31/12 20180101; Y10S 530/826 20130101 |
Class at
Publication: |
424/188.1 ;
530/350 |
International
Class: |
A61K 039/21; C07K
014/16 |
Claims
What is claimed is:
1. A Vif antagonist that binds to the multimerization domain within
a Vif protein in a cell and inhibits Vif protein multimerization,
wherein said antagonist is a peptide selected from the group
consisting of SEQ. ID. NO: 5; SEQ. ID. NO: 6; SEQ. ID. NO: 7; SEQ.
ID. NO: 8; SEQ. ID. NO: 9; SEQ. ID. NO: 10; SEQ. ID. NO: 12; SEQ.
ID. NO: 13; SEQ. ID. NO: 14; SEQ. ID. NO: 15; SEQ. ID. NO: 16; SEQ.
ID. NO: 17; SEQ. ID. NO: 18; SEQ. ID. NO: 19; SEQ. ID. NO: 20; SEQ.
ID. NO: 21; SEQ. ID. NO: 22; and SEQ. ID. NO: 23.
2. The Vif antagonist of claim 1, wherein the antagonist is SEQ.
ID. NO: 9.
3. The Vif antagonist of claim 1, wherein the antagonist is SEQ.
ID. NO:13.
4. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a Vif antagonist of claim 1.
Description
CONTINUING APPLICATION DATA
[0001] This application is a divisional of U.S. application Ser.
No. 118,575, filed Apr. 8, 2002 which claims priority to U.S.
provisional application No. 60/282,270, filed Apr. 6, 2001.
FIELD OF THE INVENTION
[0002] The present invention generally related to the fields of
molecular biology and virology and to a method for treating an
individual exposed to or infected with human immunodeficiency virus
type 1 (HIV-1) and, more particularly, to compositions that inhibit
or prevent the replicative and other essential functions of HIV-1
viral infectivity factor protein (Vif) by interactively blocking
the Vif multimerization domain.
BACKGROUND OF THE INVENTION
[0003] One approach to treating individuals infected with HIV-1 is
to administer to such individuals compounds that directly intervene
in and interfere with the machinery by which HIV-1 replicates
itself within human cells. Lentiviruses such as HIV-1 encode a
number of accessory genes in addition to the structural gag, pol,
and env genes that are expressed by all replication-competent
retroviruses. One of these accessory genes, vif (viral infectivity
factor), is expressed by all known lentiviruses except equine
infectious anemia virus. Vif protein of HIV-1 is a highly basic,
23-kDa protein composed of 192 amino acids. Sequence analysis of
viral DNA from HIV-1-infected-individuals has revealed that the
open reading frame of Vif remains intact. (Sova, P., et al., J.
Virol. 96:2557-2564, 1995; Wieland, U., et al., Virology 203:43-51,
1994; Wieland, U., et al., J. Gen. Virol. 78:393-400, 1997).
Deletion of the vif gene dramatically decreases the replication of
simian immunodeficiency virus (SIV) in macaques and HIV-1
replication in SCID-hu mice (Aldrovandi, G. M. & Zack, J. A.,
J. Virol. 70:1505-1511, 1996; Desrosiers, R. C., et al., J. Virol.
72:1431-1437, 1998), indicating that the vif gene is essential for
the pathogenic replication of lentiviruses in vivo.
[0004] In cell culture systems, vif-deficient (vif.sup.-) HIV-1 is
incapable of establishing infection in certain cells, such as H9 T
cells, peripheral blood mononuclear cells, and monocyte-derived
macrophages. This has led to classification of these cells as
nonpermissive. However, in some cells, such as C8166, Jurkat,
SupT1, and HeLa-T4 cells, the vif gene is not required; these cells
have been classified as permissive. (Gabuzda, D. H., et al., J.
Virol. 66(11):6489-95, 1992; von Schwedler, U., et al., J. Virol.
67(8):4945-55, 1993; Gabuzda, D. H., et al., J. AIDS 7(9):908-15,
1994).
[0005] As Vif is required by nonpermissive but not permissive cells
for HV-1 replication two possibilities exist. In permissive cells,
there may be a Vif cellular homologue that can replace Vif function
in the virus-producing cells; alternatively, there may be an
inhibitor(s) of viral replication in nonpermissive cells that
requires Vif to counteract its effect. (Trono, D., Cell 82:189-192,
1995). Recently, it was proposed that Vif protein is required to
counteract an unknown endogenous inhibitor(s) in the
virus-producing cells. (Madani, N., & Kabat, D., J. Virol.
72:10251-10255, 1998; Simon, J. H., et al., Nat. Med. 4:1397-1400,
1998). HIV-1 Vif can complement the function of HIV-1 Vif and
SIV.sub.AGM Vif in human nonpermissive cells, whereas it cannot
complement the function of HIV-1 and SIV.sub.AGM Vif in simian
cells. SIV.sub.AGM Vif, however, can complement the function of
HIV-1 Vif and SIV.sub.AGM Vif in simian cells but not the function
of HIV-1 and SIV.sub.AGM Vif in human cells, indicating that a
cellular cofactor(s) is involved in the action of Vif protein.
(Simon, J. H., et al., EMBO J. 17:1259-1267, 1998). Conversely,
since a Vif mutant (Vif from HIV-1.sub.F12) can inhibit wild-type
HIV-1 replication in permissive cells, a Vif homologue in the
permissive cells may exist. (D'Aloja, P., et al., J. Virol.
72:4308-4319, 1998).
[0006] It has been proposed that Vif functions in virus-producing
cells or cell-free virions and affects viral assembly. (Blanc, D.,
et al., Virology 193:186-192, 1993; Gabuzda, D. H., et al., J.
Virol. 66:6489-6495, 1992; von Schwedler, U., et al., J. Virol.
67:4945-4955, 1993). Defects of the vif gene do not have detectable
effects on viral transcription and translation or on virion
production. HIV-1 variants with a defective vif gene are able to
bind and penetrate target cells but are not able to complete
intracellular reverse transcription and endogenous reverse
transcription (ERT) in cell-free virions. (Courcoul, M., et al., J.
Virol. 69:2068-2074, 1995; Goncalves, J., et al., J. Virol.
70:8701-8709, 1996; Sova, P., & Volsky, D. J., J. Virol.
67:6322-6326, 1993; von Schwedler, U., et al., J. Virol.
67:4945-4955, 1993). When ERT is driven by the addition of
deoxyribonucleoside triphophates (dNTP) at high concentrations,
certain levels of plus-strand viral DNA can be completed. Moreover,
when vif.sup.- viruses, generated from nonpermissive cells and
harboring larger quantities of viral DNA generated by ERT, are
allowed to infect permissive cells, they can partially bypass the
block at intracellular reverse transcription through which
vif.sup.- viruses without deoxynucleoside triphosphate treatment
can not pass. Consequently, viral infectivity can be partially
rescued from the vif.sup.- phenotype. (Dornadula, G., et al., J.
Virol. 74:2594-2602, 2000).
[0007] The expression of viral components, including viral proteins
and nucleic acids, is not altered in the virions produced from
nonpermissive cells. (Fouchier, R. A., et al., J. Virol.
70:8263-8269, 1996; Gabuzda, D. H., et al., J. Virol. 66:6489-6495,
1992; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993).
Deletion of the vif gene, however, results in alterations of virion
morphology. (Borman, A. M., et al., J. Virol. 69:2058-2067, 1995;
Bouyac, M., et al., J. Virol 71:2473-2477, 1997; Hoglund, S., et
al., Virology 201:349-355, 1994). The quantity of Vif protein in
the HIV-1 virions generated from chronically infected cells is
approximately 7 to 28 molecules per virion. (Camaur, D., &
Trono, D., J. Virol. 70:6106-6111, 1996; Fouchier, R. A., et al.,
J. Virol. 70:8263-8269, 1996; Simon, J. H., et al., Virology
248:182-187, 1998). As the virion-associated Vif proteins do not
depend on the expression of viral components and the amount of Vif
in the virus-producing cells, it seems that Vif proteins are not
specifically incorporated into the virions. (Camaur, D., &
Trono, D., J. Virol 70:6106-6111, 1996; Simon, J. H., et al.,
Virology 248:182-187, 1998).
[0008] Although, it seems that Vif is not specifically incorporated
into virions, Vif is able to bind to the NCp7 domain of p55 Gag
precursors through its positively charged amino-acid enriched
C-terminus. (Bouyac, M., et al., J. Virol. 71:9358-9365, 1997;
Huvent, I., et al., J. Gen. Virol. 79:1069-1081, 1998). Vif protein
is found to co-localize with Gag precursors in the cytoplasm of
HIV-1-infected cells. (Simon, J. H., et al., J. Virol.
71:5259-5267, 1997). The molar ration of Vif to Gag precursors in
infected cells is 1:1.7, suggesting that Vif plays a structural
rather than a regulatory role in virus-producing cells. (Goncalves,
J., et al., J. Virol. 68:704-712, 1994; Simon, J. H., et al.,
Virology 248:182-187, 1998).
[0009] Vif has been shown to be an RNA-binding protein and an
integral component of a messenger ribonucleoprotein (mRNP) complex
of viral RNA in the cytoplasm of HIV-1-infected cells. The
expression of Vif in infected cells is quite high, and the majority
of Vif in virus-producing cells is in the cytoplasmic fraction;
some is associated with the cellular membrane. The Vif protein in
this mRNP complex may protect viral RNA from various endogenous
inhibitors and could mediate viral RNA engagement with HIV-1 Gag
precursors and thus could be involved in genomic RNA folding and
packaging. As such, the interaction between Vif and HIV-1 RNA plays
an important role in the late events of the HIV-1 life cycle. Given
the Vif protein's direct or indirect involvement in the viral
assembly process, it is an ideal target for anti-HIV-1
therapeutics.
[0010] Many HIV-1 proteins, including Gag, protease, reverse
transcriptase, integrase, glycoprotein 41(gp41), Tat, Rev, Vpr, and
Nef, have been shown to form dimers or multimers in vitro and in
vivo. The formation of dimers or multimers has been demonstrated to
be important for their functions in the lentiviral life-cycle.
(Frankel, A. D. & Young, J. A., Ann. Rev. Biochem. 67:1-25,
1998; Vaishnav, Y. N. & Wong-Staal, F., Annu Rev Biochem
60:577-630, 1991; Zhao, L. J., et al., J Biol Chem 269(51):32131-7,
1994; Liu, L., et al., J. Virol. 74:5310-5319, 2000). The present
invention provides evidence that Vif protein possesses a strong
tendency to self-associate and that multimerization of Vif proteins
is important for Vif function in the viral life-cycle. The present
invention is directed to a method of treating HIV-1 exposed or
infected individuals by administering a composition that inhibits
or prevents the replicative and other essential functions of Vif by
binding to, or otherwise associating with, the multimerization
domain of Vif, thereby preventing multimerization of Vif and,
consequently, HIV-1 replication.
ABBREVIATIONS
[0011] "HIV-1" means "human immunodeficiency virus type I."
[0012] "Vif" means "virion infectivity factor."
[0013] "GST" means "glutathione-S-transferease."
[0014] "CAT" means "chloramphenicol acetyltransferase."
[0015] "IP" means "immunoprecipitation."
[0016] "WB" means "Western blotting."
DEFINITIONS
[0017] The term "antagonist" as used herein, refers to a molecule
that binds to Vif protein, preferably, the multimerization domain
within Vif protein, thereby inhibiting Vif-Vif interaction and Vif
protein multimerization. Antagonists may include proteins or
peptidomimetics thereof, nucleic acids, carbohydrates, or any other
molecules, which inhibits Vif protein multimerization.
[0018] The terms "analogs," "derivatives," or "fragments" are used
interchangeably to mean a chemical substance that is related
structurally and functionally to another substance. An analog,
derivative, or fragment contains a modified structure from the
parent substance, in this case Vif protein, and maintains the
function of the parent substance, in this instance, the binding
ability to the multimerization domain of Vif protein in cellular
and animal models. The biological activity of the analog,
derivative, or fragment may include an improved desired activity or
a decreased undesirable activity. The analogs, derivatives or
fragments may be prepared by various methods known in the art,
including but not limited to, chemical synthesis or recombinant
expression. Analogs, derivatives, or fragments of the instant
invention, include, but are not limited to, synthetic or
recombinant peptides that are homologous to Vif protein or fragment
thereof (consisting of at least the sequence from amino acid
residue 144-171, preferably, 151-164, more preferably,
161-164).
DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Vif Self Association in a Cell-free System.
[0020] A. An autoradiograph illustrating that GST-Vif (lane 2) but
not GST (lane 3) can bind to in vitro translated .sup.35S-labeled
HIV-1.sub.NL4-3 Vif protein. .sup.35S-labeled HIV-1.sub.NL4-3 Vif
proteins were allowed to bind with GST-Vif conjugated beads. After
binding, the bead associated .sup.35S-labeled Vif was analyzed via
SDS-PAGE and direct autoradiography.
[0021] B. An autoradiograph showing that under native or relatively
native conditions .sup.35S-labeled HIV-1.sub.NL4-3 Vif proteins
form monomers, dimers, trimers or tetramers. In vitro translated
.sup.35S-labeled HIV-1.sub.NL4-3 Vif proteins were loaded directly
onto a 4-20% Tris-HCl gel (SDS-free) with native loading buffer
[62.5 mM Tris-HCl (pH 6.8) and 20% glycerol] plus SDS at different
concentrations. Electrophoresis was performed with a Tris-Glycine
running buffer containing 0.05% SDS, followed by
autoradiography.
[0022] FIG. 2. The Effect of Vif Mutants on Vif-Vif
Interactions.
[0023] A. A schematic showing a series of deletions along the Vif
protein generated using PCR-based mutagenesis and in vitro
translation. The in vitro translated .sup.35S-labeled
HIV-1.sub.NL4-3 Vif protein and its mutants were allowed to bind to
GST-Vif conjugated on agarose beads. The bead-associated,
.sup.35S-labeled Vif protein and its mutants were subjected to
SDS-PAGE and visualized by direct autoradiography. The ratio of
bound Vif versus the input were calculated using the ratio of
GST-Vif bound .sup.35S-labeled wild-type Vif protein and
.sup.35S-labeled wild-type Vif input as 100% (with the standard
deviations). The values were obtained by quantitation with
densitometry of the autoradiography. In most cases, the data
reflect at least five independent experiments.
[0024] B. An autoradiograph illustrating that in the presence of
0.1% SDS, .sup.35S-labeled HIV-1.sub.NL4-3 Vif protein mutants
.DELTA.151-192 and .DELTA.151-164 are unable to form multimers,
while other mutants are able to do so. In vitro translated
.sup.35S-labeled HIV-1.sub.NL4-3 Vif protein and its mutants
(50,000 cpm count for each) were loaded directly onto a 4-20%
Tris-HCl gel (SDS-free) with a loading buffer [62.5 mM Tris-HCl (pH
6.8) and 20% glycerol] plus 0.1% SDS. Electrophoresis was performed
with a Tris-Glycine running buffer containing 0.05% SDS, followed
by autoradiography.
[0025] FIG. 3. Co-immunoprecipitation Method to Study Vif-Vif
Interactions Within Cells.
[0026] Western Blots (top two panels) showing that the expression
of Vif protein tagged with c-Myc or Flag epitope at its C-terminus
in COS-1 transfected cells can be detected using A14 anti-c-Myc
polyclonal antibody and or M2 anti-Flag monoclonal antibody,
respectively. COS-1 cells were transfected with vectors harboring
Flag or c-Myc tagged Vif. After 54 hours of incubation at 5%
CO.sub.2, 37.degree. C., 20 .mu.g total cell lysates were resolved
by 15% Tris-HCl gel. A third Western Blot illustrates that
Flag-tagged Vif was co-precipitated with Myc-tagged Vif when the
cell lysates were immunoprecipitated with A14 anti-c-Myc polyclonal
antibody. For co-immunoprecipitation, the whole cell lysates from
the same batch were subjected to immunoprecipitation with A14
anti-c-Myc polyclonal antibody. Immunoprecipitates are resolved at
15% Tris-HCl gel and transferred onto a membrane and then detected
using an M2 anti-Flag antibody.
[0027] FIG. 4. Mammalian Two-hybrid System to Study Vif-Vif
Interaction.
[0028] A. A schematic map showing the plasmids utilized in the
experiments: pVif-VP, pGAL-Vif, and pSG5GalVP.
[0029] B. A gel illustrating the CAT activity of COS-1 cells
transfected with plasmids combined with various vectors. After 48
hours, cell lysates were harvested and subjected to CAT
analyses.
[0030] FIG. 5. Viral Infectivity Affected by Vif or Vif
Mutants.
[0031] A diagram depicting the CAT activity of HelaCD4-CAT cells
infected with recombinant viruses. The pCI-Neo constructs,
containing wild-type vif gene or its mutants,
pNL4-3.DELTA.env.DELTA.vif plasmid and pMD.G (containing VSV env),
were co-transfected into H9 cells to generate the pseudotyped viral
particles. After concentration via ultracentrifuge, the viral
particles were normalized by HIV-1 p24 antigen. In the presence of
polybrene (8 .mu.g/ml), the viruses were used to infect HelaCD4-CAT
cells. After 48 hours, the cell lysates were collected and
subjected to CAT analyses. Lane 1) pNL4-3; Lane 2)
pNL4-3.DELTA.env.DELTA.vif, VSV env plus wild-type vif; Lane 3)
pNL4-3.DELTA.env.DELTA.vif, VSV env, plus vif.DELTA. 151-164; Lane
4) pNL4-3.DELTA.env.DELTA.vif, VSV env, plus vif.DELTA. 144-150;
Lane 5) pNL4-3.DELTA.env.DELTA.vif, VSV env, plus pCI-Neo vector
only. The value of wild-type vif complementation was set as 100%.
The relative values of the other samples were calculated
accordingly. The figure represents three independent experiments.
Values are means.+-.standard deviations.
[0032] FIG. 6. The Relative Affinity Comparison between PXP Motif
Containing Peptides.
[0033] The GST-Vif protein, Vif mutant (deletion of 151-192 amino
acids), and GST only were placed onto the plate. The phage clones
isolated through Vif-containing column were serially diluted and
added. After incubation to allow phage-Vif binding, excess phages
were washed off. Anti-M13 phage antibody, conjugated with HRP, was
added to bind the phages that were captured by Vif. After washing,
the substrate was added and color development was allowed. The
phages captured by Vif, therefore, were semi-quantitated. OD at 405
nm equal or larger than 0.15 was considered as positive. The phage
sample number (VMI) was the same as shown in Table 1.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Vif protein of HIV-1 is essential for viral replication in
vivo and productive infection of peripheral blood mononuclear cells
(PBMC), macrophages and H9 T-cells. The molecular mechanism(s) of
Vif remains unknown and needs to be further determined. The present
invention demonstrates that like many other proteins encoded by
HIV-1, Vif proteins possess a strong tendency towards
self-association. Under relatively native conditions, Vif proteins
form multimers in vitro, including dimers, trimers, or tetramers.
In vivo binding assays, such as co-immunoprecipitation and a
mammalian two-hybrid system, demonstrate that Vif proteins interact
with each other within a cell, indicating that the multimerization
of Vif proteins is not simply due to fortuitous aggregation.
[0035] The present invention further evidences that the domain
affecting Vif self-association is located at the C-terminus of this
protein, especially the proline-enriched 151-164 region. The
sequence of this domain is AALIKPKQIKPPLP (SEQ. I.D. NO: 1).
Studies demonstrate that a Vif mutant with deletion at amino acid
positions 151-164 is unable to rescue the infectivity of
vif-defective viruses generated from H9 T-cells, implying that the
multimerization of Vif proteins is important for Vif function in
the viral life-cycle.
Methods
[0036] Plasmid Constructions
[0037] With infectious clone pNL4-3 as a template, deletion mutants
of HIV-1 Vif were generated by polymerase chain reaction
(PCR)-mediated and site-directed mutagenesis. (Zhang, H., et al.,
Proc. Natl. Acad. Sci. USA 93(22):12519-24, 1996). The
PCR-generated wild-type vif gene and its mutants were then inserted
into pCITE-4a vector (Novagen, Madison, Wis.) for in vitro
translation. The vif gene also was inserted into pGEX vector for in
vitro expression and isolation of GST-Vif fusion protein. For
studying intracellular Vif-Vif interaction, vif genes were tagged
via PCR with Flag (DYKDDDDK) (SEQ. I.D. NO: 2) or c-Myc
(EQKLISEEDL) (SEQ. I.D. NO: 3) epitope-encoding sequences at the 3'
terminus respectively. These tagged vif genes were then inserted
into the vector pCI-Neo, which contains a chimeric intron just
downstream of the CMV enhancer and immediate early promoter
(Promega, Madison, Wis.). The resulting plasmids were named
pCI-vif-c-myc or pCI-vif-flag, respectively. For mammalian
two-hybrid analysis, either pGal-Vif or pGal-Vif.DELTA.151-164 was
constructed by replacing the Hind III-BamH I fragment (containing
vp gene) of pSG5GalVP with a PCR-amplified complete vif gene or its
mutant .DELTA.151-164. The pVif-VP or pVif.DELTA.151-164-VP was
constructed by replacing the EcoRI-BglII fragment (containing gal4
gene) of pSG5GalVP with an PCR-amplified complete vif gene or its
mutant .DELTA.151-164, respectively. (Shimano, R., et al., Biochem.
Biophys. Res. Comm 242(2):313-6, 1998). The integrity of all the
constructs was confirmed by DNA sequencing.
[0038] Protein Expression and In Vitro Binding Assays
[0039] The vector pGEX, with or without the vif gene, was
transformed into BL21 competent cells (Novagen, Madison, Wis.).
After growth at 37.degree. C. to approximately 0.6 O.D., the
expression of GST or GST-Vif proteins was induced by 0.4 mM
isopropylthio-.beta.-D-galactoside (IPTG). The bacterial cells were
lyzed by adding lysing buffer (1% Triton-X-100, 0.1 mg/ml lysozyme,
2 mM EDTA, 1 mM PMSF, 2 ug/ml leupeptin, and 1 .mu.g/ml aprotinin),
followed by sonication. The sample was pelleted at 12,000 g for 10
min at 4.degree. C., and the supernatant was applied to a
glutathione-conjugated agarose bead (Sigma, St. Louis, Mo.) column.
After batch binding, the matrix was washed three times, each time
by the addition of 10 bed volumes of phosphorus-buffer saline
(PBS). The GST or GST-Vif conjugated agarose beads were then
aliquoted and stored at -20.degree. C. Conversely, .sup.35S-labeled
Vif or its mutant proteins were synthesized utilizing SPT3 kits
(Novagen, Madison, Wis.). The protocol supplied by the manufacturer
was followed. After in vitro translation, RNase A (0.2 mg/ml) was
added to stop the reaction and remove tRNAs and the in vitro
transcribed mRNA. The trichloroacetic acid (TCA)-insoluble
radioactive amino acids were quantitated in the presence of a
scintillation cocktail.
[0040] For GST pull-down assays, a GST or GST-Vif conjugated bead
slurry was mixed with .sup.35S-labeled Vif or its mutants (50,000
cpm) in a binding buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.5),
0.1% Triton-X-100]. After binding at 4.degree. C. for 1 hour, the
mixture was centrifuged at 3,000 g for 1 min, and the beads were
washed three times with binding buffer. The .sup.35S-labeled Vif
proteins were dissociated from the beads by adding SDS-containing
loading buffer and heating at 95.degree. C. for 5 minutes. The
samples were then electrophoresized in SDS-PAGE gels (15% Tris-HCl
ready gel made by Bio-Rad, Hercules, Calif.). After treatment with
the fixing buffer (10% acetic acid, 10% methanol) and then the
Amplify (Amersham-Pharmacia, Piscataway, N.J.), the gels were dried
and exposed to X-ray film or quantitatively analyzed utilizing
phosphor image (Molecular Dynamics, Sunnyview, Calif.).
[0041] A Vif-Vif binding assay was similar to the GST pull-down
assays, except that the GST or GST-Vif conjugated bead slurry was
mixed with .sup.35S-labeled Vif and the test peptides or molecules
in the binding buffer. The results were compared to that from the
GST pull-down assay, which was designated as 100%.
[0042] In addition, in vitro translated, .sup.35S-labeled Vif
(50,000 cpm) was also directly loaded onto a 4-20% Tris-Glycine gel
(SDS free) via 10% glycerol-containing loading buffer, with SDS at
various concentrations, and electrophoresized with a SDS-free
Tris-Glycine running buffer. After fixing and drying, the gel was
directly subjected to autoradiography.
[0043] Western Blotting and Co-Immunoprecipitation
[0044] The COS-1 or 293T cells were transfected with 5 .mu.g
pCI-vif-c-myc and pCI-vif-flag using calcium phosphate
precipitation method. (Zhang, H., et al., Proc. Natl. Acad. Sci.
USA 93(22):12519-24, 1996; Zhang, H., et al., J. Virol.
69(6):3929-32, 1995). After 48 hours, the cells were lyzed in a
cell lysing buffer [150 mM NaCl, 50 mM Tris-HCl (pH8.0), 5 mM EDTA,
1% Triton-X-100, 10% glycerol, 1 mM PMSF, 2 .mu.g/ml aprotinin, 2
.mu.g/ml leupeptin, and 2 .mu.g/ml pepstatin A]. For direct Western
blotting, the whole cell lysates were mixed with acetone (1:3). The
mixture was incubated on ice for 20 minutes, followed by
centrifugation at 12,000 g for 10 minutes. The pellets were then
air-dried and resuspended in SDS-containing sample buffer. The
samples were electrophoresized in SDS-PAGE gels and then
electronically transferred onto a nylon/nitrocellulose membrane.
The primary antibodies, goat anti-c-Myc antibody (A14) (Research
Antibodies, Santa Cruz, Calif.) or mouse anti-Flag antibody (M2)
(Stratagene, La Jolla, Calif.) were used to bind the samples,
respectively. The horseradish peroxidase (HPR)-conjugated anti-goat
IgG antibody or anti-mouse IgG antibody (Research Antibodies, Santa
Cruz, Calif.) were used as the secondary antibodies. A
chemilufluminescence-based system (ESL, Amersham-Pharmacia Biotech,
Piscataway, N.J.) was used to visualize the antigen-antibody
binding.
[0045] For co-immunoprecipitation, cell lysates from COS-1 or 293T
cells expressing Vif-Flag and/or Vif-c-Myc were incubated with A14
anti-c-Myc antibody (Research Antibodies, Santa Cruz, Calif.) (1
.mu.g/ml) by mixing 12 hours at 4.degree. C., followed by
incubation with protein A-conjugated Sepharose CL-4B
(Amersham-Pharmacia Biotech, Piscataway, N.J.) for an additional 2
hours. The pellet was washed three times with cell lysing buffer
and then resuspended in SDS-containing buffer, heated at 95.degree.
C., and centrifuged at 12,000 g. The supernatant was then subjected
to SDS-PAGE. After transfer onto a nylon/nitrocellulose membrane,
the samples were detected with a mouse M2 anti-Flag antibody. An
HRP conjugated anti-mouse IgG- (Research Antibodies, Santa Cruz,
Calif.) was used as a secondary antibody.
[0046] Mammalian Two-Hybrid System Assay
[0047] A mammalian two hybrid system, which was modified from the
GAL4-based yeast two hybrid assay, was used to study the
self-association of HIV-1 Vif proteins in vivo. (Shimano, R., et
al., Biochem. Biophys. Res. Comm. 242(2):313-6, 1998; Bogerd, H.,
& Greene, W. C., J. Virol. 67(5):2496-502, 1993). The procedure
was described, with some modifications, in Shimano, R., et al.,
Biochem. Biophys. Res. Comm. 242(2):313-6, 1998 and Bogerd, H.,
& Greene, W. C., J. Virol. 67(5):2496-502, 1993. Briefly, 5
.mu.g pGal-Vif and pVif-VP were co-transfected with pG5BCAT into
COS-1 cells using the Superfect transfection reagent (Qiagen,
Valencia, Calif.). Forty-eight hours post-transfection, the cells
were lyzed in reporter lysing buffer (Promega, Madison, Wis.) and
subjected to a chloramphenicol acetyltransferase (CAT) assay, as
described previously by Zhang, H., et al. in J. Virol.
69(6):3929-32, 1995.
[0048] Single-Round Viral Infectivity Assays
[0049] The biological activity of Vif mutants was evaluated by
using a single-round viral infectivity assay as described in
Dornadula, G., et al., J. Virol. 74(6):2594-602, 2000 with some
modifications. To generate recombinant HIV-1 viruses, H9 cells were
transfected with 5 .mu.g pNL4-3.DELTA.vif.DELTA.env, pMD.G
[containing VSV (vesicular stomatitis virus) envelope], and
wild-type vif gene or its mutants (in pCI-neo construct) by
electroporation. (Dornadula, G., et al., J. Virol. 74(6):2594-602,
2000; Naldini, L., et al., Proc Natl Acad Sci USA 93(21):11382-8,
1996). The electroporation (350 V, 250 .mu.F, 5.1-6.3 msec) was
performed by a gene pulser apparatus and capacitance (Bio-Rad,
Hercules, Calif.). Thereafter, conditioned medium (RPMI 1640 plus
10% fetal bovine serum) was used to maintain the transfected H9
cells. Two days after transfection, the viral particles in
supernatant were collected and pelleted via ultracentrifugation.
(Dornadula, G., et al., J Virol. 74(6):2594-602, 2000). After
normalization by HIV-1 p24 antigen level, which was detected via
enzyme-linked immunosorbent assays (ELISA, kits from DuPont), the
viruses were used to infect 5.times.10.sup.5 HeLa CD4-CAT cells.
(Ciminale, V., et al., AIDS Res. Hum. Retro. 6(11):1281-7, 1990).
Forty-eight hours post-infection, the cells were lyzed in reporter
lysing buffer (Promega, Madison, Wis.) and subjected to CAT
assays.
[0050] Phage Display Peptide Screening
[0051] Vif binding peptides displayed on M13 phages were screened
using the Ph.D.-12.TM. Phage Display Peptide Library kit (New
England Biolabs, Beverly, Mass.). Phage panning procedure was
performed according to the kit protocol with some modifications.
GST-Vif fusion protein attached on glutathione-agarose beads
(Sigma, St. Louis, Mo.) was used as target for phage panning. For
each round panning, 10.sup.11 phages were added to 10 mg GST
attached on 3 ml glutathione-agarose gel in a final volume of 6 ml
in TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) and incubated
for 1 hr at room temperature with shaking. The binding solution was
separated by centrifugation at 500 g for 10 min and the supernatant
was then added to 10 mg GST-Vif attached on 3 ml
glutathione-agarose beads. The mixture was incubated for 1 hr at
room temperature and then washed 6 times with TBST [50 mM Tris-HCl
(pH 7.5), 500 mM NaCl, 0.5% Tween-20]. The GST-Vif binding phages
were eluted by adding 3 ml of 5 mM reduced glutathione in TBS. The
eluted phages were amplified by adding 2.5 ml of the elution to 20
ml of E. coli ER2738 culture (O.D at 0.6) and incubated at 37
.degree. C. with vigorous shaking for 4.5 hr. After centrifuge, the
phages in the supernatant were precipitated by PEG/NaCl. After
washing, the phages were suspended in 200 .mu.l TBS. The titration
of the eluted or amplified phages was determined as described in
the kit protocol. After 3 round panning, individual phage plaques
from the GST or GST-Vif elution tittering plates were selected for
amplification respectively. Phage DNA was purified and
sequenced.
[0052] Determination of Binding Affinity by ELISA
[0053] A phage enzyme-linked immunosorbent assay (ELISA) was
performed to measure the relative binding affinity of phages to
GST, GST-Vif, or GST-Vif without 151-192 amino acids. One hundred
and fifty .mu.l of 100 .mu.g/ml GST and GST-Vif in 0.1 M
NaHCO.sub.3 (pH 8.6) were coated on 96 well microtiter plates
respectively and incubated at 4.degree. C. overnight. The plates
were blocked with blocking buffer (0.1 M NaHCO.sub.3, pH 8.6, 5
mg/ml BSA) for 2 hr at room temperature. The individual phage
clones in 200 .mu.l TBST were 4-fold-serially diluted (from
10.sup.11 to 10.sup.5) and added to the wells coated with GST,
GST-Vif, or GST-Vif without 151-192 amino acids and incubated for 2
hr at room temperature. After washing, HRP-conjugated anti-M13
antibody was added to bind the phages. After washing, the substrate
was added and color development was performed. The phages captured
by Vif, therefore, were semi-quantitated. OD at 405 nm equal or
larger than 0.15 was considered as positive.
[0054] Generation of Antibodies
[0055] The method of treating individuals exposed to or infected
with HIV-1 in accordance with the present invention is based on the
administration of compounds that interactively block, i.e., prevent
or inhibit, the formation of Vif multimers, thereby inhibiting Vif
function in the lentivirus life-cycle. According to the invention,
Vif proteins, its fragments or other derivatives, or analogs
thereof, may be used as an immunogen to generate antibodies that
recognize such an immunogen. Such antibodies include, but are not
limited to, single-chain, Fab fragments, and Fab expression
library. In a specific embodiment, single-chain antibodies to a
human protein are produced.
[0056] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce Vif-specific single chain antibodies. Methods
for the production of single-chain antibodies are well known to
those of skill in the art. The skilled artisan is referred to U.S.
Pat. No. 5,359,046, (incorporated herein by reference) for such
methods. A single chain antibody is created by fusing together the
variable domains of the heavy and light chains using a short
peptide linker, thereby reconstituting an antigen binding site on a
single molecule. Single-chain antibody variable fragments (scFvs)
in which the C-terminus of one variable domain is tethered to the
N-terminus of the other variable domain via a 15 to 25 amino acid
peptide or linker have been developed without significantly
disrupting antigen binding or specificity of the binding (Bedzyk et
al., 1990; Chaudhary et al., 1990). The linker is chosen to permit
the heavy chain and light chain to bind together in their proper
conformational orientation. See, for example, Huston, J. S., et
al., Methods in Enzym. 203:46-121 (1991), which is incorporated
herein by reference. These Fvs lack the constant regions (Fc)
present in the heavy and light chains of the native antibody.
[0057] An additional embodiment of the invention utilizes the
techniques described for the construction of Fab expression
libraries (Huse, et al., Science 246:1275-1281, 1989) to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity for Vif proteins, derivatives, or analogs.
[0058] Antibody fragments that contain the idiotype of the molecule
can be generated by known techniques. For example, such fragments
include but are not limited to: the F(ab').sub.2 fragment which can
be produced by pepsin digestion of the antibody molecule; the Fab'
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragment; and the Fab fragments which can be
generated by treating the antibody molecule with papain and a
reducing agent.
[0059] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art.
[0060] Intracellular Expression Systems
[0061] Single-chain antibodies can be synthesized by a cell,
targeted to particular cellular compartment, and used to interfere
in a highly specific manner with HIV-1 replication. In the present
invention, this method comprises the intracellular expression of a
single-chain antibody that is capable of binding to a Vif protein,
or derivative thereof, wherein the antibody preferably does not
contain sequences coding for its secretion. Such single-chain
antibodies will bind the target intracellularly. The antibodies of
the present invention are expressed from a DNA sequence(s) that
contains a sufficient number of nucleotides coding for the portion
of an antibody capable of binding to the target. Due to the
inherent degeneracy of the genetic code, other DNA sequences that
encode substantially the same or a functionally equivalent heavy
and light chain amino acid sequences, are within the scope of the
invention. Altered DNA sequences that may be used in accordance
with the invention include deletions, additions or substitutions of
different nucleotide residues resulting in a sequence that encodes
the same, or a functionally equivalent, gene product. The gene
product itself may contain deletions, additions or substitutions of
amino acid residues within a heavy or light chain sequence that
result in a silent change, thus producing a functionally equivalent
monoclonal antibody.
[0062] Single-chain antibody genes can be prepared using techniques
known in the art. See U.S. Pat. No. 6,072,036, which is
incorporated herein by reference. Preferably, the gene does not
encode the normal leader sequence for the variable chains. The
nucleotides coding for the binding portion of the antibody
preferably do not encode the antibody's secretory sequences (i.e.,
the sequences that cause the antibody to be secreted from the
cell). This type of design to leave out such sequences can readily
be accomplished in the selection and omission of nucleotides coding
for the antibody.
[0063] In addition, the gene is operably linked to a promoter or
promoters that will permit expression of the antibody in the
cell(s) of interest. Promoters that will permit expression in
mammalian cells are well known in the art and can readily be
selected depending on the target cell. Promoters include, but are
not limited to, CMV, a viral LTR such as the rous sarcoma virus
LTR, HIV-LTR, HTLV-1 LTR, the SV40 early promoter, E. coli lac UV5
promoter and the herpes simplex tk virus promoter. Furthermore, the
use of inducible promoters, which are also well known in the art,
in some embodiments are preferred. Then by "turning the promoter
on" one can selectively obtain the expression of the antibody. The
entire sequence(s) encoding the heavy and light chains of the
single-chain antibody and promoter is described herein as an
antibody cassette. The cassette is delivered to the cell by any of
a number of means described below, which permit intracellular
delivery of a gene. The cassette results in the intracellular
expression of the antibody. The expressed antibody can then bind to
the target antigen.
[0064] The antibodies of the present invention bind specifically to
the target, i.e., the Vif protein, or derivative thereof, and can
thus effectively inhibit Vif multimerization. To insure that the
antibodies of the present invention can compete successfully with
other molecules, they must retain at least about 75% of the binding
effectiveness of the complete antibody to that target. More
preferably, it has at least 85% of the binding effectiveness of the
complete antibody. Still more preferably, it has at least 90% of
the binding effectiveness of the complete antibody. Even more
preferably, it has at least 95% of the binding effectiveness.
[0065] Gene Therapy
[0066] The antibody cassette is delivered to the cell by any of the
known means. See for example, Miller, A. D., Nature 357:455-460
(1992); Anderson, W. F., Science 256:808-813 (1992); Wu, et al, J.
of Biol. Chem. 263:14621-14624 (1988). For example, a cassette
containing these antibody genes, such as the sFv gene, can be
targeted to a particular cell by a number of known forms of gene
therapy according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., Clinical
Pharmacy 12:488-505, 1993; Wu and Wu, Biotherapy 3:87-95, 1991;
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596, 1993;
Mulligan, Science 260:926-932, 1993; and Morgan and Anderson, Ann.
Rev. Biochem. 62:191-217, 1993; May, 1993, TIBTECH 11(5):155-215.
Methods commonly known in the art of recombinant DNA technology
that can be used are described in Ausubel et al. (eds.), 1993,
Current Protocols in Molecular Biology, John Wiley & Sons, NY;
and Kriegler, 1990, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY.
[0067] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286) (see infra), or by direct injection of naked DNA, or by
use of microparticle bombardment (e.g., a gene gun; Biolistic,
Dupont), or coating with lipids or cell-surface receptors or
transfecting agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering it in linkage to a peptide that
is known to enter the nucleus, by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see e.g., Wu and
Wu, J. Biol. Chem. 262:4429-4432, 1987) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, a nucleic acid-ligand complex can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec.
23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis
et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO
93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression by homologous recombination. (Koller &
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935, 1989; Zijlstra
et al., Nature 342:435-438, 1989).
[0068] In a preferred aspect, the therapeutic agent comprises a
nucleic acid encoding a Vif single-chain antibody, or functional
derivative thereof, that is part of an expression vector that
expresses a Vif antibody, or fragment thereof, in a suitable host.
In particular, such a nucleic acid has a promoter operably linked
to the Vif antibody coding region, the promoter being inducible or
constitutive, and, optionally, tissue-specific. In another
particular embodiment, a nucleic acid molecule is used in which the
Vif antibody coding sequences and any other desired sequences are
flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the Vif antibody nucleic acid. (Koller and Smithies,
Proc. Natl. Acad. Sci. USA 86:8932-8935, 1989; Zijlstra et al.,
Nature 342:435-438, 1989).
[0069] Delivery of the nucleic acid into a patient is direct, i.e.,
the patient is directly exposed to the nucleic acid or nucleic
acid-carrying vector. This approach is known, as in vivo gene
therapy.
[0070] Proteins, Derivatives and Analogs Thereof
[0071] The invention further relates to Vif proteins, and
derivatives (including but not limited to fragments) and analogs
thereof, which bind to the multimerization domain of Vif protein
thereby inhibiting Vif-Vif interaction and Vif protein
multimerization. Molecules comprising Vif proteins or derivatives
also are provided.
[0072] The production and use of derivatives and analogs related to
Vif are within the scope of the present invention. In a specific
embodiment, the derivative or analog is an antagonist capable of
interactively binding Vif but incapable of exhibiting the
functional activities associated with a full-length, wild-type
protein. Such derivatives or analogs that have the desired
immunogenicity or antigenicity can be used, for example, for
inhibition of Vif activity. Derivatives or analogs that lack or
inhibit a desired Vif property of interest (e.g., inhibition of
infectivity) can be used as inhibitors of such property and its
physiological correlates. A specific embodiment relates to a Vif
fragment that can be bound or otherwise associated with Vif itself,
thereby preventing or interfering with Vif multimerization.
Derivatives or analogs of Vif can be tested for the desired
activity by procedures known in the art.
[0073] In a specific embodiment of the invention, proteins
consisting of, or comprising a fragment of, a Vif protein
consisting of at least the amino acid sequence substantially
corresponding to the amino acid sequence from amino acid residue
144-177 (SEQ. ID. NO: 26), preferably, 151-164 (SEQ. ID. NO: 1),
and more preferably, 161-164 (SEQ. ID. NO: 25), are provided.
Derivatives or analogs of Vif having amino acid residues 144-171,
preferably, 151-164, more preferably, 161-164, or a sequence
substantially corresponding thereto, include but are not limited to
those molecules comprising regions that are substantially
homologous to Vif or fragments thereof (e.g., in various
embodiments, at least 60% or 70% or 80% or 90% or 95% identity over
an amino acid sequence of identical size or when compared to an
aligned sequence in which the alignment is done by a computer
homology program known in the art) or whose encoding nucleic acid
is capable of hybridizing to a coding vif sequence, under
stringent, moderately stringent, or nonstringent conditions.
[0074] "Stringent conditions" as used herein refers to those
hybridizing conditions that (Virgilio, L., et al., 1994, Proc Natl
Acad Sci USA, 91:12530-12534) employ low ionic strength and high
temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium
citrate/0.1% SDS at 50.degree. C.; (Narducci, M. G., et al., 1997,
Cancer Res, 57:5452-5456) employ, during hybridization, a
denaturing agent such as formamide, for example, 50% (vol/vol)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, 75 mM sodium citrate at 42.degree. C.; or (Virgilio,
L., et al., 1998, Proc Natl Acad Sci USA, 95:3885-3889) employ 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium pyrophosphate,
5.times. Denhardt's solution, sonicated salmon sperm DNA (50 g/ml),
0.1% SDS, and 10% dextran sulfate at 42.degree. C., with washes at
42.degree. C. in 0.2.times.SSC and 0.1% SDS.
[0075] "Moderately stringent conditions" or "nonstringent
conditions" may be identified as described by Sambrook et al.,
Molecular Cloning: A Laboratory Manual, New York: Cold Spring
Harbor Press, 1989, and include the use of washing solution and
hybridization conditions (e.g., temperature, ionic strength and %
SDS) less stringent than those described above. An example of
"moderately stringent conditions" is overnight incubation at
37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
.mu.g/mL denatured sheared salmon sperm DNA, followed by washing
the filters in 1.times.SSC at about 37-50.degree. C. The skilled
artisan will recognize how to adjust the temperature, ionic
strength, etc. as necessary to accommodate factors such as probe
length and the like. An example of "nonstringent conditions" is
overnight incubation at 37.degree. C. in a solution comprising:
5.times.SSC, 25% formamide, 5.times. Denhardts solution, 10%
dextran sulfate, and 100 g/ml denatured salmon sperm DNA followed
by washing the filters in 5.times.SSC, 0.1% SDS at room
temperature.
[0076] The Vif derivatives and analogs of the invention can be
produced by various methods known in the art. The manipulations
that result in their production can occur at the gene or protein
level. Still within the scope of the present invention, other
sterically similar compounds, called peptidomimetics, may be
formulated to mimic the key portions of the structure of Vif
protein, derivatives and analogs thereof. Such compounds may be
used in the same manner as Vif protein, derivatives and analogs
thereof and hence are also functional equivalents. The generation
of a structural functional equivalent may be achieved by the
techniques of modeling and chemical design known to those of skill
in the art. It will be understood that all such sterically similar
constructs fall within the scope of the present invention.
[0077] Additionally, the vif encoding nucleic acid sequence can be
mutated in vitro or in vivo to create and/or destroy translation,
initiation, and/or termination sequences, or to create variations
in coding regions and/or form new restriction endonuclease sites or
destroy preexisting ones to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be
used, including but not limited to, chemical mutagenesis, in vitro
site-directed mutagenesis (Hutchinson, C., et al., J. Biol. Chem
253:6551, 1978), etc.
[0078] Manipulations of the Vif sequence also may be made at the
protein level. Included within the scope of the invention are
protein fragments or other derivatives or analogs that are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0079] In addition, analogs and derivatives of Vif can be
chemically synthesized. For example, a peptide corresponding to a
portion of a Vif protein that comprises the desired domain, or
which mediates the desired activity in vitro, can be synthesized by
use of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the Vif sequence. Non-classical amino
acids include but are not limited to the D-isomers of the common
amino acids, .alpha.-amino isobutyric acid, 4amino-butyric acid,
Abu, 2-amino butyric acid, .gamma.-Abu, .epsilon.-Ahx, 6-amino
hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic
acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, .beta.-alanine, fluoro-amino
acids, designer amino acids such as .beta.-methyl amino acids,
C-.alpha.-methyl amino acids, N-.alpha.-methyl amino acids, and
amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[0080] In a specific embodiment, the Vif derivative is a chimeric,
or fusion, protein comprising a Vif protein or fragment thereof
(consisting of at least the sequence from amino acid residue
144-171, preferably, 151-164, more preferably, 161-164) joined at
its amino- or carboxy-terminus via a peptide bond to an amino acid
sequence of a different protein. In one embodiment, such a chimeric
protein is produced by recombinant expression of a nucleic acid
encoding the protein (comprising a Vif-coding sequence joined
in-frame to a coding sequence for a different protein). Such a
chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other by methods known in the art, in the proper coding frame, and
expressing the chimeric product by methods commonly known in the
art. Alternatively, such a chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
Chimeric genes comprising portions of vif fused to any heterologous
protein-encoding sequences may be constructed.
[0081] In another specific embodiment, the Vif derivative is a
molecule comprising a region of homology with a Vif protein. By way
of example, in various embodiments, a first protein region can be
considered "homologous" to a second protein region when the amino
acid sequence of the first region is at least 30%, 40%, 50%, 60%,
70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence
in the second region of an equal number of amino acids as the
number contained in the first region or when compared to an aligned
sequence of the second region that has been aligned by a computer
homology program known in the art. For example, a molecule can
comprise one or more regions homologous to a Vif domain or a
portion thereof or a full-length protein.
[0082] Also provided by the present invention are molecules
comprising one or more peptidomimetics of a Vif domain or a portion
thereof or a full-length protein.
[0083] PXP Motif-Containing Peptides
[0084] The present invention also relates to peptides containing
PXP motifs. Molecules comprising PXP motif-containing peptides also
are provided.
[0085] The PXP motif-containing peptides may be about 5 to 20 amino
acids long. By way of example, but not by way of limitation, such
PXP motif-containing peptides may include peptides with amino acid
sequence of SEQ. ID. NO: 5-23.
[0086] The production and use of PXP motif-containing peptides are
within the scope of the present invention. In a specific
embodiment, the PXP motif-containing peptides are antagonists
capable of interactively binding to the multimerization domain of
Vif protein and inhibiting Vif protein multimerization. Still
within the scope of the present invention, other sterically similar
compounds, called peptidomimetics, may be formulated to mimic the
key portions of the structure of PXP motif-containing peptide. Such
compounds may be used in the same manner as the PXP
motif-containing peptides of the invention and hence are also
functional equivalents. The generation of a structural functional
equivalent may be achieved by the techniques of modeling and
chemical design known to those of skill in the art. It will be
understood that all such sterically similar constructs fall within
the scope of the present invention.
[0087] The PXP motif-containing peptides of the invention can be
produced by various methods known in the art. For example, PXP
motif-containing peptides can be chemically synthesized by use of a
peptide synthesizer. Furthermore, if desired, nonclassical amino
acids or chemical amino acid analogs can be introduced as a
substitution or addition into the PXP motif-containing peptides.
Non-classical amino acids include but are not limited to the
D-isomers of the common amino acids, .alpha.-amino isobutyric acid,
4 amino-butyric acid, Abu, 2-amino butyric acid, .gamma.-Abu,
.epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta.-methyl amino acids, C-.alpha.-methyl amino acids,
N-.alpha.-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0088] In a specific embodiment, a PXP motif-containing peptide is
a chimeric, or fusion, protein comprising a PXP motif-containing
peptide joined at its amino- or carboxy-terminus via a peptide bond
to an amino acid sequence of a different protein. In one
embodiment, such a chimeric protein is produced by recombinant
expression of a nucleic acid encoding the protein (comprising a
coding sequence for the PXP motif-containing peptide joined
in-frame to a coding sequence for a different protein). Such a
chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other by methods known in the art, in the proper coding frame, and
expressing the chimeric product by methods commonly known in the
art. Alternatively, such a chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
Chimeric genes comprising coding sequence for PXP motif-containing
peptides fused to any heterologous protein-encoding sequences may
be constructed.
[0089] In other specific embodiment of the invention, molecules
comprising PXP motif-containing peptides are provided. A molecule
can comprise one or more PXP motif-containing peptides. A PXP
motif-containing peptides may be 5 to 20 amino acids long. By way
of example, but not by way of limitation, such PXP motif-containing
peptides may include peptides with amino acid sequences of SEQ. ID.
NO: 5-23.
[0090] Also provided are molecules comprising one or more
peptidomimetics of PXP motif-containing peptides. Such PXP
motif-containing peptides include, but are not limited to, peptides
with amino acid sequences of SEQ. ID. NO: 5-23.
[0091] Screening for Small Molecules Inhibiting Vif
Multimerization
[0092] The present invention relates to the detection of molecules
that specifically bind to Vif, thereby inhibiting its
multimerization. Such molecules will thus inhibit the HIV-1
life-cycle. In a preferred embodiment, assays are performed to
screen for molecules with potential utility as therapeutic agents
or lead compounds for drug development. The invention provides
assays to detect molecules that bind to Vif and antagonize Vif
multimerization, thereby inhibiting the activity of Vif and
subsequent replication of the lentivirus.
[0093] For example, recombinant cells expressing Vif nucleic acids
are used to recombinantly produce Vif or Vif conjugate and screen
for molecules that bind to Vif or Vif conjugate. Molecules are
contacted with the Vif or Vif conjugate, or fragment thereof, under
conditions conducive to binding, and then molecules that
specifically bind to the Vif or Vif conjugate are identified.
Methods that are used to carry out the foregoing are commonly known
in the art. By way of example, but not way of limitation, phage
peptide display assay or phage enzyme-linked immunosorbent assay
(ELISA) may be used.
[0094] In another embodiment of the present invention, molecules
that bind to Vif or Vif conjugate and inhibit Vif protein
multimerization may be identified by Vif-Vif binding assay. More
specifically, Vif-Vif binding assay comprises the steps of, 1)
conjugating Vif or Vif-containing peptides to a column or beads; 2)
applying a test molecule and labeled Vif, or fragments thereof,
that contains the multimerization domain on the Vif- or
Vif-containing peptide-conjugated column or beads; 3) washing the
column or beads and dissociating the labeled Vif, or fragments
thereof, from the column or beads; and 4) measuring and comparing
the amount of labeled Vif, or fragments thereof, that was bound to
the column or beads to determine the antagonism activity of the
molecule. By "labeled Vif or fragments thereof," it is referred to,
but not limited to, radio labeled, chemical labeled, or fluorescent
labeled.
[0095] In a specific embodiment of the present invention, Vif
and/or cell line that expresses Vif is used to screen for
antibodies, peptides, or other molecules that bind to Vif and act
as an antagonist of Vif. The antagonists of the present invention
will function in any cell. The Vif antagonists of the present
invention will bind to the multimerization domain of Vif,
preventing Vif self-association, thereby inhibiting or preventing
the replicative and other essential functions of Vif. Therefore,
Vif antagonists will inhibit or prevent a disesase state or
condition associated with lentivirus infection. Such disease states
include, but are not limited to, acquired immunodeficiency
syndrome.
[0096] Vif antagonists are identified by screening organic or
peptide libraries with recombinantly expressed Vif. These Vif
antagonists are useful as therapeutic molecules, or lead compounds
for the development of therapeutic molecules, to modify the
activity of Vif. Synthetic and naturally occurring products are
screened in a number of ways deemed routine to those of skill in
the art.
[0097] By way of example, diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries are screened for
molecules that specifically bind to Vif. Many libraries are known
in the art that are used, e.g., chemically synthesized libraries,
recombinant (e.g., phage display libraries), and in vitro
translation-based libraries.
[0098] Examples of chemically synthesized libraries are described
in (Fodor, et al., Science 251:767-773, 1991; Houghten, et al.,
Nature 354:84-86, 1991; Lam, et al., Nature 354:82-84, 1991;
Medynski, Bio/Technology 12:709-710, 1994; Gallop, et al., J.
Medicinal Chemistry 37(9):1233-1251, 1994; Ohlmeyer, et al., Proc.
Natl. Acad. Sci. USA 90:10922-10926, 1993; Erb, et al., Proc. Natl.
Acad. Sci. USA 91:11422-11426, 1994; Houghten, et al.,
Biotechniques 13:412, 1992; Jayawickreme, et al., Proc. Natl. Acad.
Sci. USA 91:1614-1618, 1994; Salmon, et al., Proc. Natl. Acad. Sci.
USA 90:11708-11712, 1993; PCT Publication No. WO 93/20242; and
Brenner & Lerner, Proc. Natl. Acad. Sci. USA 89:5381-5383,
1992).
[0099] Examples of phage display libraries are described in (Scott
& Smith, Science 249:386-390, 1990; Devlin, et al., Science,
249:404-406, 1990; Christian, R. B., et al., J. Mol. Biol.
227:711-718, 1992; Lenstra, J. Immunol. Meth. 152:149-157, 1992;
Kay, et al., Gene 128:59-65, 1993; PCT Publication No. WO 94/18318
dated Aug. 18, 1994).
[0100] In vitro translation-based libraries include, but are not
limited to, those described in PCT Publication No. WO 91/0505 dated
Apr. 18, 1991; Mattheakis, et al., Proc. Natl., Acad. Sci. USA
91:9022-9026, 1994.
[0101] By way of examples of nonpeptide libraries, a benzodiazepine
library (see e.g., Bunin, et al., Proc. Natl. Acad. Sci. USA
91:4708-4712, 1994) can be adapted for use. Peptoid libraries
(Simon, et al., Proc. Natl. Acad. Sci. USA 89:9367-9371, 1992) also
can be used. Another example of a library that can be used, in
which the amide functionalities in peptides have been permethylated
to generate a chemically transformed combinatorial library, is
described by Ostresh, et al. in Proc. Natl. Acad. Sci. USA
91:11138-11142, 1994.
[0102] Screening the libraries is accomplished by any of a variety
of commonly known methods. See, e.g., the following references,
which disclose screening of peptide libraries: Parmley & Smith,
Adv. Exp. Med. Biol. 251:215-218, 1989; Scott & Smith, Science
249:386-390, 1990; Fowlkes, et al., BioTechniques 13:422-427, 1992;
Oldenburg, et al., Proc. Natl. Acad. Sci. USA 89:5393-5397, 1992;
Yu, et al., Cell 76:933-945, 1994; Staudt, et al., Science
241:577-580, 1988; Bock, et al., Nature 355:564-566, 1992; Tuerk,
et al., Proc. Natl. Acad. Sci. USA 89:6988-6992, 1992; Ellington,
et al., Nature 355:850-852, 1992; U.S. Pat. No. 5,096,815, U.S.
Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner, et
al.; Rebar & Pabo, Science 263:671-673, 1993; and PCT
Publication No. WO 94/18318.
[0103] In a specific embodiment, screening is carried out by
contacting the library members with Vif, or fragment thereof,
immobilized on a solid phase and harvesting those library members
that bind to the Vif, or fragment thereof. Examples of such
screening methods, termed "panning" techniques, are described by
way of example in Parmley & Smith, Gene 73:305-318, 1988;
Fowlkes, et al., BioTechniques 13:422-427, 1992; PCT Publication
No. WO 94/18318 and in references cited hereinabove.
[0104] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields & Song, Nature
340:245-246, 1989; Chien et al., Proc. Natl. Acad. Sci. USA
88:9578-9582, 1991) is used to identify molecules that specifically
bind to Vif, or fragment thereof.
[0105] Therapeutic Uses
[0106] The invention provides for treatment or prevention of
various diseases, disorders, and conditions by administration of a
therapeutic compound. Such therapeutics include but are not limited
to Vif proteins and analogs and derivatives (including fragments)
thereof; antibodies thereto; nucleic acids encoding the proteins,
analogs, or derivatives; and antagonists. In a preferred
embodiment, disorders involving lentivirus infection are treated or
prevented by administration of a therapeutic that inhibits Vif
function.
[0107] Generally, administration of products of a species origin or
species reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a preferred
embodiment, a human Vif protein, derivative, or analog, or nucleic
acid, or an antibody to a human Vif protein or human Vif nucleic
acid, is therapeutically or prophylactically administered to a
human patient.
[0108] A vif polynucleotide and its protein product can be used for
therapeutic/prophylactic purposes for diseases and conditions
involving lentivirus infection, as well as other disorders
associated with the multimerization of Vif. A vif polynucleotide,
and its protein product, may be used for therapeutic/prophylactic
purposes alone or in combination with other therapeutics useful in
the treatment of acquired immunodeficiency syndrome or other
diseases and conditions caused by lentiviruses.
[0109] In specific embodiments, therapeutics that inhibit Vif
function are administered therapeutically (including
prophylactically): (1) in diseases, disorders, or conditions
involving lentiviruses, specifically HIV-1; or (2) in diseases,
disorders, or conditions wherein in vitro (or in vivo) assays
indicate the utility of Vif antagonist administration. The presence
of HIV-1 can be readily detected by any means standard in the art.,
e.g., by obtaining a patient blood sample and assaying it in vitro
for the presence of HIV-1.
[0110] Therapeutic/Prophylactic Methods
[0111] The invention provides methods of treatment and prophylaxis
by administration to a subject of an effective amount of a
therapeutic, i.e., a monoclonal (or polyclonal) antibody,
retroviral vector, or Vif antagonist of the present invention. In a
preferred aspect, the therapeutic is substantially purified. The
subject is preferably an animal, including but not limited to,
animals such as cows, pigs, chickens, etc., and is preferably a
mammal, and most preferably human.
[0112] Various delivery systems are known and are used to
administer a therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, expression by recombinant
cells, receptor-mediated endocytosis (see, e.g., Wu & Wu, J.
Biol. Chem. 262:4429-4432, 1987), construction of a therapeutic
nucleic acid as part of a retroviral or other vector, etc. Methods
of introduction include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, and oral routes. The compounds are administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local. In addition, it may be desirable to introduce
the pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir.
[0113] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, the implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration is by direct injection at the site (or former site)
of a malignant tumor or neoplastic or pre-neoplastic tissue.
[0114] In a specific embodiment where the therapeutic is a nucleic
acid encoding a protein therapeutic the nucleic acid is
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot, et al., Proc. Natl.
Acad. Sci. U.S.A. 88:1864-1868, 1991), etc. (supra). Alternatively,
a nucleic acid therapeutic can be introduced intracellularly and
incorporated within host cell DNA for expression by homologous
recombination (supra).
[0115] Pharmaceutical Compositions
[0116] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a therapeutic and a pharmaceutically acceptable
carrier or excipient. Such a carrier includes, but is not limited
to, saline, buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. The carrier and composition can be
sterile. The formulation will suit the mode of administration.
[0117] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc.
[0118] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
also includes a solubilizing agent and a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it is be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline is
provided so that the ingredients are mixed prior to
administration.
[0119] The therapeutics of the invention are formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0120] The amount of the therapeutic of the invention that will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and is
determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation also
will depend on the route of administration, and the seriousness of
the disease, disorder, or condition and is decided according to the
judgment of the practitioner and each patient's circumstances.
However, suitable dosage ranges for intravenous administration are
generally about 20-500 micrograms of active compound per kilogram
body weight. Suitable dosage ranges for intranasal administration
are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0121] Suppositories generally contain active ingredient in the
range of 0.5% to 10 k by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0122] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) is a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
Results
[0123] Vif Proteins Can Form Multimers In Vitro
[0124] To examine whether Vif proteins have a tendency towards
self-association, GST-Vif was expressed in BL 21 bacterial cells
and isolated onto glutathione-conjugated agarose beads. The
GST-Vif-conjugated beads were then incubated with in vitro
translated, .sup.35S-labeled Vif proteins. After binding, the
bead-associated .sup.35S-labled Vif was analyzed by SDS-PAGE,
followed by direct autoradiography. The autoradiograph of the bound
.sup.35S-labled Vif illustrates that GST-Vif (lane 2), but not GST
(lane 3), binds to .sup.35S-labeled, in vitro translated Vif
protein, indicating a Vif-Vif interaction (FIG. 1A).
[0125] To further evaluate the tendancy of Vif proteins to
self-associate, in vitro translated, .sup.35S-labeled HIV-1 Vif
proteins were directly loaded onto a Tris-Glycine-native gel
(SDS-free) with loading buffers containing 10% glycerol only or SDS
at various concentrations. Electrophoresis performed with a 4-15%
Tris-Glycine running buffer shows that, at the native or relatively
native conditions, the .sup.35S-labeled Vif proteins migrate as
monomers (23 Kd), dimers (46 Kd), trimers (69 Kd), or tetramers (92
Kd) (FIG. 1B). With the increment of concentrations of SDS in the
loading buffer, the major form of Vif eventually becomes a monomer
(23 Kd). When the sample was heated at 95.degree. C. for 5 minutes,
all the multimers of Vif proteins disappeared, implying that the
Vif-Vif binding is not covalent. Since, prior to the sample
loading, .sup.35S-labeled, in vitro translated HIV-1 Vif protein
was treated with RNase A to remove possible RNA contamination, the
Vif-Vif binding was RNA-independent.
[0126] The Binding Site for Vif Multimerization is Located in the
C-Terminus
[0127] To determine the binding sites for Vif multimerization, a
series of deletions in Vif protein are generated through PCR-based
mutagenesis, followed by in vitro translation in the presence of
.sup.35S-methionine. These Vif mutants were then allowed to bind to
GST-Vif fusion protein conjugated on agarose beads. After binding,
the bead-associated, .sup.35S-labeled Vif protein and its mutants
were subjected to SDS-PAGE and visualized by direct
autoradiography. FIG. 2A presents the results. Vif protein severely
loses the Vif-Vif binding activity with deletion of the C-terminus,
while deletion at amino acid positions 151-164 significantly
decreases the binding ability (FIG. 2A). This result is confirmed
by native multimer formation assay. In the presence of 0.1% SDS,
Vif mutants .DELTA.151-192 and .DELTA.151-164 were unable to form
multimers, while other mutants retained the ability to multimerize
(FIG. 2B).
[0128] It is notable that there are several positively-charged
amino acids in the 151-164 fragment. The mutants that substitute
these positively-charged amino acids as generated by Goncalves et
al. (Goncalves, J., et al., J. Virol. 69(11):7196-204, 1995) have
been examined for this Vif-Vif binding. However, all these mutants
still contain Vif-Vif binding ability (data not shown). It is also
notable that there are several prolines (P156, P161, P162, P164) in
this fragment. Among these prolines, P161 is highly conserved in
various strains of HIV-1 or SIV. Further investigation demonstrates
that deletion of .sup.161PPLP.sup.164 (aa 161-164 in Vif protein,
SEQ. ID. NO: 25) significantly impairs the capability of Vif
proteins to interact each others. Moreover, a highly conerved
motif, SLQYLAL (SEQ. ID. NO: 4) (amino acid positions 144-150 for
HIV-1.sub.NL4-3), is close to this domain.
[0129] The domain for Vif multimerization, therefore, is located at
the C terminus, more particularly, amino acid positions 144-171 of
HIV.sub.NL4-3 Vif protein and has the amino acid sequence of SEQ.
ID. NO: 26.
[0130] Vif to Vif Interactions Within a Cell
[0131] To examine whether Vif self-association also occurs
intracellularly, a co-immunoprecipitation method was utilized. The
Vif protein was tagged with either c-Myc (SEQ. I.D. NO: 3) or Flag
epitope (SEQ. I.D. NO: 2) at its C-terminus and expressed in COS-1
cells. The expression of c-Myc-tagged Vif and Flag-tagged Vif was
detected via Western blotting with mouse anti-c-Myc epitope
antibody or goat anti-Flag epitope antibody, respectively (FIG. 3,
top two panels). To study Vif-Vif interaction, the cell lysates
were immunoprecipitated with anti-Myc antibody and then subjected
to SDS-PAGE, followed by Western blotting. The goat anti-Flag
antibody was used to detect Flag-tagged Vif. The results are shown
in FIG. 3, bottom panel. The Flag-tagged Vif is co-precipitated
with Myc-tagged Vif when mouse anti-Myc antibody was utilized for
the immunoprecipitation, implying a Vif-Vif interaction within a
cell (FIG. 3, bottom panel).
[0132] Alternatively, the in vivo Vif to Vif interaction was
examined by the mammalian two-hybrid system. A fusion protein
composed of VP16 and Gal4 is able to activate Gal4-reseponse
element-contained E1b promoter. Gal4 functions as a DNA-binding
domain, while VP16 functions as a DNA activation domain. HIV-1 Vif
protein is allowed to replace the VP16 or Gal4 domain, respectively
(FIG. 4A). If the interaction between Vif proteins takes place, the
VP16 and Gal4 domains are brought together and the
Gal4-binding-sequence-contained in the E1b promoter is activated.
CAT analysis revealed that, like Rev-Rev interactions, Vif in
Vif-VP16 fusion protein binds to Vif in the Gal4-Vif fusion protein
and activates the expression of CAT (lane 6) (FIG. 4B). As
controls, pGal-Vif or pVif-VP alone were unable to activate CAT
expression (lanes 3 & 4, FIG. 4B). FIG. 4B also shows that Vif
mutant .DELTA.151-164, which does not have the ability to interact
with Vif protein in other systems, does not interact with Vif in
this system (lane 7).
[0133] Deletion of the Vif-Vif Binding Domain Severely Decreases
the Vif Function in the Viral Life Cycle.
[0134] As mentioned previously, Vif functions in the late stages of
the HIV-1 life-cycle and is required by "non-permissive" cells,
such as PBMC, macrophages, and H9 T-cells, for HIV-1 replication.
(Gabuzda, D. H., et al., J. Virol. 66(11):6489-95, 1992; Blanc, D.,
et al., Virology 193(1):186-92, 1993; von Schwedler, U., et al., J.
Virol. 67(8):4945-55, 1993). To investigate the physiological
significance of Vif multimerization, the ability of Vif mutant
.DELTA.151-164 to complement Vif function in the viral life-cycle
was examined. Vif mutant .DELTA.151-164 was used because it is
unable to form multimers in cell-free systems and within cells. To
this end, a single-round viral infectivity assay was adapted.
Wild-type Vif or its mutants, were expressed in the
"non-permissive" H9 T-cells. At the same time, pseudotyped (with
VSV envelope) HIV-1 viruses, without vif and env in their genome,
were generated from these cells. After ultracentrifugation for
enrichment, the recombinant viruses were allowed to infect the
target cells (Hela CD4-CAT), which harbor an expression cassette
containing HIV-1 LTR promoter-driven CAT gene. The viral
infectivity was measured by the level of CAT gene expression in the
target cells, which is driven by the HIV-1 Tat protein expressed by
the newly-synthesized proviruses. FIG. 5 demonstrates that, when
the wild-type vif gene is expressed in the vif-defective HIV-1
virus-producing "non-permissive" H9 T-cells, the viral infectivity
reaches a high level (lane 2). When Vif .DELTA.151-164 is expressed
in the vif-defective HIV-1 virus-producing "non-permissive" H9
T-cells, however, the viral infectivity is unaltered (lane 3)
compared to the vif-defective HIV-1 viruses (lane 4) (FIG. 5).
These data indicate that the 151-164 deletion severely decreases
the function of Vif protein and makes it unable to rescue the
infectivity of the vif-defective HIV-1 viruses generated from
"non-permissive" T-cells. The results demonstrate that
multimerization of Vif proteins is required for Vif function.
[0135] Peptides Containing PXP Motif Inhibit Vif-Vif Interaction by
Binding to PPLP Domain
[0136] To further identify peptides that bind to the Vif protein
multimerization domain, thereby inhibiting Vif-Vif interaction and
viral infectivity of HIV-1 virus, a set of 12-mer peptides
containing a PXP motif (Table 1, SEQ. ID. NO: 5-20) was
constructed, which structure is shared by the .sup.161PPLP.sup.164
domain (SEQ. ID. NO: 25) of Vif protein. Through phage peptide
display method, it was demonstrated that these peptides bind to
purified HIV-1 Vif protein at high affinity (FIG. 6). Some of these
peptides were synthesized and were added into the reaction system
for Vif-Vif binding. As shown in Table 1, peptides containing PXP
motif such as LPLPAPSFHRTT (VMI9, SEQ. ID. NO: 13) or SNQGGSPLPRSV
(VMI7, SEQ. ID. NO: 11) can significantly inhibit Vif-Vif
interaction.
[0137] Further experiments demonstrated that PXP motif-containing
peptides were unable to bind to .sup.161PPLP.sup.164
domain-deleted-VIF protein, thereby evidencing that the
.sup.161PPLP.sup.164 domain plays a key role in Vif multimerization
and that PXP motif-containing peptides block the multimerization of
Vif through binding to the .sup.161PPLP.sup.164 domain of Vif
protein.
[0138] A set of synthesized Vif peptides, Vif155-166 (SEQ. ID. NO:
21), Vif157-171 (SEQ. ID. NO: 23), Vif161-175 (SEQ. ID. NO: 22),
and Vif117-131 (SEQ. ID. NO: 24) were screened for their ability to
block the Vif-Vif interaction in vitro. As shown in Table 1, three
peptides, Vif155-166 (SEQ. ID. NO: 21), Vif157-171 (SEQ. ID. NO:
23), and Vif161-175 (SEQ. ID. NO: 22), which contain the
.sup.161PPLP.sup.164 domain, were able to inhibit the Vif-Vif
interaction, further supporting that the .sup.161PPLP.sup.164
domain is responsible for Vif multimerization.
1TABLE 1 Inhibitory Effect of Peptides containing PXP Motif upon
Vif-Vif Interaction .sup.35S-Vif binds with GST-Vif (%) SEQ. ID.
NO: peptide Mean .+-. SD No peptide 100 5 (VMI1) SNFASITTPRPH ND 6
(VMI2) WPTNPTTVPVPS ND 7 (VMI3) LTSDTYFLPVPA ND 8 (VMI4)
SLHWPVSHPPPP ND 9 (VMI5) SVSVGMKPSPRP 36.3 + 5.1 10 (VMI6)
WHSQRLSPVPPA ND 11 (VMI7) SNQGGSPLPRSV 19.0 + 2.2 12 (VMI8)
SEPHLPFPVLPH ND 13 (VMI9) LPLPAPSFHRTT 22.0 + 6.2 14 (VMI10)
YPLPHPMWSMLP ND 15 (VMI11) TMTPPPTSVRGT ND 16 (VMI12) TPLPTIRGDTGT
ND 17 (VMI13) GPPPHHRDYHGP ND 18 (VMI14) YPAPIKVLLPNS ND 19 (VMI15)
SPYPMALFPLHN ND 20 (VMI16) SPYPSWSTPAGR ND 21 (Vif155-166)
KPKKIKPPLPSV 57.1 + 8.7 22 (Vif161-175) PPLPSVTKLTEDRWN 70.2 + 5.5
23 (Vif157-171) KKIKPPLPSVTKLTE 49.2 + 2.5 24 (Vif117-131)
ESAIRKAILGHIVSP 94.5 + 11.2
Discussion
[0139] The formation of dimers or multimers by many HIV-1 proteins,
e.g., Gag, protease, reverse transcriptase, integrase, glycoprotein
41(gp41), Tat, Rev, Vpr, and Nef, has been shown to be important
for their functions in the lentiviral life-cycle. (Frankel, A. D.
& Young, J. A., Ann. Rev. Biochem. 67:1-25, 1998; Vaishnav, Y.
N. & Wong-Staal, F., Annu Rev Biochem 60:577-630, 1991; Zhao,
L. J., et al., J Biol Chem 269(51):32131-7, 1994; Liu, L., et al.,
J. Virol. 74:5310-5319, 2000). In addition, multimerization is
critical to the biological activity of many prokaryotic and
eukaryotic proteins and is a common mechanism for the functional
activation/inactivation of proteins. The present invention
demonstrates that HIV-1 Vif proteins form dimers or multimers and
that such multimerization is essential for Vif function in the
viral life-cycle. The evidence reveals that in vitro translated
.sup.35S-lableled Vif proteins are able to form multimers in the
native environment. Conversely, GST-Vif fusion proteins, rather
than GST proteins, which are generated from a bacterial expression
system, are able to bind to the in vitro translated
.sup.35S-labeled Vif proteins. Further, results of
co-immunoprecipitation and a mammalian two hybrid system
demonstrate a Vif-Vif interaction intracellularly. These in vitro
and in vivo data strongly imply that Vif proteins are able to form
multimers. Deletion of the domain essential for Vif-Vif binding
severely decreases the function of Vif in the "non-permissive"
cells, evidencing further that multimerization of Vif is important
for its function in the HIV-1 life-cycle.
[0140] The domain for Vif multimerization is located in a
positively-charged amino acid- and proline-enriched fragment (amino
acid positions 144-171) and has the amino acid sequence of SEQ.
I.D. NO: 26. (FIG. 2). The positively-charged amino acids in this
region are not responsible for the Vif-Vif interaction. However,
the prolines, more particularly, the .sup.161PPLP.sup.164 domain is
responsible for Vif multimerization (FIG. 6 and Table 1). Based on
this, a set of PXP motif-containing peptides are identified as
inhibitors of Vif protein multimerization. It is notable that a
highly conserved motif, SLQYLAL (SEQ. I.D. NO: 4) (amino acid
positions 144-150 for HIV-1.sub.NL4-3), is close to this domain. It
also has been shown that serine165 is phosphorylated by the
mitogen-activated protein kinase (p44/42) of Vif and that this
phosphorylation is important for Vif function. (Yang, X., &
Gabuzda., D., J. Bio. Chem. 273(45):29879-87, 1998). As these
residues are close to the domain for multimerization, it is
possible that the multimerization of Vif proteins is regulated by
phosphorylation in the virus-producing cells.
[0141] Interestingly, the positively-charged amino acids (replaced
in B4 and B7 mutants) in the C-terminus of Vif are responsible for
Vif-NCp7 binding in vitro. (Bouyac, M., et al., J. Virol.
71(12):9358-65, 1997). Recent studies demonstrate not only that
HIV-1 Vif is an RNA binding protein and an integral component of an
mRNP complex of viral RNA in the cytoplasm but also that it could
be involved in the viral RNA packaging process. (Zhang, H., et al.,
J. Virol. 74;8252-8261, 2000). In contrast to interactions with
NCp7 via its C-terminus, Vif binds to RNA via its N-terminus. When
RNA is mixed with Vif or Gag separately, more RNA binds to Vif than
to Gag; in contrast, when Vif protein is mixed together with RNA
and NCp7, RNA only binds to Gag. (Zhang, H., et al., J. Virol.
74;8252-8261, 2000). This "displacement" may be due to various
mechanisms; however, as the domains for Vif multimerization and for
Vif-NCp7 binding are quite close in location or possibly overlap,
it is possible that the interaction between Vif and Gag, as well as
the interactions between Vif, RNA, and Gag, is regulated by Vif
multimerization.
[0142] In summary, Vif proteins possess a strong tendency to
self-associate, forming dimers and multimers. The domain affecting
self-association is located at the C-terminus of the protein,
specifically the .sup.161PPLP.sup.164 domain. The PXP
motif-containing peptides block the multimerization of Vif through
binding to the .sup.161PPLP.sup.164 domain of Vif protein. The
evidence reveals that a Vif mutant with deletion at amino acid
positions 151-164 is unable to rescue the infectivity of
vif-defective viruses generated from H9 T-cells, implying that the
multimerization of Vif proteins is important for Vif function in
the lentivirus life-cycle.
[0143] While this invention has been described with a reference to
specific embodiments, it will obvious to those of ordinary skill in
the art that variations in these methods and compositions may be
used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the claims.
Sequence CWU 1
1
26 1 14 PRT Artificial Sequence Fragment of vif protein sequence 1
Ala Ala Leu Lys Ile Pro Lys Gln Ile Lys Pro Pro Leu Pro 1 5 10 2 8
PRT Artificial Sequence Fragment of vif protein sequence 2 Asp Tyr
Lys Asp Asp Asp Asp Lys 1 5 3 10 PRT Artificial Sequence Fragment
of c-Myc protein sequence 3 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10 4 7 PRT Artificial Sequence Fragment of vif protein sequence
4 Ser Leu Gln Tyr Leu Ala Leu 1 5 5 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 5 Ser Asn Phe Ala Ser Ile
Thr Thr Pro Arg Pro His 1 5 10 6 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 6 Trp Pro Thr Asn Pro Thr
Thr Val Pro Val Pro Ser 1 5 10 7 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 7 Leu Thr Ser Asp Thr Tyr
Phe Leu Pro Val Pro Ala 1 5 10 8 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 8 Ser Leu His Trp Pro Val
Ser His Pro Pro Pro Pro 1 5 10 9 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 9 Ser Val Ser Val Gly Met
Lys Pro Ser Pro Arg Pro 1 5 10 10 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 10 Trp His Ser Gln Arg Leu
Ser Pro Val Pro Pro Ala 1 5 10 11 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 11 Ser Asn Gln Gly Gly Ser
Pro Leu Pro Arg Ser Val 1 5 10 12 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 12 Ser Glu Pro His Leu Pro
Phe Pro Val Leu Pro His 1 5 10 13 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 13 Leu Pro Leu Pro Ala Pro
Ser Phe His Arg Thr Thr 1 5 10 14 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 14 Tyr Pro Leu Pro His Pro
Met Trp Ser Met Leu Pro 1 5 10 15 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 15 Thr Met Thr Pro Pro Pro
Thr Ser Val Arg Gly Thr 1 5 10 16 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 16 Thr Pro Leu Pro Thr Ile
Arg Gly Asp Thr Gly Thr 1 5 10 17 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 17 Gly Pro Pro Pro His His
Arg Asp Tyr His Gly Pro 1 5 10 18 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 18 Tyr Pro Ala Pro Ile Lys
Val Leu Leu Pro Asn Ser 1 5 10 19 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 19 Ser Pro Tyr Pro Met Ala
Leu Phe Pro Leu His Asn 1 5 10 20 12 PRT Artificial Sequence
Synthetic peptide containing PXP motif 20 Ser Pro Tyr Pro Ser Trp
Ser Thr Pro Ala Gly Arg 1 5 10 21 12 PRT Artificial Sequence
Fragment of vif 21 Lys Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val
1 5 10 22 15 PRT Artificial Sequence Fragment of vif 22 Pro Pro Leu
Pro Ser Val Thr Lys Leu Thr Glu Asp Arg Trp Asn 1 5 10 15 23 15 PRT
Artificial Sequence Fragment of vif 23 Lys Lys Ile Lys Pro Pro Leu
Pro Ser Val Thr Lys Leu Thr Glu 1 5 10 15 24 15 PRT Artificial
Sequence Fragment of vif 24 Glu Ser Ala Ile Arg Lys Ala Ile Leu Gly
His Ile Val Ser Pro 1 5 10 15 25 4 PRT Artificial Sequence Fragment
of vif protein 25 Pro Pro Leu Pro 1 26 31 PRT Artificial Sequence
Fragment of vif protein 26 Lys Val Gly Ser Leu Gln Tyr Leu Ala Leu
Ala Ala Leu Ile Thr Pro 1 5 10 15 Lys Lys Ile Lys Pro Pro Leu Pro
Ser Val Thr Lys Leu Thr Glu 20 25 30
* * * * *