BACKGROUND OF THE INVENTION Human immunodeficiency virus type I (HIV-1) Vpr is a phosphorylated nuclear protein that is highly conserved among primate lentiviruses. In addition to facilitating the nuclear uptake of the viral pre-integration complex in non-dividing cells, Vpr prevents the passage of HIV-infected cells through mitosis at the G2 stage of the cell cycle to provide an optimized environment for maximal levels of viral expression and replication. The cell cycle arrest mediated by Vpr can be observed in human and primate cells as well as in fission yeast. Furthermore, Vpr has been shown to induce mitochondrial dysfunction in human and yeast cells that lead to growth arrest and cell killing in yeasts and apoptosis in human cells. The occurrence of Vpr activities in human and primate cells from a variety of tissues, as well as in yeasts indicates that Vpr interacts with highly conserved cellular processes to cause its effects.

The human immunodeficiency virus 1 (HIV-1) vpr gene product is a small (14 kDa), phosphorylated nuclear protein that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus (SIV) (for reviews see 1; 2). Several functions of Vpr have been demonstrated in vitro, underlining the importance of this protein for HIV replication and pathogenesis. Vpr is packaged into viral particles suggesting that it may play a role in early events during HIV-1 infection (3; 4). Indeed, experimental evidence indicates that Vpr

increases HIV-1 replication in non-dividing cells such as macrophages, possibly by facilitating, with other viral components, nuclear import of the large viral pre-integration complex (5-13). Vpr localizes predominantly to the nucleus in a variety of cell types and has been found to contain two non- canonical nuclear localization signals located in the N- terminus and the C-terminal regions of the protein, respectively (8; 9; 11 ; 14; 15).

Vpr also has been shown to promote cell differentiation and growth arrest at the G2/M phase of the cell cycle (16-21). This property of Vpr has been proposed to enhance viral replication because HIV-1 transcription is presumably more active during the G2 phase of the cell cycle (22-24). Vpr-mediated cell cycle G2 arrest can be observed in cells from distantly related eukaryotes including human and fission yeast (Schizosaccharomyces pombe, [S. pombe]) and was shown to occur through inhibitory phosphorylation of Cdc2/Cdkl (18; 19; 25; 26). In all eukaryotic cells, entry into mitosis is regulated by the phosphorylation status of threonine 14 and tyrosine 15 on Cdc2/Cdkl, which is phosphorylated by Mytl and Weel protein kinases during G2 and rapidly dephosphorylated by the Cdc25 phosphatase to trigger entry into mitosis. Both Weel and Cdc25 activities are themselves regulated at the level of their subcellular localization as well as by upstream kinase/phosphatase networks (27). Recent genetic studies with fission yeast suggest that Vpr induces cell cycle G2 arrest through a pathway involving protein phosphatase 2A (PP2A) and acting on both Weel and Cdc25 (29) (30). These observations indicate that Vpr appears to target a well-conserved cellular pathway controlling the G2 checkpoint during cell cycle.

However, although Vpr has been shown to interact with various host proteins (31-34), the molecular mechanism underlying Vpr- induced cell cycle G2 arrest in HIV-1 infected cells remains undefined.

In addition to nuclear targeting and cell cycle G2 arrest activities, Vpr has also been shown to differentially regulate the occurrence of apoptosis in human cells. In particular, it has been reported that during active HIV-1 replication, the ability of Vpr to arrest cells in the G2 phase finally results in cell killing by apoptosis (36 ; 24).

Moreover, several studies showed that Vpr is capable of regulating either positively or negatively, apoptosis depending on the level of protein expression or the state of immune activation (37-39). Vpr thus clearly plays an important role in HIV-mediated pathogenesis.

There thus remains a need to modulate Vpr-induced function. Given the role of Vpr in the pathogenesis of HIV, Vpr represents a target for the modulation of pathogenesis of HIV and related virus.

SUMMARY OF THE INVENTION The invention relates to agents or compounds which are capable of binding to, and/or modulating (e. g. inhibiting) at least one biological activity relating to, a Vpr or a functional derivative thereof.

In an aspect, the invention provides a substantially pure compound capable of (a) binding to Vpr or a functional derivative thereof (b) modulating Vpr-related activity or (c) both (a) and (b), the compound comprising a polypeptide comprising two W residues. In an embodiment, the two W residues are adjacent to one another in the polypeptide. In an embodiment, the polypeptide further comprises a hydrophobic amino acid, wherein the hydrophobic amino acid is located in the portion of the polypeptide which is C-terminal to the two W residues. In an embodiment, the hydrophobic amino acid is

an aromatic amino acid. In an embodiment, the aromatic amino acid is selected from the group consisting of W and F.

In an embodiment, the polypeptide comprises the sequence WWX, wherein X is an amino acid and cD is a hydrophobic amino acid. In an embodiment, the hydrophobic amino acid is an aromatic amino acid. In an embodiment, the aromatic amino acid is selected from the group consisting of W and F. In a further embodiment, the hydrophobic amino acid is F.

The invention further provides a substantially pure compound capable of (a) binding to Vpr or a functional derivative thereof (b) modulating Vpr-related activity or (c) both (a) and (b), the compound comprising a polypeptide comprising the sequence WXSF, wherein X is an amino acid. In an embodiment, X is selected from the group consisting of Y and M.

The invention further provides a substantially pure compound capable of (a) binding to Vpr or a functional derivative thereof (b) modulating Vpr-related activity or (c) both (a) and (b), the compound being of Formula I: Z1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - X10 - X11 - X12 - X13 - X14 - X15 - X16 - X17 - X18 - Z2 (I) wherein Xi is absent or S or E or F or T or A or Q or W or R or G or V or an analogue thereof; X2 is absent or I or S or C or V or G or T or F or Y or L or R or A or an analogue thereof; X3 is absent or R or S or G or Q or P or A or F or V or E or Y or M or an analogue thereof; X4 is W or an analogue thereof; X5 is W or Y or M or A or an analogue thereof;

X6 is L or V or W or G or A or K or E or S or Y or C or R or an analogue thereof; X ? is absent or W or V or F or L or an analogue thereof; X8 is absent or S or C or H or G or L or A or W or K or an analogue thereof; Xg is absent or R or G or V or Q or S or C or an analogue thereof; X10 is absent or G or L or an analogue thereof; X1l is absent or S or R or an analogue thereof; X12 is absent or G or D or an analogue thereof; X13 is absent or K or P or an analogue thereof; X14 is absent or G or an analogue thereof; X15 is absent or N or an analogue thereof; X16 is absent or K or an analogue thereof; X17 is absent or L or an analogue thereof; X18 is absent or N or an analogue thereof; Z1 is selected from the group consisting of: (a) an N-terminal group the formula H2N-, RHN-or, RRN- ; (b) one or more amino acids; and (c) both (a) and (b); Z2 is selected from the group consisting of: (d) a C-terminal <BR> <BR> <BR> <BR> group of the formula-C (O) OH, -C (O) R, -C (O) OR, -C (O) NHR, - C (O) NRR; (e) one or more amino acids; and (f) both (d) and (e); R at each occurrence is independently selected from (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, substituted (C1-C6) alkyl, substituted (C1-C6) alkenyl, or substituted (C1-C6) alkynyl ; and "-"is a covalent linkage.

In an embodiment, Z1 is selected from the group consisting of glutathione S-transferase (GST), the polypeptide KGLSGP, and GST-KGLSGP. In an embodiment, Z2 is a V residue.

The invention further provides a substantially pure synthetic compound or recombinant compound capable of (a) binding to Vpr or a functional derivative thereof (b)

modulating Vpr-related activity or (c) both (a) and (b), the compound having a domain of Formula II: - X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - X10 - X11 - X12 - X13 -X14-X15-X16-X17-X18- (II) wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X1O, X11/X12t X13/X14/ X15, X16, X17, Xig and"-"are as defined above.

In embodiments, the compound is selected from the group consisting of: SIRWWL; ESRWWV; FCSWWW; CGWWVWSRGSGK; TVQWWV; AVPWWV; QGSWWV ; TAWWVV; FFWWLF; WCRWWL; RYVWWL ; GGWWGF; SWWLFC; SEWWVW ; VWWLLGCLRDPGNKLN; RWWAFH; RCGWWK; RWWEWG; LYWWVW; GRFWWV ; RWWWFC; WYSFLG; VFMWWW; RGWWWV; AWMSFL; CWWSFL ; WWYFAQ; PWACVW; WWSFKS ; AFWWVF; and WGVWWR.

In embodiments the domain is selected from the group consisting of: SIRWWL; ESRWWV; FCSWWW; CGWWVWSRGSGK; TVQWWV; AVPWWV; QGSWWV; TAWWVV; FFWWLF; WCRWWL; RYVWWL; GGWWGF; SWWLFC; SEWWVW; VWWLLGCLRDPGNKLN; RWWAFH; RCGWWK; RWWEWG; LYWWVW; GRFWWV; RWWWFC; WYSFLG; VFMWWW; RGWWWV; AWMSFL; CWWSFL; WWYFAQ; PWACVW; WWSFKS; AFWWVF; and WGVWWR.

In an embodiment, the functional derivative consists essentially of the polypeptide corresponding to residues 52 to 96 of HIV-1 Vpr.

In an embodiment, the Vpr is derived from a lentivirus. In an embodiment, the lentivirus is a primate lentivirus. In an embodiment, the primate lentivirus is selected from the group consisting of HIV-1, HIV-2 and SIV.

In an embodiment, the compound is capable of inhibiting the Vpr-related activity.

The invention further provides a method of preventing or treating a Vpr-related disease in a subject, the method comprising administering to the subject an effective amount of the above-mentioned compound.

The invention further provides a method of diagnosis of a Vpr-related disease in a subject, comprising determining whether a component of a sample from the subject binds to the above-mentioned compound.

The invention further provides a method of prognosticating a Vpr-related disease in a subject, the method comprising determining a first level of binding of the above- mentioned compound to a component of a sample from the subject, wherein comparison of the first level to a second level of binding of the above-mentioned compound to a component of a sample from the subject determined at an earlier time is used to prognosticate the disease.

In an embodiment, the disease is associated with an infection of the subject by a lentivirus. In an embodiment, the lentivirus is a primate lentivirus. In an embodiment, the primate lentivirus is selected from the group consisting of HIV-1, HIV-2 and SIV. In embodiments, the sample is selected from a cell, tissue or body fluid of the subject. In an embodiment, the cell, tissue or body fluid is blood. In an embodiment, the cell is an immune cell. In an embodiment, the method is carried out in vitro. In an embodiment, the method is carried out in vivo. In an embodiment, the subject is a mammal, in a further embodiment a primate, in a further embodiment, a human.

The invention further provides a method of modulating (e. g. inhibiting) Vpr-related activity in a sample, the method comprising contacting the sample with the above- mentioned compound.

In an embodiment, the above-mentioned Vpr- related activity is selected from the group consisting of: cellular apoptosis; cell death; inhibition of cell growth; cell cycle arrest; Vpr nuclear import; and lentiviral pathogenesis in an animal. In an embodiment the animal is a

mammal, in a further embodiment a primate, in a further embodiment a human.

The invention further provides a method of detecting Vpr or a functional derivative thereof in a sample, the method comprising contacting the sample with the above-mentioned compound, wherein binding of the compound to a component of the sample is indicative that the sample comprises Vpr or a functional derivative thereof The invention further provides a pharmaceutical composition comprising the above-mentioned compound in admixture with a pharmaceutically acceptable carrier.

The invention further provides a commercial package comprising the above-mentioned compound, together with instructions for a process selected from the group consisting of: modulating Vpr-related activity; preventing or treating a Vpr-related disease; diagnosing a Vpr-related disease; prognosticating a Vpr-related disease; and detecting the presence of Vpr or a functional derivative thereof in a sample.

The invention further provides a use of the above- mentioned compound for a process selected from the group consisting of: modulating Vpr-related activity; preventing or treating a Vpr-related disease; diagnosing a Vpr-related disease; prognosticating a Vpr-related disease; and detecting the presence of Vpr or a functional derivative thereof in a sample.

The invention further provides a use of the above- mentioned compound or composition for preparation of a medicament for preventing or treating a Vpr-related disease.

The invention further provides a method of identifying a modulator of a Vpr-related activity in vivo comprising: incubating a yeast cell capable of expressing a Vpr or a functional derivative thereof and in which a Vpr- related activity is detectable, with a candidate compound or

library thereof; determining whether the detectable Vpr- related activity is modulated; and identifying a candidate compound as a modulator of Vpr-related activity in vivo when the Vpr-related activity is measurably different in the presence of the compound as compared to in the absence thereof.

The invention further provides a method of identifying a compound capable of Vpr binding, the method comprising: incubating a yeast cell capable of expressing a Vpr or a functional derivative thereof and in which a Vpr- related activity is detectable, with a candidate compound or library thereof; determining whether the detectable Vpr- related activity is modulated; and identifying a candidate compound as a compound capable of Vpr binding when the Vpr- related activity is measurably different in the presence of the compound as compared to in the absence thereof. In an embodiment, the Vpr-related activity is an inhibition of cell growth.

The invention further provides a modulator of a Vpr function in a cell. In an embodiment, the modulator overcomes or inhibits at least one Vpr function in the cell. In an embodiment, inhibitor is a peptide according to the present invention.

The invention further provides a method of inhibiting or overcoming a Vpr-dependent inhibitory growth function in a cell comprising an administration to the cell of an efficient amount of the above-mentioned modulator.

The invention further provides a genetic selection assay to identify modulators of a Vpr biological function in vivo comprising: incubating a yeast cell which expresses Vpr or functional part thereof and in which a function of Vpr or functional part thereof is detectable, with a candidate compound or library thereof; detecting whether the detectable Vpr function is modulated; and identifying a candidate

compound as a modulator of Vpr function in vivo when the Vpr function is measurably different in the presence of the compound as compared to in the absence thereof. In an embodiment, the Vpr is HIV-1 Vpr.

In an embodiment, the Vpr function is a growth inhibiting function.

The invention further provides an isolated nucleic acid comprising a sequence that encodes the above-mentioned polypeptide or domain; as well as a corresponding vector comprising the nucleic acid operably linked to a transcriptional regulatory element. The invention further provides a host cell comprising the vector; as well as a method of producing the polypeptide and/or domain, comprising culturing the host cell under conditions permitting expression of the polypeptide and/or domain.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows one embodiment of a genetic selection of GST- peptide inhibiting Vpr-mediated yeast growth defect. A) HP16 strain of S. cerevisiae yeast was transformed with plasmids p424Gall-Vpr or p424Gall and grown in selective/Vpr non- inducible (trp-, raf+) or inducible (trp-, gal+) medium for two days. Yeast growth was then monitored by measuring each yeast cell culture density by spectrophotometric analysis at a wavelength of 600 (OD600). B) Yeast cultures grown in either Vpr non-inducible or inducible selective media were collected and lysed in NP-40 lysis buffer (in the presence of glass beads). Expression of Vpr in cell lysates was detected by western blotting with anti-Vpr antibodies. C) HIV-1 Vpr expressing HP16 yeasts were transformed with a GST-fused hexameric peptide library and selected in agar plates in Vpr inducible conditions (trp, ura, gal+). After two rounds of selection on agar plates, the growing clones were cultured in

doubly selected/Vpr-inducible liquid medium (trp, ura, gal+) and the growth of each yeast clone was evaluated after three days of incubation by measuring the cell density by spectrophotometry at a wavelength of 600 (OD600). The asterisk indicates that C10 was found to express two distinct GST peptides. These data are representative of results obtained in two independent experiments. D) Expression of GST-peptides and Vpr in yeast. HP16 yeasts co-transformed with Vpr and each GST-peptides were first cultured in Vpr-non-inducible selective medium for 3 days and then, grown in Vpr-inducible selective medium overnight. Yeasts were then collected, lysed and similar amounts of proteins (500 ug) were analyzed by SDS- PAGE and western blotting using specific anti-GST (upper panel) or anti-Vpr antibodies (lower panel). M: non- transformed yeast. The GST-p9 and plO were respectively expressed from two distinct GST-peptide PGK plasmids which contained DNA fragments encoding each of the two GST-peptides found in Clone 10 (C10) (as indicated in panel C).

Figure 2 shows the amino acid sequence of inhibitory GST- peptides according to certain embodiments of the invention. A) Plasmids encoding GST-peptides were isolated from yeast clones growing in the presence of Vpr and their nucleotide sequence was determined at the junction of GST and the peptide library.

Panel A shows a schematic structure of a GST-fused peptide with the deduced amino acid sequence of the peptide moieties below. The peptide library was linked to the C-terminus of the GST protein through a linker comprising six amino acids (KGLSGP [SEQ ID NO : 35] ) while the peptide C-terminus was linked, to a valine residue. B) Schematic representation of the computer-predicted amphipathic structure of peptide-4-16 and-18. Hydrophobicity plots were determined according to Kyte and Doolittle using the MacVector software (International Biotechnologies, New Haven, CT, U. S. A).

Figure 3 shows that selected GST-peptides inhibit specifically the effect of Vpr on cell growth and morphology in budding yeast. A) A panel of plasmids encoding GST-peptide or the GST control (as indicated on the left side of the panel) were transformed into S. cerevisiae HP16 strain that contained either p424Gall-Vpr or p424Gall (as indicated). Yeast co- transformants were selected and grown in non-inducible selective medium (trp-, ura-, Raf+) for two days. Similar amounts of yeast (0.5 OD600) were then serially (. lOx) diluted, spotted onto either Vpr non-inducible selective agar plates (trp-, ura-, Raf+) (left panel) or Vpr-inducible selective agar plates (trp-, ura-, gal+) (right panel) and incubated for 3 to 5 days to evaluate their growth rates. This data is representative of results obtained in two independent experiments. B) The S. cerevisiae HP16 strain expressing either GST alone (a), or both Vpr and GST (b) GST-pl6 (c) or GST-pl8 (d) were cultured in Vpr-inducible selective medium for 3 days and examined by light phase microscopy.

Figure 4 shows that selected GST-peptides interact directly with HIV-1 Vpr. HP16 yeast cells co-expressing HIV-1 Vpr and GST or GST-peptide (as indicated) were radio-labeled. with 35S- translabel for 6 hours and lysed with CHAPS lysis buffer. GST or GST-peptides complexes were then pulled down with glutathione-sepharoseT"-4B. Following extensive washes, protein complexes were eluted with 100 mM of gluthatione and the radiolabeled GST, GST-peptide complexes were separated by SDS-PAGE and revealed by autoradiography (panel A). In parallel, total amounts of Vpr in each sample were immunoprecipitated with anti-Vpr antibodies and analyzed by SDS-PAGE and autoradiography (panel B). As controls, yeast cells expressing or not Vpr (panels A and B, lanes 6 and 7) were also labeled and analyzed using the same procedure. The

positions of GST, GST-peptide and Vpr are indicated on the right side of the autoradiogram.

Figure 5 shows the effect of Vpr-binding GST-peptides on Vpr- mediated apoptosis and cell cycle G2 arrest upon infection of Jurkat cells with VSV-G-pseudotyped HIV-1 virus. CD4+ Jurkat T cell populations expressing GST-peptides including GST-p4,- pl2,-pl8 as well as a GST control were generated by electroporation of plasmids expressing GST or GST-peptides and selection with hygromycin B. (A) GST-peptide mRNA expression was detected by reverse transcription-PCR amplification using GST specific primers. To test the effect of each GST-peptide on Vpr-mediated apoptosis and cell cycle G2 arrest, each GST- peptide-expressing Jurkat cell population was infected with R+ or R VSV-G pseudotyped HIV-1 viruses at m. o. i. of 0.125 and 0.25. At 48 h post-infection, infected cells were collected and analyzed for Vpr-mediated apoptosis (B) and cell cycle G2 arrest (C). The data are representative of results obtained in three independent experiments. M, molecular mass markers. NC, negative control (no mRNA included).

Figure 6 shows that the expression of GST-peptides inhibits Vpr-mediated cell cycle G2 arrest in human 293T cells. A) Expression of GST and GST-peptides, including GST-p4,-pl2,- pl6 and-pl8 in 293T cells was detected by radio- immunoprecipitation using anti-GST antibodies. GST-p4 showed a slower migration (lane 2), while GST alone exhibited a faster migration (lane 6). B) Inhibitory effect of GST-peptides on Vpr-mediated G2 arrest. 293T cells were co-transfected with R- /GFP-or R+/GFP expression plasmids and different GST-peptide constructs (as indicated). GFP-positive cells were sorted by FACS and cell cycle profiles were analyzed by PI staining and flow cytometry 48h post-transfection. Dose-dependent inhibitory effect of GST-pl8 on Vpr-5 mediated G2 arrest. 293T

cells were co-transfected with R+/GFP and GST-p expression plasmids at different molar ratio (as indicated). At 48h post- transfection, a sample (1/4) of the cell population was used to analyze the cell cycle profile (C) while the remaining portion (3/4) of cells were used to perform radio- immunoprecipitation to detect Vpr and GST-pl8 proteins (as indicated) (D).

Figure 7 shows the intracellular localization of HIV-1 Vpr and GST-peptides. COS-7 cells were transfected with GST or GST-peptide alone (indicated in a to c) or co-expressed with GST, GST-pl6 or-pl8 and Vpr expression plasmids (indicated in d to i). Forty eight hours post-transfection, cells were fixed with acetone, incubated with goat anti-GST and/or rabbit anti- Vpr antibodies followed by labeling with fluorescein- conjugated mouse anti-goat and/or rhodamine-conjugated mouse anti-rabbit antibodies. Following several washes with PBS, cells were observed at 100x with oil emulsion on a Zeiss fluorescence microscope. A representative cell staining is shown for each transfection.

Figure 8 shows GST-pull down assay results evaluating the binding efficiency of GST-hexameric peptide fusions as described in Example 17.

Figure 9: Amino acid sequences of Vpr52-96 and anti-Vpr GST- peptides according to embodiments of the invention. The amino acid sequence of Vpr52-96 (SEQ ID NO: 36), pl8 (SEQ ID NO : 37) and control (SEQ ID NO : 38) peptides are presented. Vpr52- 96 corresponds to a polypeptide comprising the C-terminal part (45 amino acids) of HIV-1 Vpr protein. This peptide was shown to induce apoptosis by a mechanism that involves defects in mitochondrial membrane permeability (Jacotot, et al., 2001.

J Exp Med 193: 509-519). A domain containing a HFRIGCRHSRIG

(SEQ ID NO : 39) amino-acid sequence (underlined) was shown to be critical for Vpr pro-apototic activity. The pl8 peptide (13 amino acids) was chemically synthesized and is derived from the anti-HIV-1 GST-pl8 peptide fusion protein. The GST- pl8 protein was shown to inhibit Vpr-mediated cell cycle G2 arrest and apoptosis possibly by preventing critical protein- protein interactions required for Vpr biological activities (Yao X-J. et al., 2002. J. Biol Chem. 277: 48816-48826). The control peptide is identical to the pl8 peptide except for three tryptophan residues (W) that were substituted for alanine (A) residues, as indicated.

Figure 10: Inhibitory effect of pl8 peptide on Vpr52-96 induced apoptosis in Jurkat T cells.

A). To evaluate the apoptosis induced by Vpr52-96 peptide, 1 x 106 Jurkat cells were incubated with different concentration of Vpr52-96 polypeptide at 37°C for one hour. Cells were then washed and incubated in RPMI-1640 complete medium for 4 hours.

Apoptotic cells were detected using the Annexin V-FITC/ Propidium Iodide (PI) assay as described in (Yao X-J. et al., 2002. J. Biol Chem. 277: 48816-48826) as recommended by the manufacturer (Boehringer Mannheim Inc). Briefly, 0.5 x 106 Jurkat cells were washed once with PBS and resuspended in Annexin V binding buffer (2.5 g/ml of Annexin V-FITC, 10 mM HEPES-NaOH (pH7.4), 150 mM NaCl, 5mM KC1, 1 mM MgCl2,1. 8 mM CaCl2 and 1 g/ml PI). After 10-15 min incubation, cells were washed twice with binding buffer, resuspended in binding buffer and analyzed by FACScan. The percentage of annexin+/PI- (apoptotic cells) and annexin+/PI+ (dead cells) are presented.

B). To test the inhibitory effect of the pl8 peptide on Vpr52-96-induced apoptosis in Jurkat cells, 1 pM of R52-96 peptide was pre-incubated with different concentrations of pl8 or control peptide at 37°C for 30 min. The peptide-polypeptide Vpr 52-96 mix was then incubated with 1 x 106 Jurkat cells for

one hour at 37°C. Following several washes, Jurkat cells were incubated in RPMI-1640 complete medium for 4 hours, Apoptotic cells were detected by the Annexin V-FITC/PI assay as recommended by the manufacturer (Boehringer Mannheim Inc) and analyzed by FACS. The percentages of annexin+/PI-cells (apoptotic) in presence of increasing concentrations of pl8 or control peptides are presented.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawings, which is exemplary and should not be interpreted as limiting the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to lentiviral infections such as AIDS and related diseases and to the causative agents thereof, lentiviruses and more particularly HIV and related viruses. The present invention also relates to modulators of the Vpr-based function in cells, to assays for screening and identifying same and to the use of such modulators and derivatives thereof to treat or prevent this Vpr-based growth arrest and/or apoptosis in lentiviruses.

In a further embodiment, the invention relates to genetic selection of peptide inhibitors of Vpr, e. g. human immunodeficiency virus type 1 Vpr.

In the present study, we utilized a yeast glutathione-S-transferase (GST) -fused hexameric peptide library screening system to select a panel of GST-fused peptides that have the capacity of inhibiting to different extent the Vpr-mediated growth arrest phenotype in S. cerevisiae budding yeasts. Sequence analysis reveals that a common di-tryptophan amino acid motif is conserved in the

inhibitory peptides, suggesting that this motif is critical for the ability of GST-peptides to interfere with HIV-1 Vpr activity. Mechanistic analyses indeed reveal that the peptide- mediated growth arrest inhibition is not the result of a lack of synthesis nor degradation of Vpr, but is mediated via a direct interaction between GST-peptide fusion proteins and Vpr. Expression of Vpr-binding GST-fused peptides in human 293 T cells is shown herein to inhibit Vpr-mediated cell cycle G2 arrest. Furthermore, intracellular localization analysis revealed that the inhibitory GST-peptides co-localized with HIV-1 Vpr in mammalian cells and interfered with the protein nuclear translocation. This yeast genetic selection system represents a novel approach to identify peptide inhibitors of Vpr (e. g. HIV-1 Vpr) biological activities and provides information about amino acid motifs that may be present in Vpr-interacting cellular factors or downstream effectors.

It is herein demonstrated that particular agents (e. g. peptides) can modulate, for example, overcome the growth inhibition properties of Vpr.

The present invention, in one embodiment, relates to these Vpr-inhibiting peptides or compounds. In another embodiment, the invention relates to a genetic selection assay to identify modulators of a Vpr biological function in vivo.

In an embodiment, such modulators are inhibitors of a Vpr function. In yet another embodiment, the present invention relates to the use of agents or compounds identified as such or derivatives of such agents, to treat or prevent a lentiviral infection and more particularly an HIV infection.

The present invention also relates to the manufacture of a medicament to treat or prevent such lentiviral infections comprising a mixing of the above-noted agent together with a pharmaceutical carrier. In a particular embodiment of the present invention, the agent is derivatized or modified

according to known methods prior to a mixing with a pharmaceutical carrier.

Herein, we expressed a library of glutathione-S- transferase (GST) -fused unconstrained hexamer peptides in Saccharomyces cerevisiae to identify GST-peptides capable of reestablishing cell proliferation under conditions in which Vpr was expressed. After re-selection, a number of selected GST-peptides, but not GST control, were found to overcome to different extent Vpr-mediated growth arrest in budding yeasts.

Amino-acid analysis of the inhibitory peptide sequences revealed the conservation of a di-tryptophan (diW) motif. Di- tryptophan-containing GST-peptides were found to directly interact with Vpr in GST pull-down assays, and their level of interaction correlated well with their ability to overcome Vpr-mediated growth arrest. Vpr-binding GST-peptides were also found to inhibit Vpr-mediated G2 arrest in mammalian cells. Furthermore, they co-localized with Vpr and interfered with its nuclear translocation. Overall, this study defines a class of di-tryptophan-containing peptides that directly interacts with HIV-1 Vpr and inhibits its biological activities in budding yeast and human cells.

While the present invention has been exemplified with peptides, the present invention should not be so limited, since it will be recognized by a person of ordinary skill that the genetic screening assays of the present invention can be screened with libraries of compounds at large, and should thus not be limited to libraries of peptides.

In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow.

Nucleotide sequences are presented herein by single strand, in the 5'to 3'direction, from left to right, using the one letter nucleotide symbols as commonly used in the art

and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.

Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).

The term"molecule"or"agent"is used herein in a broad sense and is intended to include natural molecules, synthetic molecules, and mixture of natural and synthetic molecules. The term"molecule"is also meant to cover a mixture of more than one molecule such as for example pools or libraries of molecules. Non-limiting examples of molecules include chemicals, biological macromolecules, cell extracts and the like. The term"compound"is used herein interchangeably with molecule and is similarly defined.

As used herein the recitation"indicator cells" refers to cells that express a Vpr protein or part thereof which is sufficient to enable an observation and determination of a modulation of a Vpr-dependent biological activity in vivo. Such indicator cells can be used in the screening assays of the present invention. In certain embodiments, the indicator cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of Vpr. As exemplified herein, the indicator cells are yeast cells. In one particular embodiment, the indicator cell is a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (Ausubel et al. , 1994,

supra) and can be used to test a compound or a library thereof. In one embodiment, a reporter gene encoding a selectable marker or an assayable protein can be operably linked to a control element such that expression of the selectable marker or assayable protein is dependent on an interaction of Vpr with a predetermined factor or as in the case of peptides with an expression library thereof. The indicator cells of the present invention could be used to rapidly screen at high-throughput a vast array of test molecules.

A cell (e. g. a host cell or indicator cell), tissue, organ, or organism into which has been introduced a foreign nucleic acid (e. g. exogenous or heterologous DNA [e. g. a DNA construct] ), is considered"transformed","transfected", or "transgenic". A transgenic or transformed cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing a transgenic organism as a parent and exhibiting an altered phenotype resulting from the presence of a recombinant nucleic acid construct. A transgenic organism is therefore an organism that has been transformed with a heterologous nucleic acid, or the progeny of such an organism that includes the transgene. The transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained on a episomal element such as a plasmid. With respect to eukaryotic cells, a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA. Transfection

methods are well known in the art (Sambrook et al., 1989, supra; Ausubel et al. , 1994 supra).

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (such as resistance to antibiotics) may be introduced into the host cells along with the gene of interest. As used herein, the term"selectable marker"is used broadly to refer to markers which confer an identifiable trait to the indicator cell. Non-limiting example of selectable markers include markers affecting viability, metabolism, proliferation, morphology and the like. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the peptide compound or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (cells that have incorporated the selectable marker gene will survive, while the other cells die).

The term"in vivo"is used herein to refer especially to an effect observed in a cell. This terminology thus relates to a physiologically-relevant effect of a Vpr on a cell. Of course, this definition should not be interpreted as excluding"in vivo"in the sense of an animal, for example.

As exemplified herein below in one embodiment, at least one of the Vpr interaction domains, peptides or compounds of the present invention may be provided as a fusion protein. The design of constructs therefor and the expression and production of fusion proteins are exemplified herein and are well known in the art (Sambrook et al. , 1989, supra; and Ausubel et al. , 1994, supra).

Non-limiting examples of such fusion proteins include a hemaglutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions. In certain embodiments, it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused. Such protease cleavage sites between two heterologously fused polypeptides are well known in the art.

In certain embodiments, it might also be beneficial to fuse the interaction domains of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell. Signal peptides from diverse organisms are well known in the art. Bacterial OmpA and yeast Suc2 are two non-limiting examples of proteins containing signal sequences. In certain embodiments, it might also be beneficial to introduce a linker (commonly known) between the interaction domain and the heterologous polypeptide portion.

As used herein, the terms"molecule"or"compound" are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term "molecule"therefore denotes for examples macromolecules, cell or tissue extracts (from plants or animals). Non-limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents.

The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling. The terms"rationally selected"or "rationally designed"are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention. More particularly, such rationally selected or designed compounds can be chosen based on the configuration of the peptides of the present invention.

As will be understood by the person of ordinary skill,

macromolecules having non-naturally occurring modifications are also within the scope of the term"molecule". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above. Similarly, in a preferred embodiment, the peptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain. The molecules identified in accordance with the teachings of the present invention have a therapeutic value reducing the infectivity of HIV (and other lentiviruses) and for the treatment of AIDS.

As used herein, agonists and antagonists of Vpr biological activity also include potentiators of known compounds with such agonist or antagonist properties. In one embodiment, antagonists of the growth arrest property of Vpr, for example, can be detected by contacting the indicator cell with a compound or mixture or library of molecules for a fixed period of time and assessing the growth of the cell.

In one embodiment, the level of gene expression of a reporter gene (e. g. the level of luciferase, or (3-gal, produced) within the treated cells can be compared to that of the reporter gene in the absence of the molecule (s). The difference between the levels of gene expression indicates whether the molecule (s) of interest agonizes the aforementioned interaction. The magnitude of the level of reporter gene product expressed (treated vs. untreated cells) provides a relative indication of the strength of that molecule (s) as an agonist. The same type of approach can also be used in the presence of an antagonist (s).

Alternatively, an indicator cell in accordance with the present invention can be used to identify antagonists in the presence of an agonist.

It shall be understood that the"in vivo" experimental model can also be used to carry out an"in vitro" assay. For example, cellular extracts from the indicator cells can be prepared and used in an in vitro test.

For certainty, as used herein"Vpr"refers herein to polypeptides or nucleic acid molecules from lentiviruses, and particularly primate lentiviruses, e. g. HIV-1, HIV-2, SIV, and to different isolates thereof. More particularly, the Vpr sequence of the present invention should preferably be associated with a biological activity which is assessable in a cell.

For certainty, the sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) interaction domain. While a"defective"Vpr could be used, the Vpr used should retain at least one biological activity of the present invention: It will be clear to the person of ordinary skill that whether an interaction domain of the present invention, variant, derivative, or fragment thereof retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.

The invention thus relates to compounds/peptides/ peptide comppounds capable of modulating and/or binding to a Vpr or a Vpr-related compound, and their use in modulating, e. g. inhibiting, Vpr-related activity, such as growth arrest, apoptosis and lentiviral pathogenesis. In embodiments, the compounds of the invention may be used for prevention and/or treatment of Vpr-related disease.

"Vpr-related compounds"refers to compounds which are structurally and/or functionally related to a Vpr. Such compounds include homologs, variants or fragments of a Vpr

protein which retain Vpr activity, as well as"functional derivatives"of Vpr. Such compounds may comprise a peptide which is substantially identical to a Vpr protein or fragment thereof, e. g. substantially identical to the HIV-1 Vpr protein sequence (or a fragment thereof) set forth below and in SEQ ID NO : 41, to the HIV-2 Vpr protein sequence (or a fragment thereof) set forth below and in SEQ ID NO : 43, or to the SIV Vpr protein sequence (or a fragment thereof) set forth below and in SEQ ID NO: 45. Similarly, such compounds include peptides or proteins encoded by a nucleic acid sequence which is substantially identical to, or is related by hybridization criteria (see below) to a nucleic acid sequence capable of encoding a Vpr, such as the HIV-1 Vpr cDNA set forth below and in SEQ ID NO : 40, the HIV-2 Vpr cDNA set forth below and in SEQ ID NO : 42, or the SIV Vpr cDNA set forth below and in SEQ ID NO : 44. Such compounds further include the region of residues 52-96 of HIV-1 Vpr as described in the Examples below.

Therefore, in an aspect the invention provides a method for preventing and/or treating a Vpr-related disease in a subject, by administering to the subject a compound of the invention. In an embodiment, the subject is a mammal, in a further embodiment, a human.

"Vpr-related disease"as used herein refers to any disease in which Vpr activity may play a role in its onset and pathogenesis. In an embodiment, such a disease is associated with a lentiviral infection, in an embodiment, a lentiviral infection. Such lentiviruses include HIV, e. g. HIV-1 and HIV- 2, and SIV. In an embodiment such a disease is AIDS.

The terms"Vpr activity", "Vpr-related activity"or "biological activity of Vpr"refer to any detectable and/or measurable activity associated with Vpr or a functional derivative thereof. More particularly, it refers to any detectable activity which can be determined in a cell. A non-

limiting example of such a cellular activity includes growth arrest, inhibition of passage through the G2 stage, cell cycle arrest, cell killing, apoptosis, induction of a cascade of phosphorylation/phosphatase events involving cell cycle genes, binding to factors involved in the cell cycle, or in growth arrest, or apoptosis, or mitochondrial membrane permeability, effect on compartmentalization of cell cycle regulators, and lentiviral pathogenesis. Preferably, the activity which is measured is of physiological relevance. Vpr biological activity is not limited, however, to these most important biological activities herein listed or known. Biological activities may also include simple binding or pKa analysis of Vpr with compounds, substrates, interacting proteins, and the like. For example, measuring the effect of a test compound on its ability to increase or inhibit such Vpr binding or interaction is measuring a biological activity of Vpr according to this invention. Vpr biological activity thus also includes any standard biochemical measurement of Vpr such as conformational changes, phosphorylation status or any other feature of the protein that can be measured with techniques known in the art. Of course, if a biochemical measurement or binding activity is measured, a confirmation or verification of the effect on a physiologically relevant activity should be assessed.

Generally, high throughput screens for Vpr candidates or test compounds or agents (e. g. , peptides, peptidomimetics, small molecules or other drugs) may be based on assays which measure biological activity of Vpr. The invention therefore provides a method (also referred to herein as a"screening assay") for identifying modulators, which have a stimulatory or preferably an inhibitory effect on Vpr or functional part thereof biological activity or expression, or which bind to or interact with Vpr protein, or which have a stimulatory or preferably inhibitory effect on, for example,

the expression or activity of Vpr interacting proteins (targets) or substrates.

As exemplified herein below, the interaction domains of the present invention (which for example include Vpr and other interacting factors including the peptides of the present invention) can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds or an improvement of their activity.

In one aspect the invention provides nucleic acids that encode peptide compounds of the invention. Such nucleic acids may be introduced into cells for expression using standard recombinant techniques for stable or transient expression. Nucleic acid molecules of the invention may include any chain of two or more nucleotides including naturally occurring or non-naturally occurring nucleotides or nucleotide analogues.

Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term"recombinant" means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term"recombinant"when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.

The term"recombinant"when made in reference to genetic composition refers to a gamete or progeny or cell or genome with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated

at a different location in nature. Referring to a nucleic acid construct as'recombinant'therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i. e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.

The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms.

Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.

As used herein, "isolated nucleic acid molecule", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA and RNA molecules purified from their natural environment.

The term"DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.

The terminology"amplification pair"refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or

nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.

As used herein, the term"physiologically relevant" is meant to describe a biological function which occurs, is observable or is assayable in its natural setting or at least in a cell.

Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al. , 1989, Molecular Cloning-A Laboratory Manual, 2nd Edition, CSH laboratories; Ausubel et aI., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons lnc., N. Y.).

The term"DNA"molecule or sequence (as well as sometimes the term"oligonucleotide") refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), often in a double- stranded form, and comprises or includes a"regulatory element"according to the present invention, as the term is defined herein. The term"oligonucleotide"or"DNA"can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5'to 3'direction.

A"substantially identical"sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, as discussed herein, or by one or more non-conservative substitutions, deletion, or insertions located at positions of the sequence that do not destroy the biological function of the test compound. Such a sequence can be at least 60% or 75%, or more generally at least 80%, 85%, 90%, or 95%, or as much as 99% identical at the amino acid or nucleotide level to the sequence used for comparison. Sequence identity can be readily measured using publicly available sequence analysis software (e. g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, or BLAST software available from the National Library of Medicine).

Examples of useful software include the programs, Pile-up and PrettyBox. Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications.

"Homology"and"homologous"refers to sequence similarity between two peptides or two nucleic acid molecules.

Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is"homologous"to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term'homologous'does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional

motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.

An"unrelated"or"non-homologous"sequence shares less than 40% identity, though preferably less than about 25 % identity, with sequences of the invention.

Substantially complementary nucleic acids are nucleic acids in which the"complement"of one molecule is substantially identical to the other molecule. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U. S. A. ). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www. ncbi. nlm. nih. gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are

extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci.

USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P (N) ), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. "Nucleic acid hybridization"refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure.

Hybridization to filter-bound sequences under moderately

stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0. 1% SDS at 42°C (see Ausubel, et al.

(eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. , and John Wiley & Sons, Inc. , New York, at p. 2.10. 3; Sambrook et al. , supra).

Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0. 1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).

Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2"Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23: 295 and Moran et al. , 1987, Nucleic Acids Res. , 14: 5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.

The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic

acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.

Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection.

Furthermore, it enables automation. Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides.

It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

The above-mentioned labels, markers and labelling methods may in embodiments also be used in respect of the detection, diagnostic and prognostic methods described herein, whereby the label is associated with (e. g. attached to) a compound of the invention and thus may be used to detect its presence, e. g. in a mixture or binding complex.

As used herein, the term"gene"is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A"structural gene"defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein.

A"heterologous" (i. e. a heterologous gene) region of a DNA molecule is a subsegment segment of DNA within a larger segment that is not found in association therewith in

nature. The term"heterologous"can be similarly used to define two polypeptidic segments not joined together in nature. Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, p-galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.

The term"vector"is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.

The term"expression"defines the process by which a structural gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.

The terminology"expression vector"defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host. The cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences. Typically, expression vectors are prokaryote specific or eukaryote specific although shuffle vectors are also widely available.

Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuffle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue- specificity elements, and/or translational initiation and termination sites.

The DNA construct can be a vector comprising a

promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule. "Promoter"refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. For purposes of the present invention, the promoter is bound at its 3'terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

Eukaryotic promoters will often, but not always, contain "TATA"boxes and"CCAT"boxes. Prokaryotic promoters contain- 10 and-35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine- Dalgarno sequences, which serve as ribosome binding sequences during translation initiation.

In another aspect of the invention, an isolated nucleic acid, for example a nucleic acid sequence encoding a peptide compound of the invention, or homolog, fragment or variant thereof, may further be incorporated into a recombinant expression vector. In an embodiment, the vector will comprise transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence capable of encoding a peptide compound, polypeptide or domain of the invention. A first nucleic acid sequence is "operably-linked"with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence

if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element"is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked.

The recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for selectable markers and reporter genes are well known to persons skilled in the art.

A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism.

Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms "host cell"and"recombinant host cell"are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms"transformation"and"transfection"refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral- mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing DNA into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy for a Vpr-related disease.

As used herein, the designation"functional derivative"denotes, in the context of a functional derivative of a sequence whether an nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivatives or may be prepared synthetically.

Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. In one particular embodiment, the sequence of a

peptide of the present invention is modified so as to render same more resistant to degradation. Methods and modifications of peptides to suit particular needs are well-known in the art. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid as chemico- physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives"is intended to include"fragments", "segments","variants","analogs"or"chemical derivatives"of the subject matter of the present invention.

Thus, the term"variant"refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.

The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.

As used herein, "chemical derivatives"is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i. e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.

The term"allele"defines an alternative form of a gene which occupies a given locus on a chromosome.

As commonly known, a"mutation"is a detectable

change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist. The result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.

In one aspect, the invention provides compounds/peptides/peptide compounds, such as compounds capable of Vpr modulation and/or binding, that are purified, isolated or substantially pure. A compound is"substantially pure"when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75% or over 90%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesised or produced by recombinant technology will generally be substantially free from its naturally associated components. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i. e. , covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis.

Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.

Compounds of the invention can be prepared, for example, by replacing, deleting, or inserting an amino acid residue of a compound capable of Vpr modulation and/or binding or a domain of the invention, with other conservative amino

acid residues, i. e. , residues having similar physical, biological, or chemical properties, and screening for biological function. It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. The peptides, ligands and domains of the present invention also extend to biologically equivalent peptides, ligands and domains that differ from a portion of the sequence of novel ligands of the present invention by conservative amino acid substitutions. As used herein, the term"conserved amino acid substitutions"refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e. g. , within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about-1.6 such as Tyr (-1.3) or Pro (- 1.6) s are assigned to amino acid residues (as detailed in United States Patent No. 4,554, 101, incorporated herein by reference): Arg (+3.0) ; Lys (+3.0) ; Asp (+3.0) ; Glu (+3. 0) ; Ser (+0.3) ; Asn (+0.2) ; Gln (+0.2) ; Gly (0); Pro (-0.5) ; Thr (-0.4) ; Ala (-0.5) ; His (-0.5) ; Cys (-1.0) ; Met (-1.3) ; Val (- 1.5) ; Leu (-1.8) ; Ile (-1.8) ; Tyr (-2.3) ; Phe (-2.5) ; and Trp (-3. 4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is

substituted for another having a similar hydropathic index (e. g. , within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5) ; Val (+4.2) ; Leu (+3.8) ; Phe (+2.8) ; Cys (+2.5) ; Met (+1.9) ; Ala (+1. 8); Gly (-0.4) ; Thr (-0.7) ; Ser (-0.8) ; Trp (-0.9) ; Tyr (-1.3) ; Pro (-1.6) ; His (-3.2) ; Glu (-3.5) ; Gln (-3.5) ; Asp (-3.5) ; Asn (-3.5) ; Lys (-3.9) ; and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally- occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally- occurring non-genetically encoded amino acids include beta- alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N- methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3- benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine, 4- fluorophenylalanine, penicillamine, 1,2, 3,4-tetrahydro- isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid,

p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta- amino valeric acid, or 2,3-diaminobutyric acid.

In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge.

Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al.

(J. Mol. Bio. 179: 125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys.

Non-genetically encoded hydrophobic amino acids include t- butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as-OH,-SH,-CN,-F,-Cl,-Br,-I,-NO2,- <BR> <BR> <BR> <BR> NO,-NH2,-NHR,-NRR,-C (O) R,--C (O) OH, -C (O) OR, -C (O) NH2,-<BR> <BR> <BR> <BR> <BR> <BR> <BR> C (O) NHR, -C (O) NRR, etc. , where R is independently (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkenyl, substituted (C1-C6) alkenyl, (C1-C6) alkynyl, substituted (C1-C6) alkynyl, (C5-C20) aryl, substituted (C5-C20) aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl.

Genetically encoded aromatic amino acids include Phe, Tyr, and

Tryp, while non-genetically encoded aromatic amino acids include phenylglycine, 2-napthylalanine, beta-2- thienylalanine, 1, 2,3, 4-tetrahydro-isoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine3- fluorophenylalanine, and 4-fluorophenylalanine.

An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i. e. , the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met, while non-genetically encoded apolar amino acids include cyclohexylalanine. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile, while non-genetically encoded aliphatic amino acids include norleucine.

A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.

Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln, while non-genetically encoded polar amino acids include citrulline, N-acetyl lysine, and methionine sulfoxide.

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic

amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.

The above classifications are not absolute and an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.

Conservative changes can also include the substitution of a chemically derivatised moiety for a non- derivatised residue, by for example, reaction of a functional side group of an amino acid. Thus, these substitutions can include compounds whose free amino groups have been derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Similarly, free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides, and side chains can be derivatized to form 0-acyl or 0-alkyl derivatives for free hydroxyl groups or N-im-benzylhistidine for the imidazole nitrogen of histidine. Peptide analogues also include amino acids that have been chemically altered, for example, by methylation, by amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, or ethylene diamine, or acylation or methylation of an amino acid side chain (such as acylation of the epsilon amino group of lysine). Peptide analogues can also include replacement of the amide linkage in the peptide with a substituted amide (for example, groups of the formula-C (0)-NR, where R is (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, substituted (C1-C6)

alkyl, substituted (C1-C6) alkenyl, or substituted (C1-C6) alkynyl) or isostere of an amide linkage (for example,-CH2NH-, -CH2S,-CH2CH2-,-CH=CH- (cis and trans),-C (O) CH2-,-CH (OH) CH2- , or-CH2SO-).

The term"alkyl"refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, etc. The alkyl groups can be (C1-C6) alkyl, or (C1-C3) alkyl. A"substituted alkyl"has substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups), and esters (including alkyloxycarbonyl-and aryloxycarbonyl groups) ), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters),-CF3, - CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls,-CF3,-CN, and the like.

The terms"alkenyl"and"alkynyl"refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. An"alkenyl" is an unsaturated branched, straight chain, or cyclic hydrocarbon radical with at least one carbon-carbon double bond. The radical can be in either the cis or trans conformation about the double bond (s). Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, pentenyl, hexenyl, etc. An"alkynyl"is an unsaturated branched, straight chain, or cyclic hydrocarbon radical with at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, etc.

In alternative embodiments, compounds capable of Vpr modulation and/or binding can be produced by substitution of either/or (i) side chains, (ii) backbone, or (iii) ionic interaction within a peptide. Additionally, structural or functional analogs can include 1) homologs of the peptidic compounds capable of Vpr modulation and/or binding generated by peptidomimicry and 2) analogs where the sequence/structure of the compounds capable of Vpr modulation and/or binding is introduced in a larger protein to convey Vpr binding to that protein.

The compounds capable of Vpr modulation and/or binding, peptides and domains of the invention may be covalently linked, for example, by polymerisation or conjugation, to form homopolymers or heteropolymers. Spacers and linkers, typically composed of small neutral molecules, such as amino acids that are uncharged under physiological conditions, can be used. Linkages can be achieved in a number of ways. For example, cysteine residues can be added at the peptide termini, and multiple peptides can be covalently

bonded by controlled oxidation. Alternatively, heterobifunctional agents, such as disulfide/amide forming agents or thioether/amide forming agents can be used.

Peptides or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesis using solution or solid phase synthesis methodology.

Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA technology using standard methods.

As the compounds of the invention have been shown herein to possess Vpr binding activity, the compounds of the invention may be used to detect the presence of a Vpr or Vpr- related compound in a sample. In an embodiment such a sample is a biological sample, such as a cell (e. g. an immune cell) tissue or body fluid of a subject, including blood. The compounds of the invention may also be used for diagnosing a Vpr-related disease, and/or for prognosticating a Vpr-related disease, in the latter case based on determining the level of Vpr in a sample from a subject and comparing to a corressponding Vpr level determined in the subject at an earlier time.

In an embodiment, the level of Vpr measured in a sample of a subject may be compared to an established standard of Vpr.

In an embodiment, the level of Vpr may be compared to a corresponding level of Vpr measured in a cell tissue or body fluid of a control subject. In an embodiment, the control subject is an age-and/or weight-matched subject.

The peptide compounds of the invention may be introduced into a cell by the introduction of suitable peptide-encoding nucleic acids into the cell. Various methods of introducing nucleic acids into a cell may be used, examples of which are described below. Methods such as the gene

therapy methods discussed below may be used in this regard.

The method may also comprise administering to an area or tissue a cell comprising such a peptide-encoding nucleic acid, via for example transplantation or introduction of a suitable cell or precursor thereto (e. g. a stem cell) comprising such a peptide-encoding nucleic acid.

A further aspect of the present invention is the treatment of a Vpr-related disease by administering to a subject a nucleic acid molecule encoding a peptide compound of the invention. Suitable methods of administration include gene therapy methods.

A nucleic acid of the invention may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e. g. , Acsadi et al. (1991) Nature 332: 815-818 ; Wolff et al. (1990) Science 247: 1465-1468). A delivery apparatus (e. g. , a"gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e. g. , from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem.

263: 14621; Wilson el al. (1992) J. Biol. Chem. 267: 963-967; and U. S. Pat. No. 5,166, 320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel el al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850;

Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2122- 2126).

Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D.

(1990) Blood 76: 271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds. ) Greene Publishing Associates, (1989), Sections 9.10-9. 14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include . psi. Crip,. psi. Cre,. psi. 2 and. psi. Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl.

Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl.

Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc.

Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc.

Natl. Acad. Sci. USA 88: 8039-8043; Ferry et al. (1991) Proc.

Natl. Acad. Sci. USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl.

Acad. Sci. USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci.

USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104- 4115; U. S. Pat. No. 4,868, 116; U. S. Pat. No. 4,980, 286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

For use as a gene therapy vector, the genome of an adenovirus may be manipulated so that it encodes and expresses a peptide compound of the invention, but is inactivated in terms of its ability to replicate in a normal lytic viral life

cycle. See for example Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; and Rosenfeld et al. (1992) Cell 68: 143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e. g. , Ad2, Ad3, Ad7 etc. ) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad.

Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin el al. (1992) Proc. Natl. Acad. Sci. USA 89: 2581- 2584).

Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes.

AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol.

(1992) 158: 97-129). AAV may be used to integrate DNA into non- dividing cells (see for example Flotte et al. (1992) Am. J.

Respir. Cell. Mol. Biol. 7: 349-356; Samulski et al. (1989) J.

Virol. 63: 3822-3828; and McLaughlin et al. (1989) J. Virol.

62: 1963-1973). An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4: 2072-2081; Wondisford et al.

(1988) Mol. Endocrinol. 2: 32-39; Tratschin et al. (1984) J.

Virol. 51: 611-619; and Flotte et al. (1993) J. Biol. Chem.

268: 3781-3790). Lentiviral gene therapy vectors may also be adapted for use in the invention.

General methods for gene therapy are known in the art. See for example, U. S. Pat. No. 5,399, 346 by Anderson et al. A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al.

Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W. , et al., Blood 78: 1132-1139 (1991); Anderson, Science 288: 627-9 (2000); and, Cavazzana-Calvo et al., Science 288: 669-72 (2000)).

The invention further relates to transplantation methods, to introduce into a subject a cell comprising a nucleic acid capable of encoding a peptide compound of the invention. The nucleic acid may be present in a vector as described above, the vector being introduced into the cell in vitro, using for example the methods described above. In an embodiment, the cell is autologous, and is obtained from the subject. In embodiments, the cell is allogeneic or xenogeneic.

In embodiments, the therapeutic and diagnostic methods may be used in conjunction with each other. For example, a subject suffering from a Vpr-related disease may be identified or diagnosed using a diagnostic method and then subsequently treated using a therapeutic method. Further, the therapeutic method may be used for treatment in conjunction with the diagnostic or prognostic method which is used to monitor the progress of the treatment.

The invention further provides a use of a compound of the invention for the modulation (e. g. inhibition) of Vpr activity, preventing or treating a Vpr-related disease, diagnosing and/or prognosticating a Vpr-related disease, and/or detecting the presence of Vpr in a sample.

The invention further provides a use of an Vpr or a Vpr-related compound for the preparation of a medicament for the prevention and/or treatment of a Vpr-related disease.

The invention further provides commercial packages comprising a compound of the invention, or the above-mentioned composition, together with instructions for the modulation (e. g. inhibition) of Vpr-related activity, preventing or treating a Vpr-related disease, diagnosing and/or prognosticating a Vpr-related disease, and/or detecting the presence of Vpr in a sample.

In accordance with the present invention, there is also provided a method for identifying, from a library of compounds, a compound with therapeutic effect on lentiviral infections and more particularly on Vpr-based growth dependent defects in cells comprising providing a screening assay comprising a measurable biological activity of Vpr protein or biologically active fragment thereof; contacting the screening assay with a test compound; and detecting if the test compound modulates the biological activity of Vpr protein; wherein a test compound which overcomes or inhibits the biological activity is a compound with this therapeutic effect.

Compounds of the invention can be provided alone or in combination or conjugation with other compounds (for example, toxins, growth factors, anti-apoptotic agents, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to a mammal, e. g. a human. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from diseases such as AIDS. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.

Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration,

formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

If desired, treatment with a compound according to the invention may be combined with more traditional therapies for the disease such as, for example, nucleotide analogs and protease inhibitors.

For therapeutic or prophylactic compositions, the compounds may be administered to an individual in an amount sufficient to stop or slow the growth arrest or destruction of cells (such as due to lentiviral infection). Amounts considered sufficient will vary according to the specific compound used, the mode of administration, the stage and severity of the disease, the age, sex, and health of the individual being treated, and concurrent treatments. As a general rule, however, dosages can range from about microgram to about 100 mg per kg body weight of a patient for an initial dosage, with subsequent adjustments depending on the patient's response.

In general, compounds of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i. e. , the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.

The invention provides pharmaceutical compositions (medicaments) containing (comprising) a compound of the invention. In one embodiment, such compositions include a compound of the invention, in a therapeutically or prophylactically effective amount sufficient to inhibit a Vpr- related disease, and a pharmaceutically acceptable carrier. A "therapeutically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a compound of the invention, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.

A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A"prophylactically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting Vpr- related disease onset or progression. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according

to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

As used herein"pharmaceutically acceptable carrier" or"excipient"includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.

Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.

Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.

The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols

such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, a compound of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound (e. g. a compound of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. In accordance with an alternative aspect of the invention, a compound of the invention, may be formulated with one or more additional compounds that enhance its solubility.

For administration to humans (or other mammal), the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (e. g. type of agent...), the response and condition of the patient as well as the severity of the disease.

Compositions within the scope of the present invention should contain one active agent identified in accordance with the present invention in an amount effective to achieve an inhibitory effect on a lentivirus, HIV or related viruses while avoiding adverse side effects. For the administration of polypeptides, antagonists, agonists and the like, the amount administered should be chosen so as to avoid adverse side effects. The dosage of the agent identified in accordance with the present invention will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0. 001 to 50 mg/kg/day will be administered to the mammal. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed. , Mack Ed.).

EXAMPLES The present invention is illustrated in further detail by the following non-limiting examples.

Example 1: Plasmids, antisera and chemicals The H1V-1 Vpr yeast expression plasmid (p424Gall-Vpr) was constructed by inserting a PCR-generated BamHI-BamHI fragment containing Vpr sequence (44) into a high-copy yeast expression plasmid p424Gall, which harbors a galactose- inducible Gall promoter and a tryptophan selection marker (45). A library of unconstrained hexameric peptides fused to

the C-terminus of a GST inert carrier protein (>108 independent transformants) was generated in a plasmid containing a phosphoglycerate kinase (PGK) promoter and a URA3 selectable marker (see below for a description). The constitutive expression of GST-peptide fusion proteins is under the control of a PGK promoter (46). To generate a plasmid capable of expressing GST-peptides in mammalian cells, an EcoRI-XbaI fragment encoding GST-peptide was obtained from phosphoglycerate kinase-based yeast expression plasmids and cloned into the mammalian cell expression vector pHebo (70), which contains a SL3-3 murine leukemia retrovirus long terminal repeat (441/+33; +1 = site of transcription initiation (71) or pcDNA3.1 (Invitrogen). pHebo contains the Epstein-Barr virus origin P and directs the expression of the Escherichia. coli gene encoding hygromycin B phosphotransferase (HygR) using the herpes simplex virus thymidine kinase promoter and polyadenylation site. The Vpr/Green Fluorescent Protein (GFP) dual-expression plasmids, SVCMV-R+-GFP and SVCMV-R--GFP, were constructed by inserting a BamHI-BglII fragment containing a CMV promoter-GFP-polyA cassette derived from the pQBI25 plasmid, (Quantum Biotechnologies lnc) into the BamHl site of SVCMV-R+ or SVCMV- R-expression plasmids (44). These plasmids were used to analyze Vpr-mediated G2 arrest in transient expression assays.

The HIV-1 envelope-defective proviral constructs HxBRUR+/Env and HxBRUR/Env as well as the vesicular stomatitis virus envelope G glycoprotein expression plasmid, SVCMV-VSV-G, used in this study were previously described (24).

The rabbit anti-Vpr polyclonal serum was raised against bacterially expressed recombinant Vpr as described previously (47). The goat antibody directed against GST was purchased from Amersham Pharmacia Biotech Inc. Fluorescein- conjugated mouse anti-goat antibody and rhodamine-conjugated mouse anti-rabbit antibody were respectively purchased from

Sigma Inc and Jackson ImmunoResearch Laboratories Inc.

Galactose, raffinose, glucose and propidium iodide (PI) were purchased from Sigma Inc. The annexin V-fluorescein isothiocyanate kit was purchased from Roche Molecular Biochemicals.

Example 2: Hexamer library A peptide library was synthesized from an oligonucleotide containing 18 randomized nucleotides with SfiI and XbaI restriction sites at the 5'and 3'ends, respectively, 5'AGTAGGCCTGAGCGGCCCTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKGTCTAGAGG AT CCGC (SEQ ID NO : 46; 28, 48). Randomized codons, designated N-N-K where N is either A, C, G, T and K is G or T, produce a population of peptide sequences as described (48,28, 48).

From the NNK motif, 32 possible codons can be generated that encode all amino acids and only one of the 3 possible stop codons. An oligonucleotide with complementarity to the 3'end of the library oligonucleotide (5'GCGGATCCTCTAG [SEQ ID NO : 47] ) was annealed and the complete complementary strand was synthesized using the Klenow enzyme in the presence of all four deoxynucleotide-triphosphates. The resulting double- stranded product was restricted with SfiI and XbaI enzymes and inserted into identically restricted GST expression plasmid.

The library was transformed into F. coli and plasmid DNAs were harvested by alkaline lysis of bacterial colonies.

The library is composed of greater than 1 x 108 independent transformants and at least 95% of the plasmids contained hexamer-encoding GST fusions (S. Kurtz, K. Esposito, W. Tang, R. Menzel, supra). The hexamers are joined to the C-terminus of GST by a linker composed of Gly-Leu-Ser-Gly-Pro (SEQ ID NO : 48) residues. The nucleotide sequence at the GST-hexamer fusion junction is: AAA GGC CTG AGC GGC CCT (NNK) 6 GTC TAG A

(SEQ ID NO : 49). Thus the peptide sequence at the fusion junction is: K-G-L-S-G-P- (X) 6-V-stop (SEQ ID NO : 50).

Example 3: Yeast strains and genetic selection of anti-Vpr GST-peptides The S. cerevisiae yeast strain used in this study was the protease-deficient HP16 strain (MATa ura3-52 his3al leu2 trpla63 prbl-1122 pep4-3 prcl-407) (49). Plasmid transformation was performed using the lithium acetate method (50). To test for Vpr expression and Vpr-mediated phenotypic changes, HP16 cells transformed with p424Gall-Vpr were grown in selective medium (SC-trp; Adams et al. , 1997) containing galactose (2%). To co-express Vpr and the GST-hexamer peptides in yeast, the HP16 yeast strain harboring p424Gall-Vpr plasmid was re-transformed with the GST-peptide library, plated on solid medium with galactose selecting for Ura and Trp independence. After 6-8 days at 30°C, colonies were further analyzed. Positive GST-peptide plasmid DNAs were rescued as described previously (51). Sequencing analysis of GST-peptide was performed with an ABI PRISRM 310 Genetic analyzer (Applied Biosystems Inc. ) according to the manufacturer's instructions and the primer used for sequencing was 5'-TATAGCATGGCCTTGCAGG- 3' (SEQ ID NO : 51) corresponding to GST nucleotide position 593 to 611.

Example 4: Cell lines, transfections and cell cycle analysis Human CD4+ Jurkat T cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin and streptomycin. Jurkat cell lines stably expressing GST-peptides (GST, GST-p4,-pl2, and-pl8) were established by electroporating 10 ug of the linearized pHebo construct encoding the corresponding GST-peptide. Drug- resistant Jurkat cells were selected with growth medium containing hygromycin B at a concentration of 500 ug/ml.

Human epithelial 293T cells and the African green monkey kidney COS-7 cell line, were maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin and streptomycin. All cells were maintained at 37°C and 5% C02. For transfection of 293 cells and COS-7 cells, the standard calcium phosphate co-precipitation technique was used, as previously described (44). To analyze the cell cycle profile of transfected cells, 293T cells were co-transfected with Vpr/GFP dual-expression plasmid and increasing concentrations of GST-peptide expression plasmids.

Example 5: Virus Preparation and Viral Infection VSV-G pseudotyped HIV-1 virus preparations were generated by co-transfection of 293T cells with 10 pg of envelope-defective HIV-1 proviral DNA and 15 ug of the VSV-G expression plasmid SVCMV-VSV-G using the calcium phosphate co- precipitation method (15). Forty-eight hours post- transfection, cell-free supernatants were collected and ultracentrifuged at 45,000 rpm in a Beckman 60 Ti rotor for 1 h to pellet pseudotyped virus. Virus was resuspended in RPMI medium and filtered through a 0. 45-um-pore-size filter (Costar, Cambridge, MA). Virus stocks were titrated using the MAGI assay (47). To infect Jurkat cells, 0.25 x 106 cells were incubated with VSV-G pseudotyped HIV-1 virus at different multiplicities of infection (m. o. i. ) for 12 h. Infected cells were washed, cultured for another 36 h, and harvested for cell cycle analysis and detection of apoptosis.

Example 6: Cell Cycle Analysis and Annexin V/Propidium Iodide Double Staining To. detect apoptosis in infected cells, the annexin V-fluorescein isothiocyanate assay was performed as recommended by the manufacturer (Roche Molecular Biochemicals, Inc. ). Briefly, 0.25 x 106 infected cells were washed once

with PBS and then resuspended in annexin V binding buffer (2.5 ug/ml annexin V-fluorescein isothiocyanate, 10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KC1, 1 mM MgCl2, 1.8 mM CaCl2, and 1 ug/ml propidium iodide). After 10-15 min of incubation, stained cells were washed twice with binding buffer, resuspended in binding buffer containing 1% paraformaldehyde, and subsequently analyzed by FACScan. To perform cell cycle analysis, infected Jurkat cells were washed once with PBS and resuspended in 80% ethanol for 30 min on ice. For the 293T cells co-transfected with Vpr/GFP dual-expression plasmid and the GST-peptide expressor plasmids, GFP-positive cells were sorted out by fluorescence-activated cell sorter at 48 h post- transfection, washed once with PBS, and re-suspended in 80% ethanol for 30 min on ice. After an additional wash, cells were treated with 180 units/ml RNase A and subsequently stained with 30 ug/ml propidium iodide in 1 ml of PBS at 37 °C for 30 min. The DNA content was then analyzed by FACScan using the Consort 30 software. At least 10,000 events were collected for flow cytometry. Data acquisition and analysis were performed with the Cell Quest software (BD Biosciences). Samples were gated to exclude debris and clumps, and electronic compensation was used to remove residual spectral overlap. The mathematical model MODFIT was used to calculate the proportions of cells in the G2/M phases and G1 phase of the cell cycle. For simplicity, G2/M: G1 ratios have been provided.

Example 7: Immunoblot analysis, metabolic labeling and GST pull-down assay To examine expression of Vpr and/or GST-peptide fusions in yeast, HP16 co-transformants grown in suspension were pelleted by centrifugation at 1,500 rpm for 10 mm and lysed in lysis buffer (50mM Tris-HC1 pH 7.4, 400mM NaCl, 0. 5% NP-40) using three cycles of vortexing in the presence of

glass beads for 2 min at 4°C. Similar amounts of protein, as measured by protein quantitation assay (Bio-Rad kit), were loaded and separated using sodium dodecyl sulfate (SDS) -12. 5% polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (0. 45uM pore size; Bio-Rad Inc. ) by electroblotting. The membrane was incubated with goat polyclonal antibodies against GST or rabbit polyclonal anti- Vpr antibodies overnight at 4°C and then probed at room temperature with horseradish peroxidase-linked sheep anti-goat or anti-rabbit antibodies (Amersham Pharmacia Biotech Inc) for 3 hours. The membrane was washed extensively, and revealed using a sensitive enhanced chemiluminescence detection system (ECL detection kit, Amersham).

To detect the interaction of Vpr and GST-peptide in yeast, HP16 yeast cells expressing GST-peptide and Vpr were radio-labeled with-translabel (ICN Inc. ) and incubated for 6 hours at 30°C. Following labeling, yeast cells were lysed in 1.5 ml of CHAPS buffer (250mM NaCl, 25mM Tris-HCl pH 7.4, 5mM EDTA, 1% CHAPS (3 ( (3-cholamidopropyl)- dimethylammonio)-1-propane-sulfonate [Sigma Chemical Co.]) supplemented with a protease inhibitor cocktail (Roche) by vortexing with 0.6g of glass beads. Following a centrifugation at 30,000 rpm for 10 min at 4°C, supernatants were collected and used for GST pull-down assay (9). Briefly, 200 pl of yeast lysate was mixed with 700 pl of column buffer (20mM Tris-Cl pH 7.4, 200mM NaCl, 1mM EDTA supplemented with a protease inhibitor cocktail) and incubated with 80ul glutathione- Sepharose 4B beads (Amerham Pharmacia Biotech lnc) for 2 hours at 4°C. The gluthatione Sepharose 4B beads were sedimented by centrifugation, washed 3 times in 500p1 of column buffer and the radiolabeled protein complexes were eluted with 100 pi of glutathione buffer (lOOmM gluthathione (Roche), 120mM NaCl, 100mM Tris-HCI pH 8.5) by shaking at 4°C for 1 hour. Eluted protein complexes were loaded onto 12. 5%

SDS-PAGE and the presence of GST-peptides and Vpr was revealed by autoradiography. Meanwhile, to detect the total amounts of Vpr, 200 pl of yeast lysate was immunoprecipitated with anti- Vpr antibodies as described (55). Radiolabeled immunocomplexes were separated on a 12. 5% SDS-PAGE and analyzed by autoradiography.

Example 8: mRNA Measurements by Semi-quantitative Reverse Transcription-PCR To detect GST or GST-peptide mRNA expression in selected Jurkat T cell populations (p4, pl2, or pl8), total mRNAs from 2 x 106 cells were isolated using the High Pure RNA Isolation Kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. GST or GST-peptide mRNAs were then specifically amplified from 0.5 ug of each RNA sample using the QiagenO One-Step reverse transcription-PCR kit (Qiagen Inc) following the manufacturer's instructions. The 3'-primer used for reverse transcription and PCR was 5'- TCCAGGCACATTGGGTCCATGTA-3' (SEQ ID NO: 75), whereas the 5'- primer used for PCR was 5'-CAGTCTATGGCCATCATACGT-3' (SEQ ID NO : 76). This pair of primers corresponded to GST nucleotide positions 455 and 747 and was designed to amplify a 315-bp PCR product. Amplified PCR products were analyzed on a 1% agarose gel.

Example 9: Immunofluorescence Analysis and Laser Confocal Microscopy COS-7 cells were transfected with pcDNA-GST-peptide expression plasmids or co-transfected with pcDNA-GST-peptide and SVCMV-Vpr plasmids. Cells were fixed in acetone for 30 min at 4 °C 48 h post-transfection. Fixed cells were then incubated with goat anti-GST antibodies or/and rabbit anti-Vpr polyclonal serum in PBS containing 2% skim milk powder (Carnation, Nestle) for 12 h and labeled using a fluorescein-

conjugated mouse anti-goat antibody and/or a rhodamine- conjugated mouse anti-rabbit antibody. After several washes with PBS, cells were observed on a Zeiss fluorescence microscope using a 100X objective and oil emulsion. Confocal laser microscopy was performed on Zeiss LSM 410 (Carl Zeiss) equipped with a Plan-APOCHROMAT 63x oil immersion objective and an argon/krypton laser. The fluorescein isothiocyanate images were obtained by scanning the cells with the 488-nm laser and filtering the emission with 515-540-nm band-pass. For the lissamine/rhodamine images, the 568-nm laser was used in combination with a 575-640 nm band-pass filter. For each cell studied, additive signal through the cell whole thickness was first digitized. Then the confocal serial sections were scanned and analyzed.

Example 10: Selection of GST-fused hexameric peptides that inhibit Vpr mediated growth arrest in S. cerevisiae It has been shown that expression of HIV-1 Vpr in budding yeast causes cell growth arrest and cell killing by a mechanism that appears to involve mitochondrial dysfunction (40,35, 43). S. cerevisiae was chosen as a host to carry-out a genetic selection of agents interfering with Vpr biological activity. The strategy of screening and identifying such agents with S. cerevisiae was exemplified in a first stage with peptides for several reasons which include: 1) Vpr induces a well defined phenotype in budding yeast that constitutes a very stringent biological screen; 2) peptide interfering with Vpr biological activity in this system may provide valuable information on the molecular mechanism underlying Vpr-mediated cell killing and mitochondrial dysfunction; 3) the peptide library was initially designed and constructed for expression in budding yeast given the availability of proven expression, induction and selection

systems ; and 4) S. cerevisiae is a genetically well characterized organism whose genome DNA sequence is now readily available.

We generated a yeast expression plasmid (p424Gall- Vpr) encoding HIV-1 Vpr under the control of the galactose- inducible GAL1 promoter and transformed HP16 S. cerevisiae strain to confirm that Vpr induced a growth arrest in this budding yeast under our experimental conditions. In parallel, the empty p424Gall vector was transformed as a negative control. After growing for two days in Vpr non-inducible selective medium (Trp-, 2% raffinose (raf+)), yeast cells transformed with either the p424Gall-Vpr or p424Gall plasmid showed comparable growth rates (Fig. 1A, left panel). However, when grown in the Vpr inducible medium (Trp-, 2% galactose (gal+)), yeast cells transformed with p424Gall-Vpr exhibited a significant growth defect when compared to cells transformed with the p424Gall control plasmid (Fig. 1A, right panel). To test whether this effect correlated with Vpr induction, Vpr expression in both p424Gall-Vpr and p424Gall transformed yeasts was measured by western blotting using anti-Vpr antibodies. Results of Figure 1 B reveal that Vpr expression is only detected in the sample derived from p424Gall-Vpr transformed yeast cells grown in Vpr inducible medium (lane 4). These results confirm that expression of HIV-1 Vpr in S. cerevisiae HP16 cells induces a growth defect.

We next used this yeast system to screen a GST-fused hexameric peptide (GST-peptide) library to genetically select peptides inhibiting Vpr-induced growth arrest. The construction and the organization of the GST-fused hexameric peptide library are described in Example 2. The peptide library (a library of 1x108 GST-peptides) was transformed into HPI6 yeast cells harboring the p424Gall-Vpr expression plasmid. Transformants containing both Vpr and GST-peptide expression plasmids were selected for tryptophan and uracil

independence in medium supplemented with galactose (2%). After 6 to 8 days of culture, approximately fifty growing colonies were randomly selected from approximately 10 x 106 transformants. After multiple steps of re-selection, fourteen yeast clones, designated Cl, C2, C3, C4, C5, C6, C7, C10*, C11, C12, C13, C16, C17 and C18, were shown to proliferate effectively in Vpr-inducible medium (trp-, ura-, gal+), while yeasts expressing both Vpr and GST alone exhibited a strong growth defect. (Fig. 1C and data not shown for C17). These results indicate that these fourteen yeast clones presumably expressing a GST-peptide, overcame to different extent Vpr- induced growth arrest.

False positive results are common to genetic selections and screens that rely on transcription of one component of the genetic system. A likely false positive result in our system is a GST-peptide that interferes with Vpr expression from the p424Gall-Vpr plasmid. To test this possibility, each Vpr and GST-peptide co-transformed yeast clones were transferred to galactose-containing media for 1 day (to induce Vpr expression), cell lysates were prepared and the amounts of Vpr and GST peptides were evaluated by Western blot analysis. Results with anti-GST antibody reveal that similar amounts of GST or each of GST-peptides were expressed in co-transformed yeast cells (Fig. 1D, upper panel). In parallel, immunoblotting with anti-Vpr antibodies clearly shows that abundant and comparable amounts of Vpr are also detected in each co-transformant, but not in non-transformed yeast (Fig. 1D, lower panel). These results demonstrate that expression of the selected GST-peptides did not affect Vpr expression when they are co-expressed in yeast.

Example 11: Sequence analysis reveals that a common double- tryptophan motif is conserved in all selected GST-fused peptides

Plasmid DNAs encoding GST-peptides were rescued and their nucleotide sequence at the junction between GST and the hexameric peptide library was determined. We found that the plasmid isolated from yeast clone C10 encoded two GST-fused peptides, each of them being driven by their own PGK promoter.

After separation and subcloning into the PGK-GST plasmid, the clones were re-designated GST-p9 and GST-p10 respectively (as shown in Fig. 1D (lower panel) and Fig. 2A). Sequence analysis reveals that all fifteen selected GST-peptides (GST-p) contain a conserved di-tryptophan (di-W) motif. The presence of this motif within peptides may thus be critical for their ability to inhibit Vpr-mediated growth arrest activity (Fig. 2A).

Interestingly, the amino-acid sequence of four GST-peptides, GST-plO, GST-pl3, GST-pl6 and-pl7, was found to contain a WxxF motif, previously reported to be critical for Vpr interactions (52). Nevertheless the previously undescribed diW motif was still well preserved in these four peptides.

Another observation was that most peptides were rich in hydrophobic amino acids, especially at their C-terminus (Fig. 2A), suggesting that, in addition to the diW motif, the hydrophobicity of the peptides may also be required for their inhibitory activity. Interestingly, even though GST-pl8 and GST-p4 contain a WWxW sequence, GST-pl8 exhibits a stronger inhibitory activity toward Vpr-mediated growth arrest than GST-p4 (Fig. 10 and Fig. 3A). Computer analysis predicts that GST-p4 exhibits a high hydrophilicity at the C-terminus as compared to both GST-pl8 and GST-pl6 which contain a hydrophobic C-terminal sequence (Fig. 2B). We conclude from these results that the presence of a diW motif and the preservation of a hydrophobic C-terminal may be important parameters governing the peptides anti-Vpr activity. Further, extension of the C-terminus relative to the diW motif position, such as in GST-p4, may also affect the peptide inhibitory activity. Therefore, it appears that the peptide

inhibitors may in embociments contain one or more of: the diW motif, and an additional hydrophobic C-termini and/or an extension at the C-terminus, which may further increase the peptide inhibitory activity.

The present invention exemplified with peptides also enables the determination of a consensus sequence comprised in the peptides enabling an inhibition of the growth-inhibiting function of Vpr. This consensus sequence is shown to be WWx, wherein x is any amino acid and is a hydrophobic amino acid (including glycine), W being the standard one-letter code for tryptophan. In an embodiment, the peptide comprises the consensus WWOO, in a further embodiment, xWWOO, in a further embodiment, xxWWOO, wherein x is any amino acid and is a hydrophobic amino acid (including glycine). Of course, as shown in Figure 2A, the peptides can be comprised in longer peptides or have the sequence xWWOt or xxWWOO, wherein x is any amino acid and is a hydrophobic amino acid (including glycine). In embodiments, the hydrophobic amino acid is an aromatic amino acid, such as W or F. In any event, the genetic screen of the present invention is demonstrated to enable the selection of agents which modulate (in this case inhibit or overcome) the growth-inhibiting function of Vpr in a cell.

Example 12: Selected GST-peptides specifically inhibit the effect of Vpr on cell growth and morphology without affecting Vpr expression To test whether the selected GST-peptides affect specifically HIV-1 Vpr activity or alternatively have a general effect on cell proliferation, we selected a panel of eight representative diW-containing GST-peptides, including GST-pl,-p4,-p9,-plO,-pl2,-pl6,-pl7,-pl8 and re- transformed purified plasmid DNAs encoding these eight anti- Vpr GST-peptides into HP16 yeast strain harboring p424Gall-

Vpr. Suspensions of transformants of similar cell densities were then serially diluted (lOx), spotted onto either a Vpr non-inducible plate (raffinose) or a Vpr-inducible plate (galactose) and their growth evaluated following an incubation of 3-5 days (Fig. 3A). In the absence of Vpr expression, yeast cells constitutively expressing each GST-peptide or the GST control grew at similar rate (Fig. 3A, a), thus indicating that expression of these GST-peptides per se had no general effect on yeast proliferation. As expected, when HIV-1 Vpr expression was induced, yeast co-expressing Vpr and the GST control exhibited a profound growth defect while yeast expressing the GST control only, grew efficiently (Fig. 3A, b, compare lanes 2 and 3 to lane 1). In contrast, co-expression of the selected GST-peptides was shown to overcome the yeast growth defect mediated by Vpr albeit to a different extent (Fig. 3A, b, compare lanes 4-11 to lanes 2 and 3). GST-pl8,- pl7,-pl6 and-pl2 exhibited the strongest inhibition while, interestingly, GST-p4 displayed the weakest inhibitory activity. These results strongly indicate that the selected GST-peptides specifically inhibit the activity of Vpr mediating budding yeast cell growth arrest.

In addition to mediating cell growth arrest, Vpr has also been shown to induce cell morphology changes in S. cerevisiae (53) (43). To determine whether the most potent inhibitory GST-peptides (GST-pl8 or GST-pl6) could interfere with this Vpr effect, HPI6 yeast cells either expressing the GST control, or co-expressing Vpr and GST or GST-pl6 or-pl8, were cultured in Vpr-inducible medium for 3 days and examined by light microscopy. Results show that expression of the GST control did not affect cell morphology (Fig. 33, a) as compared to non-transformed cells (data not shown). In contrast, profound morphological changes were observed when Vpr was co-expressed with GST. These Vpr-induced morphological changes were highly polymorphic, including enlarged, spherical

and shrinked cells (Fig. 33, b). When Vpr was co-expressed with GST-pl6 or GST-pl8, most cells exhibited a normal size and morphology and very few shrinked cells were present in the cultures (Fig. 33, c and d). These results indicate that, in addition to inhibiting Vpr-mediated growth arrest, GST-pl8 and GST-pl6 can also strongly attenuate Vpr-mediated morphological changes in budding yeast.

Example 13: Interaction of di-W containing GST-peptides with Vpr In an attempt to elucidate the mechanism (s) underlying the GST-peptide inhibitory effect, we investigated the ability of a panel of representative di-W containing GST- peptides, including GST-p4, GST-pl2, GST-pl6 and GST-pl8, to interact with Vpr in yeast cells by GST pull-down. These GST- peptides were primarily selected on the basis of their inhibitory effect on Vpr-mediated growth arrest. HP16 yeast cells co-expressing HIV-1 Vpr and GST-p4, GST-pl2, GST-pl6, GST-pl8 or GST were radiolabeled, lysed with CHAPS lysis buffer and their GST-peptides purified with glutathione- sepharoseTM, as described in Example 5. Pelleted radiolabeled GST or GST-peptides complexes were then eluted with gluthatione, separated by SDS-PAGE and analyzed by autoradiography. Results of Figure 4A reveal that similar amounts of GST or GST-p4, GST-pl2, GST-pl6 and GST-pl8 were eluted from the glutathione-sepharoseTM beads. While no Vpr was co-eluted with GST (Fig. 4A, lane 5), detectable amounts of Vpr were pulled-down by GST-pl8, GST-pl6, GST-pl2, and GST-p4 (Fig. 4A, lanes 1 to 4). Furthermore, no protein corresponding to Vpr was pulled-down by these GST-peptides when yeast cells were grown in Vpr non-inducing conditions therefore confirming that the co-purified 14 kDa band is indeed Vpr (data not shown). Interestingly, GST-pl8 was found to pull-down the largest amount of Vpr (quantitative analysis of the protein

bands by scanning densitometry indicates that the ratio of bound Vpr over total Vpr is approximately 2-3 fold higher for GST-pl8 as compared to GST-pl2 and-pl6, respectively) while the amounts of Vpr associated with GST-p4 were the lowest (Fig. 4, compare lane 1 to lanes 2-4). The absence of protein bands other than Vpr in the GST-peptide complexes strongly suggests that the interaction of diW-containing GST-peptides with Vpr is direct. As expected, no radioactive bands corresponding to Vpr or GST peptides were detected in lysates prepared from Vpr yeast transformants grown under Vpr inducing or non-inducing conditions (Fig. 4A, lanes 6-7). To rule-out the possibility that the different amounts of Vpr bound to GST-peptide are due to variable level of Vpr expression, the same samples were immunoprecipitated with anti-Vpr antibodies and the immunocomplexes were analyzed by SDS-PAGE and autoradiogaphy. Results show that comparable amounts of Vpr were expressed in each yeast transformants grown in Vpr- inducing conditions (Fig. 4B lanes 1-5 and 7). Overall, these results clearly indicate that GST-pl8, GST-pl6, GST-pl7 and GST-p4 interact directly with Vpr and among them, GST-pl8 shows the highest Vpr-binding efficiency.

Example 14: Expression of Vpr-binding GST-Peptides in the Human CD4+ Jurkat T Cell Line Alleviates Vpr-mediated Apoptosis and Cell Cycle G2 Arrest upon Infection with VSV-G- pseudotyped HIV-1 Virus To investigate whether Vpr-binding GST-peptides could impair Vpr-mediated apoptosis and cell cycle G2 arrest in human CD4+ T cells, we generated CD4+ Jurkat T cell populations expressing GST-peptides including GST-p4,-pl2, and-pl8 as well as the GST control. Each cell population was analyzed for its ability to express the corresponding transgene by semiquantitative reverse transcription-PCR since detection of GST-peptide fusion proteins by Western blotting

using anti-GST antibodies did not lead to clear results. This is likely due to the fact that GST-peptide fusion protein expression in these Jurkat cell populations was at the limit of immunoblot detection levels. Results of Fig. 5A reveal that GST-p4,-pl2, and-pl8 and GST mRNAs were detected in the corresponding Jurkat T cell populations, indicating that the transgenes were adequately transcribed. Expression of the GST- peptides did not appear to affect Jurkat cell growth or morphology as compared with the GST-expressing Jurkat cell population control (data not shown).

To test the effect of each GST-peptide on Vpr- mediated apoptosis and cell cycle G2 arrest, each GST-peptide expressing Jurkat T cell population was infected with Vpr+ (HxBruR+/E) or Vpr (HxBruR/E) VSV-G pseudotyped HIV-1 viruses at m. o. i. of 0.125 and 0.25. Infected cells were collected 48 h post-infection and analyzed for cell cycle and apoptosis.

The use of VSV-G-pseudotyped virus allowed us 1) to efficiently infect each cell population and 2) to examine the effect of GST-peptides on Vpr-mediated apoptosis and cell cycle G2 arrest in the absence of viral spread and without the complication of envelope-mediated cell death. Results from Fig. 5B clearly show that expression of GST-peptides had no significant effect on the percentage of apoptotic cells resulting from infection with Vpr-defective pseudotyped virus at both m. o. i. As previously reported, expression of Vpr was found to enhance apoptosis as revealed by the increased number of annexin V-positive apoptotic cells in Vpr+-pseudotyped virus-infected GST-expressing Jurkat cell control cultures at both 0.125 and 0.25 m. o. i. In contrast, Jurkat cell populations expressing GST-pl2 or pl8 exhibited a significant reduction of annexin V-positive cells upon infection with Vpr+-pseudotyped virus as compared with the GST control especially at the lower m. o. i. (0.125) (Fig. 5B). GST-p4 did not show any significant inhibition of Vpr-mediated apoptosis.

In parallel, Vpr-mediated cell cycle G2 arrest was also evaluated. Results of Fig. 5C clearly show that, whereas GST- pl2 or p-18 did not affect the cell cycle profile of Vpr- pseudotyped HIV-1-infected Jurkat cell populations, expression of these GST-peptides attenuated Vpr-mediated cell cycle G2 arrest during Vpr+ HIV-1 infection as compared with the GST control (Fig 5C). Jurkat cells expressing GST-p4 were also found to be less susceptible to Vpr-mediated cell cycle G2 arrest as compared with the GST-expressing Jurkat cell control, suggesting that GST-p4 retained some ability to inhibit Vpr activity on the cell cycle. Overall, these results indicate that expression Vpr-binding GST-peptides in human Jurkat T cells alleviates the apoptosis and the cell cycle G2 arrest mediated by HIV-1 Vpr. Similar results were obtained when GST-peptides where expressed in HeLa cells and infected with VSV-G pseudotyped HIV-1 virus (data not shown) or when 293T cells transiently overexpressing Vpr and GST-peptides where analyzed for Vpr-mediated cell cycle G2 arrest (Fig. 6).

Example 15: Intracellular localization of HIV-1 Vpr and selected GST-peptides Previous studies have shown that HIV-1 Vpr localizes in the nucleus of mammalian cells when expressed in the absence of other viral proteins (44,54, 55). To investigate the effect of Vpr-binding GST-peptides on Vpr nuclear localization, we expressed GST or GST-pl6 or GST-pl8 individually or co-expressed them with Vpr in COS-7. COS-7 cells have been previously shown to be responsive to Vpr- mediated cell cycle G2 arrest and apoptosis (56). They also have the interesting feature of having a well delineated nucleus and cytoplasm which facilitates intracellular localization analysis using immunofluorescence. Cells were fixed, labeled with anti-GST and/or anti-Vpr antibodies and analyzed by indirect immunofluorescence forty-eight hours

posttransfection. When GST was expressed alone in COS-7 cells, the protein exhibited a diffuse staining pattern and localized both in the cytoplasm and the nucleus (Fig. 7, a). In contrast, the GST-peptide fusions, GST-pl8 and GST-pl6, were clearly found to be excluded from the nucleus and were shown to primarily accumulate in a perinuclear region within the cytoplasm (Fig. 7, b and c), suggesting that the presence of diW motif-containing peptides at the C-terminus of GST prevents GST diffusion into the nucleus. When GST was co- expressed with Vpr, co-staining results showed that GST was still distributed both in the nucleus and the cytoplasm (Fig.

7, d), while Vpr was predominantly located in the nucleus (Fig. 7, g). Interestingly, even though both proteins were located in the nucleus, there was no apparent co-localization (Fig. 7 d and g). In contrast, when GST-pl6 or GST-pl8 were co-expressed with Vpr, a clear co-localization was observed (Fig. 7, e and h; f and i). In the presence of GST-pl8, Vpr nuclear localization pattern drastically changed (compare i and g). Intensive Vpr staining was observed at the periphery of the nucleus rather than in the nucleoplasm as seen when Vpr was expressed with GST. Conversely, GST-pl8 that had primarily a cytoplasmic localization when expressed alone, showed a punctuated staining pattern at the periphery of the nucleus like Vpr (compare f with c and i). Similarly, the localization pattern of GST-pl6 changed in the presence of Vpr (compare e and b). GST-pl6 that was found to accumulate in a perinuclear region of the cytoplasm, now, co-localized with Vpr primarily in the nucleoplasm and the nuclear periphery as well as in the cytoplasm as punctuated staining (compare e with b and h).

These results indicate that expression of Vpr-binding GST- peptides with Vpr results in drastic changes at the level of the proteins subcellular localization. Interestingly, GST-p4 which was shown to interact with Vpr with a lower efficiency, was not found to provoke change in Vpr subcellular

localization to such extent (data not shown). The fact that Vpr and GST-pl6 and GST-pl8 co-localized provides additional evidence that diW-containing GST-peptides interact with Vpr intracellularly. accumulation of Vpr at the level of the nuclear periphery in the presence of GST-pl 8 or at the level of the cytoplasm (punctuated staining) and nuclear periphery with GST-pl6 suggests that these peptides by interacting with Vpr may interfere with its nuclear translocation.

Example 16: Further genetic selection of hexameric peptide sequences that inhibit HIV-1 Vpr-mediated growth arrest in Saccharomyces cerevisiae Using the same approach described above, we have further screened the GST-hexameric peptide fusion library for hexameric peptides that inhibit Vpr-mediated growth arrest in Saccharomyces cerevisiae. Table 1 lists the amino sequence of the additional hexameric peptides that were selected. The sequence of peptides 18 and 20 are indicated as reference. (+ = inhibitory potential ;-= no inhibitory potential : +/- ambiguous) Table 1: Identification of additional hexameric peptides peptide # SEQ ID Activity Sequence NO: 18 52 + S E W W V W V 20 - - stop 21 16 + V W W L L G C L R D P G N K L N 22 53 + R W W A F H V 23 54 + R C G W W K V 32 55 + R W W E W G V 34 56 + L Y W W V W V 35 57 + G R F W W V V 40 58 + R W W W F C V 43 59 + W Y S F L G V 45 60 + V F M W W W V 46 61 + R G W W W V V 47 62 +/- A W M S F L V B1 63 + C W W S F L V B2 64 + W W Y F A Q V B3 65 + P W A C V W V B4 66 + W W S F K S V B5 67 - L M R G V G V B6 68 + A F W W V F V M4 69 + W G V W W R V M7 70 - I L R S S C V

Example 17: Determination of the amino-acid sequence conscensus motif required to achieve optimal binding to Vpr and maximum inhibition of Vpr-mediated growth arrest in yeast.

Using the GST-pull down assay described above, the binding efficiency of GST-hexameric peptide fusions isolated in the further round of selection (Table 1) was evaluated, with the results shown in Figure 8. Briefly, HP16 yeast strain was transformed with a plasmid expressing a specific GST-peptide fusion as well as with a plasmid encoding Vpr (+) or Rpv (-; Vpr gene inserted in the opposite orientation). Cells were radiolabeled in a medium containing 35S methionine as well as galactose to induce the Gal I promoter. Cells were lysed in CHAPS buffer and 200 pl of lysate was subjected to a GST pull-down (A) or an immunoprecipitation using an anti-Vpr antibody. Following SDS-PAGE, precipitated proteins were revealed by autoradiography.

Table 2 summarizes the binding data obtained, supporting the WWX (aromatic) as the motif within the hexameric peptide capable of conferring Vpr binding and inhibition of Vpr-mediated growth arrest in yeast. Activity = ability to inhibit Vpr-mediated growth arrest in yeast; Y : inhibitory potential; N : no inhibitory potential; +/- : ambiguous. Binding = Ability to interact with Vpr by GST- pull-down. Qualitative scale relative to the GST-20 negative control :-= negative ; + = weak interaction; ++ to +++ = strong interaction; +/- : ambiguous. The WWX motif is underlined. As shown in Table 2, it was determined that three peptide sequences (B1-B2 and B3) interact with Vpr with greater efficiency than peptide 18 (which has a WWXW motif). These three peptide sequences share a WWXF motif.

Table 2: Analysis of peptides with respect to Vpr binding and activity Binding Activity Peptide # SEQ ID Sequence NO: + +/- 4 4 * + Y 12 71 R Y V W W L V + Y 16, 17 72 S W W L F C V ++ Y 18 52 S E W W V W V - N 20 - stop = Y 21 16 V W W L L G C L R D P G N K L N +/- Y 34 56 L Y W W V W V - Y 46 61 R G W W W V V +/- N 47 62 A W M S F L V +++ Y B1 63 C W W S F L V +++ Y B2 64 W W Y F A Q V + Y B3 65 P W A C V W V +++ Y B4 66 W W S F K S V - N B5 67 L M R G V G V - +/- M8 74 G F W W W L V

Example 18: Effect of a synthetic peptide comprising the WWXO motif on the cytotoxic activity of a polypeptide corresponding to the C-terminal region of HIV-1 Vpr It has been reported that the direct addition of recombinant Vpr or polypeptides corresponding to the C- terminal region of Vpr (Vpr 52-96) to mammalian cell lines of different type (CD4+ T cell lines. , neuronal cell lines) or to budding yeast induced cell death presumably by apoptosis and mitochondrial toxicity (Jacotot et al. J. Exp Med. , 193,509- 519,2001 ; Piller et al. PNAS 95,4595-4600, 1998). We tested the effect of a synthetic peptide containing the WWX motif (pl8) on the apoptosis induced by a polypeptide corresponding to Vpr 52-96. The amino acid sequence of the pl8 peptide, a control peptide and Vpr 52-96 are described in Figure 9.

The data of Figure 10 reveals that peptide pl8 exhibited a stronger attenuation of the apoptotic activity mediated by Vpr 52-96 as compared to a similar peptide contaning substitutions in the WWX motif. This data provide evidence that small peptides containing the WWXO motif can alleviate the pro-apoptotic effect of Vpr. Small molecules that mimic the structural features of this motif should also interfere with the pro-apoptotic effect of HIV-1 Vpr. Finally, given that pl8 attenuated the pro-apoptotic effect of a polypeptide corresponding to the C-terminal region of HIV-1 Vpr, we believe that peptides containing a WWX motif interact with a domain of Vpr located at the C-terminal of Vpr, specifically between amino acids 52 and 96.

Example 19: In this study, we have taken advantage of the growth arrest phenotype induced by Vpr in budding yeast to screen a GST-fused hexameric peptide library for GST-peptide fusions

capable of inhibiting Vpr cell growth arrest activity. A number of GST-peptides that had the capacity to overcome Vpr- mediated growth arrest were identified using the exemplified genetic selection system. Given that the G2 to M transition of budding yeast is regulated differently than in fission yeast or mammalian cells-inhibitory phosphorylation of the cyclin- dependent kinase Cdc28 of budding yeast does not play a major role in this transition (57,58) (43), it is unlikely that the selected peptides overcome Vpr-mediated budding yeast growth arrest by interfering with specific interactions between Vpr and cellular components regulating inhibitory phosphorylation of the cyclin-dependent kinase and the G2-M transition as was demonstrated in mammalian cells and fission yeast (18,19, 25).

Rather, expression of Vpr in budding yeast induces growth defect and cell killing by a mechanism that appears to involve mitochondrial membrane permeabilization (42).

Using the GST pull-down binding assay, we have clearly showed that a set of representative GST peptides interacted directly with Vpr, however, with different efficiencies. The growth arrest inhibitory effect mediated by the GST-peptides correlated very-well with their Vpr binding efficiency. GST-pl8 was found to have a strong inhibitory effect on Vpr-mediated growth arrest (Fig. 3A) and was shown to strongly bind Vpr (Fig. 4A). In contrast, GST-p4 was found to have the weakest effect on Vpr-mediated growth arrest (Fig.

3A) and was shown to have the lowest Vpr binding efficiency (Fig. 4A). These results strongly suggest that the interaction of the GST-peptides with Vpr interferes with its ability to induce a growth arrest in budding yeasts. Of interest, having identified different sequences with different Vpr binding affinities and different Vpr-dependent biological function enables a dissection of the structure function relationship of these peptides and their critical determinants.

Sequence analysis of the GST-peptides exhibiting anti-Vpr activity revealed that they all contained a conserved double-tryptophan (di-W) motif, strongly suggesting that the di-W motif within the peptides is critical for anti-Vpr activity as well as for their ability to interact with Vpr. In addition, the most potent GST-peptides displayed a stretch of hydrophobic residues at their C-terminus (Fig. 2). Studies of pl8 lacking the GST moiety, including the pl8 control peptide (in which W residues were replaced with A residues), demonstrate that the observed activity correlates well with the identified motifs. A number of the peptides identified harbored a previously reported WxxF motif of a Vpr-interacting domain (52). Several studies have also shown that fusion of WxxF motif to heterologous protein allowed these fusion proteins to be targeted into HIV-1 viral particles via an interaction between Vpr and the WxxF-containing protein (52,59, 60). Surprisingly, our sequencing and functional analyses reveal the conservation of a diW motif in peptides that were selected for their ability to restablish yeast cell growth. In contrast, the presence of a phenylalanine in the context of a WxxF motif did not appear to be absolutely necessary (Fig. 2 and 3). This difference may reflect the different experimental designs that were used to select these Vpr-binding peptides. BouHamdan et al. used a phage display approach to select peptides that had the ability to bind recombinant Vpr in in vitro cell free conditions (52) whereas the genetic system exemplified in the present study selects peptides on the basis of their ability to inhibit a Vpr biological activity, intracellularly as opposed to a selection solely based on binding. It is possible that the presence of phenylalanine in the WxxF motif can substitute for the second tryptophan in the diW motif to provide Vpr binding affinity in vitro. However, since our selection assay is based on the interference of a Vpr biological activity in a cellular

context, rather than inhibition of protein-protein interactions only, it is possible that the presence of a phenylalanine residue downstream of the diW motif in the peptide, while contributing to Vpr-binding affinity, is not absolutely required for anti-Vpr activity. The diW motif and the hydrophobic properties of the peptides may be necessary to ensure that the inhibitory peptides reach the cellular compartment where Vpr interacts with critical cellular partner (s) or alternatively may promote steps following Vpr binding that lead to inhibition of Vpr-mediated growth arrest.

These peptide properties cannot be selected by using phage display given that the assay is based on a peptide-recombinant protein interaction that occurs in a cell-free system.

Cell cycle G2 arrest is one of the main functions of Vpr during HIV-I infection. This Vpr mediated cell cycle arrest, in addition to providing an intracellular milieu favorable for HIV viral expression and production, is also believed to contribute to HIV-1 pathogenesis. Interestingly, diW-containing GST peptides that were shown to interact with Vpr in yeast cells, including GST-p4, GST-pl2, GST-pl6 and GST-pl8 interfered with Vpr mediated G2 arrest in different human cell lines as well as during HIV-1 infection (Figures 5 and 6). The degree of inhibition of Vpr-mediated G2 arrest was again found to correlate with the efficiency of Vpr binding. Moreover, we have found that, in addition to inhibiting Vpr-mediated cell cycle G2 arrest, GST-pl6 and-pl8 impaired Vpr ability to induce apoptosis during VSV-G pseudotyped HlV-1 infection of HeLa and Jurkat cells (data not shown). Of note, preliminary data using stable expression of GST peptides showed that in a Vpr+ cell line, the GST peptides could overcome the Vpr-dependent apoptotic effect. Another interesting observation of our study is that Vpr-binding GST- peptides (including GST-pl6 and GST-pl8) exhibited a change of subcellular localization from the cytoplasm to the nuclear

membrane and the nucleoplasm, in the presence of Vpr and co- localized with Vpr (Fig. 6). Conversely, expression of GST peptides GST-pl6 and pl8 interfered with Vpr nuclear accumulation since Vpr was found mainly in a region close to the nuclear membrane. Interestingly, GST-p4, a peptide that had a minimal hydrophobic C-terminal and was found to bind Vpr with weak efficiency, did not co-localize with Vpr and did not provoke significant change in Vpr nuclear localization. These results provide evidence that some peptides (GST-pl8 and-pl6) interact with Vpr in mammalian cells. The requirement of Vpr nuclear localization for the protein cell cycle G2 arrest or proapoptotic activities still remains controversial (55,62- 64). However, the ability of diW-containing peptides to directly interact with Vpr and as a consequence act as dissociative inhibitors of critical protein-protein interactions is more likely to be responsible for the peptides general anti-Vpr activity given their effect on multiple Vpr biological activities.

Since different functions of Vpr may optimize HIV-1 replication and contribute to HIV-1 pathogenesis in vivo, this protein has been proposed to be a target for the development of antiviral strategies. Several studies have reported different strategies to inhibit Vpr functions during HIV-1 replication, such as Vpr dominant mutant (R73S) (65), antagonist of the glucocorticoid receptor (RU486) (66) and pentoxifylline (67). In this study, we took advantage of a genetic selection system in budding yeast, that allows the in vivo identification of hexameric peptide inhibitors that have functional relevance since they are inhibiting a function of the target protein, in this case Vpr, intracellularly. We hereby identified a class of polypeptides which have the ability to directly bind and inhibit multiple Vpr biological activities intracellularly, providing a novel approach to modulate (e. g. inhibit) HIV-1 Vpr functions.

Peptide motifs examined in this study are summarized in Table 3, and alternative peptides according to embodiments of the invention are provided in Table 4.

Table 3: Peptide motifs examined in this study Peptide SEQ ID Sequence* NO: 1 1 S I R W W L 2 2 E S R W W V 3 3 F C S W W W 4 4 C G W W V W S R G S G K 5 5 T VQ W W V 6 6 A V P W W V 7 7 Q G S W W V 9 8 T A W W V V 10 9 F F W W L F 11 10 W C R W W L 12 11 R Y V W W L 13 12 G G W W G F 16, 17 13 S W W L F C 18f 14 S E W W V W 18 control# 15 S E A A V A 21 16 V W W L L G C L R D P G N K L N 22 17 R W W A F H 23 18 R C G W W K 32 19 R W W E W G 34 20 L Y W W V W 35 21 G R F W W V 40 22 R W W W F C 43 23 W Y S F L G 45 24 V F M W W W 46 25 R G W W W V

47 26 A W M S F L B1 27 C W W S F L B2 28 W W Y F A Q B3 29 P W A C V W B4 30 W W S F K S B5 31 L M R G V G B6 32 A F W W V F M4 33 W G V W W R M7 34 I L R S S C M8 73 G F W W WL *All peptide motifs were studied using a version having an attached N-terminal GST-KGLSGP<BR> moiety, and all except P-4 and p-21 were studied having an attached C-terminal V residue.<BR> <P>#p-18 was also examined in a version lacking GST moiety.<BR> <P>#p-18 control examined in a version lacking GST moiety.

Table 4: Alternative peptides according toembodiments of the invention Peptide SEQ ID Sequence NO: 1 1 S I R W W L 2 2 E S R W W V 3 3 F C S W W W 4 4 C G W W V W S R G S G K 5 5 T V Q W W V 6 6 A V P W W V 7 7 Q G S W W V 9 8 T A W W V V 10 9 F F W W L F 11 10 W C R W W L 12 11 R Y V W W L 13 12 G G W W G F 16, 17 13 S W W L F C 18 14 S E W W V W 21 16 V W W L L G C L R D P G N K L N 22 17 R W W A F H 23 18 R C G W W K 32 19 R W W E W G 34 20 L Y W W V W 35 21 G R F W W V 40 22 R W W W F C 43 23 W Y S F L G 45 24 V F M W W W 46 25 R G W W W V 47 26 A W M S F L B1 27 C W W S F L B2 28 W WY F A Q B3 29 P W A C V W B4 30 W W S F K S B6 32 A F W W V F M4 33 W G V W W R M8 73 G F W W W L X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18

Example 20: Non-limiting examples of Vpr nucleic acid and polypeptide sequences HIV-1 Vpr (pNL4.3 clone) MEQAPEDQGPQREPYNEWTLELLEELKSEAVRHFPRIWLHNLGQHIYETYGDTWAGVEAI IR ILQQLLFIHFRIGCRHSRIGVTRQRRARNGASRS (SEQ ID NO : 41) ACCESSION M19921 Adachi, A. , Gendelman, H. E. , Koenig, S. , Folks, T., Willey, R. , Rabson, A. and Martin, M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.

J. Virol. 59 (2), 284-291 (1986) HIV-2 Vpr (ROD clone) MAEAPTELPPVDGTPLREPGDEWIIEILREIKEEALKHFDPRLLIALGKYIYTRHGDTLE GA RELIKVLQRALFTHFRAGCGHSRIGQTRGGNPLSAIPTPRNMQ (SEQ ID NO : 43)

ACCESSION M15390 Clavel, F. , Guyader, M. , Guetard, D. , Salle, M. , Montagnier, L. and Alizon, M. Molecular cloning and polymorphism of the human immune deficiency virus type 2 Nature 324 (6098), 691-695 (1986) SIV Vpr (mac239 clone) MEERPPENEGPQREPWDEWVVEVLEELKEEALKHFDPRLLTALGNHIYNRHGDTLEGAGE LI RILQRALFMHFRGGCIHSRIGQPGGGNPLSAIPPSRSML (SEQ ID NO : 45) ACCESSION M33262 Kestler, H., Kodama, T. , Ringer, D. , Marthas, M. , Pedersen, N. C., Lackner, A. , Regier, D. , Sehgal, P., Daniel, M., King, N. and Desrosiers, R. Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus. Science 248 (4959), 1109-1112 (1990).

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims,

the word"comprising"is used as an open-ended term, substantially equivalent to the phrase"including, but not limited to". The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

Throughout this application, various references are referred to describe more fully the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

REFERENCES 1. Bukrinsky, M. , and Adzhubei, A. (1999) Rev Med Virol 9, 39-49.

2. Zhao, Y. , and Elder, R. T. (2000) Front Biosci 5, D905- 916.

3. Cohen, E. A. , G. Dehni, J. G. Sodroski, and W. A.

Haseltine. (1990) J. Virol. 64,3097 3099.

4. Paxton, W. , R. I. Connor, and N. R. Landau. (1993) J.

Virol. 67,7229-7237.

5. Heinzinger, N. K. , Bukinsky, M. I., Haggerty, S. A., Ragland, A. M. , Kewalramani, V. , Lee, M. A. , Gendelman, H.

E. , Ratner, L. , Stevenson, M. , and Emerman, M. (1994) Proc Natl Acad Sci U S A 91,7311-7315.

6. Gallay, P. , Hope, T. , Chin, D. , and Trono, D. (1997) Proc Natl Acad Sci U S A 94,9825-9830.

7. Nie, Z. , Bergeron, D. , Subbramanian, R. A. , Yao, X. J., Checroune, F. , Rougeau, N. , and Cohen, E. A. (1998) J Virol 72,4104-4115.

8. Subbramanian, R. A. , Yao, X. J. , Dilhuydy, H. , Rougeau, N. , Bergeron, D. , Robitaille, Y. , and Cohen, E. A. (1998) J Mol Biol 278,13-30.

9. Vodicka, M. A. , Koepp, D. M. , Silver, P. A. , and Emerman, M. (1998) Genes Dev 12,175-185.

10. Popov, S. , M. Rexach, G. Zybarth, N. Reiling, M-A, Lee, L. Ratner, C. M. Lane, M. S. Moore, G. Blobel, and M.

Bukrinsky. (1998) EMBO J. 17,909-917.

11. Fouchier, R. A. , Meyer, B. E. , Simon, J. H. , Fischer, U., Albright, A. V. , Gonzalez-Scarano, F. , and Malim, M. H.

(1998) J Virol 72,6004-6013.

12. Zennou, V. , Petit, C. , Guetard, D. , Nerhbass, U., Montagnier, L. , S and Oharneau, P. (2000) Cell 101,173-185.

13. Bouyac-Bertoia, M. , Dvorin, J. D. , Fouchier, R. A., Jenkins, Y. , Meyer, B. E. , Wu, L. I. , Emerman, M. , and Malim, M. H. (2001) Mol Cell 7,1025-1035.

14. Jenkins, Y. , McEntee, M. , Weis, K. , and Greene, W. C.

(1998) J Cell Biol 143,875-885.

15. Sherman, M. P. , de Noronha, C. M. , Heusch, M. I. , Greene, S. , and Greene, W. 0. (2001) J Virol 75,1522-1532.

16. Levy, D. N. , L. S. Fernandes, W. V. Williams, and D. B.

Weiner. (1993) Cell 72,541-550.

17. Rogel, M. E. , L. I. Wu, and M. Emerman. (1995) J. Virol.

69,882-888.

18. He, J. , Choe, S. , Walker, R. , Di Marzio, P. , Morgan, D.

0., and Landau, N. R. (1995) J Virol 69,6705-6711.

19. Re, F. , D. Braaten, E. K. Franke, and J. Luban. (1995) J. Virol. 69,6859-6864.

20. Jowett, J. B. , Planelles, V. , Poon, B. , Shah, N. P., Chen, M. L. , and Chen, 1. 5. (1995) J Virol 69,6304-6313.

21. Bartz, S. R. , M. E. Rogel, and M. Emerman. 1996. (1996) J.

Virol. 70,2324-2331.

22. Goh, W. C. , M. E. Rogel, C. M. Kinsey, S. F. Michael, P. N.

Fultz, M. A. Nowak, B. H. Hahn, and M. Emerman. (1998) Nat.

Med. 4,65-71.

23. Forget, J. , Yao, X. J. , Mercier, J. , and Cohen, E. A.

(1998) J Mol Biol 284,915-923.

24. Yao, X. -J. A. J. Mouland, R. A. Subbramanian, J. Forget, N.

Rougeau, D. Bergeron, and E. A. Cohen. (1998) J. Virol.

72, 4686-4693.

25. Zhao, Y. , Cao, J., O'Gorman, M. R. , Yu, M. , and Yogev, R. (1996) J Virol 70,5821-5826.

26. Elder, R. T. , Yu. M. , Chen, M. , Edelson, S. , Zhao Y.

(2000) Virus Res. 68,161-173.

27. Coleman, T. R. , and Dunphy, W. G. (1994) Curr Opin Cell Biol 6,877-882.

28. Kurtz, S. , Esposito, K. , Tang, W., and Menzel, R. (2003) Biotechnol. Bioeng. , 82,38-46.

29. Masuda, M. , Nagai, Y. , Oshima, N. , Tanaka, K. , Murakami, H. , lgarashi, H. , and Okayama, H. (2000) J Virol 74,2636- 2646.

30. Elder, R. T. , Yu, M. , Chen, M. , Zhu, X. , Yanagida, M., and Zhao, Y. (2001) Virology 287,359-370.

31. BouHamdan, M. , Benichou S. , Rev, F. , Navarro, J. M., Agostini, I., Spire, B. , Camonis, J. , Slupphaug, G. R. , Sire, J. (1996) J Virol. 70,697-704.

32. Gragerov, A. , Kino, T. , Ilyina-Gragerova, G. , Chrousos, G. P. , and Pavlakis, G. N. (1998) Virology 245,323-330.

33. Withers-Ward, E. S. , Jowett, J. B. , Stewart, S. A. , Xie, Y. M. , Garfinkel, A. , Shibagaki, Y. , Chow, S. A. , Shah, N., Hanaoka, F. , Sawitz, D. G. , Armstrong, R. W. , Souza, L. M., and Chen, I. S. (1997) J Virol 71,9732-9742.

34.. Mahalingam, 5., Ayyavoo, V. , Patel, M. , Kieber-Emmons, T. , Kao, G. D. , Muschel, R. J. , and Weiner, D. B. (1998) Proc Natl Acad Sci U S A 95,3419-3424.

35. de Noronha, C. M. , Sherman, M. P. , Lin, H. W. , Cavrois, M. V. , Moir, R. D. , Goldman, R. D. , and Greene, W. C. (2001) Science 294,1105-1108.

36. Stewart, S. A. , Poon, B. , Jowett, J. B. , and Chen, I. S.

(1997) J Virol 71,5579-5592.

37. Ayyavoo, V. , Mahboubi, A. , Mahalingam, S. , Ramalingam, R. , Kudchodkar, S. , Williams, W. V. , Green, D. R. , and Weiner, D. B. (1997) Nat Med 3,1117-1123.

38. Conti, L. , Rainaldi, G. , Matarrese, P. , Varano, B., Rivabene, R. , Columba, S. , Sato, A. , Belardelli, F., Malorni, W. , and Gessani, (1998) J Exp Med 187,403-413.

39. Fukumori, T. , Akari, H. , Yoshida, A. , Fujita, M. , Koyama, A. H. , Kagawa, S. , Adachi, A. (2000) Microbes Infect. 2,1011- 1017.

40. Jacotot, E. , Ravagnan, L. , Loeffler, M. , Fern, K. F., Vieira, H. L. , Zamzami, N. , Costantini, P., Druillennec, S. , Hoebeke, J. , Briand, J. P., Irinopoulou, T. , Daugas, E., Susin, S. A. , Oointe, D. , Xie, Z. H. , Reed, J. C. , Roques, B.

P. , and Kroemer, G. (2000) J Exp Med 191,33-46.

42. Macreadie, I. G. , Thorburn, D. R. , Kirby, D. M. , Oastelli, L. A. , de Rozanio, N. L. , and Azad, A. A. (1997) FEBS Lett 410,145-149.

43. Gu, J. , Emerman, M. , and Sandmeyer, 5. (1997) Mol Cell Biol 17,4033-4042.

44. Yao, X. -J., R. A. Subbramanian, N. Rougeau, F. Boisvert, D. Bergeron, and E. A. Cohen. (1995) J. Virol. 69,7032-7044.

45. Mumberg, D. , Muller, R. , Funk, M. (1994) Nucl. Acids.

Res. 22,5767-5768.

46. Tang, W. , Ruknudin, A. , Yang, W. P. , Shaw, S. Y., Knickerbocker, A. , and Kurtz, 5. (199S) Mol Biol Cell 6,1231- 1240.

47. Lavallée, C. X. -J. Y. A. L. , H. Gottlinger. W. A.

Haseltine, and E. IS A. Cohen. (1994) J. Virol. 68,1926- 1934.

48. Scott, J. K. and Smith, G. P. (1990) Science 249,386- 390.

49. Park, H. 0., Chant, J. and Herskowitz, I. (1993) Nature 365,269-274.

50. Gietz, D. , St Jean, A, Woods, R. A., Schiestl, R. H.

1992. (1992) Nucleic Acids Res. 20,1425-1430.

51. Hoffman, C. S. a. W. , F. (1987) Gene S7,267-272.

52. BouHamdan, M. , Xue Y. , Baudat, Y. , Hu B. , Sire, J., Pomerantz R. J. , Duan L. X. (1998) J Biol Chem. 273: 8009-16.

273,8009-8016.

53. Macreadie, I. G. , Castelli, U. A. , Hewish, D. R., Kirkpatrick, A. , Ward, A. C. , and Azad, A. A. (1995) Proc Natl Acad Sci U S A 92,2770-2774.

54. Lu, Y. -L., P. Spearman, and L. Ratner. (1993) J. Virol.

67,6542-6550.

55. Subbramanian, R. A. , Kessous-Elbaz, A. , Lodge, R., Forget, J. , Yao, X. J. , Bergeron, D. , and Cohen, E. A.

(1998) J Exp Med 187,1103-1111.

56. Nishino, Y. , Myojin, T. , Kamata, M. , and Aida, Y. (1997) Biochem Biophys Res Commun 232,550-554.

57. Amon, A. , Surana, U. , Muroff, I., and Nasmyth, K. (1992) Nature 355,368-371.

58. Sorger, P. K. , and Murray, A. W. (1992) Nature 355,365- 368.

59. Kulkosky, J. , BouHamdan, M. , Geist, A. , Pomerants, R. J. (1999) Virology 255, 77-85.

60. Okui, N. , Sakuma, R. , Kobayashi, N. , Yoshikura, H., Kitamura, T. , Chiba, J. , Kitamura, Y. (2000) Hum. Gene Ther.

11,537-546.

62. Chen, M. , Elder, R. T. , Yu, M., O'Gorman, M. G. , Selig, U. , Benarous, R. , Yamamoto, A. , and Zhao, Y. (1999) J Virol 73,3236-3245.

63. Zhou, Y. , Lu, Y. , and Ratner, U. (1998) Virology 242, 414-424.

64. Zhou, Y. , and Ratner, U. (2000) J Virol 74,6520-6527.

65. Sawaya, B. E. , Khalili, K. , Rappaport, J. , Serio, D., Chen, W. , Sninivasan, A. , Amini, 5. 1999. (1999) Gene Therapy.. 6,947-950.

66. Kino, T. , Gragerov, A. , Kopp, J. B. , Staube, R. H., Pavlakis, G. N. , Chrousos, G. P. (1999) J. Exp. Med. 189,51- 62.

67. Poon, B. , Jowett, J. B. , Stewart, S. A. , Armstrong, R.

W. , Rishton, G. M. , and Ohen, I. S. (1997) J Virol 71,3961- 3971.

68. Gallouzi IE, S. J. (2001) Science.

69. Morris, M. C. , Depollier, J. , Mery, J. , Heitz, F. , and Divita, G. (2001) Nat Biotechnol 19,1173-1176.

70. Piller, S. C. , Ewart, G. D. , Jans, D. A. , Gage, P. W., and Cox, C. B. (1999) J Virol 73,4230-4238.

71. Sugden, B. , Marsh, K. , and Yates, J. (1985) Mol. Cell.

Biol. 5,410-413.

72. Cohen, E. A. , Terwilliger, E. F. , Jalinoos, Y. , Proulx, J. , Sodroski, J. G. , and Haseltine, W. A. (1990) J. Acquired Immune Defic. Syndr. 3,11-18.