Vpr AND Vpx PROTEINS OF HIV

Abstract: This invention relates to a biologically active peptide fragment of the Vpr protein of human immunodeficiency virus, to pharmaceutical compositions comprising these peptides or biologically active analogues thereof, to antagonists of the peptides, and to pharmaceutical compositions comprising these antagonists and to therapeutic and screening methods utilising compounds and compositions of the invention. In one preferred embodiment, the invention provides an antagonist of the Vpr protein of human immunodeficiency virus (HIV), or of a biologically active fragment or analogue thereof, comprising at least one amino acid sequence motif selected from HFRIG and HSRIG which has the ability to inhibit one or more activities mediated by Vpr, selected from the group consisting of growth arrest, cell replication arrest, cytotoxicity, cytoskeletal disruption, and effects on the endoplasmic reticulum. The invention also relates to use of Vpr protein, or a biologically active fragment or analogue thereof comprising the consensus sequence, in treatment of conditions mediated by cellular proliferation or caused by eukaryotic pathogens.

Description: Vpr and Vpx proteins of HIV This invention relates to a biologically-active peptide fragment of the Vpr protein of human immunodeficiency virus, to pharmaceutical compositions comprising these peptides or biologically-active analogues thereof, to antagonists of the peptides, and to pharmaceutical compositions comprising these antagonists and to therapeutic and screening methods utilising compounds and compositions of the invention. Background of the Invention HIV-1, the causative agent of AIDS, is a complex retrovirus like other primate lentiviruseε, having genes tat, rev, vif, vpr, vpu , and nef that are not found in simple retroviruses. While the functions of tat and rev are fairly well understood, the remainder, often referred to as auxiliary genes because they are not essential for in vi tro infectivity of the virus, have poorly understood roles in pathogenesis. HIV-1 viral protein R (Vpr) (Wong-Staal et al, 1987) is a virion-associated protein (Cohen et ai, 1990a; Yuan et al, 1990) . There have been reports that HIV-1 Vpr is a weak transcriptional activator (Ogawa et al , 1990; Cohen et al, 1990b) and that it binds to the HIV-1 Gag protein (Lu et al, 1993; Paxton et al, 1993; Lavallee et al, 1994) . Although Vpr is not essential for virus replication in established cell lines (Dedera et al, 1989; Cohen et al, 1990b), there is evidence to suggest that it may have a critical function for viral replication in primary macrophages (Balotta et al, 1993; Matsuda et al, 1993) . Because of its association with the virion, it has been suggested that Vpr has an early role in HIV-1 infection, possibly in penetration or uncoating of the virus (Cohen et al, 1990a; Yuan et al , 1990; Yu et al , 1990) . Vpr is one of the most highly conserved proteins of HIV-1, and exists as Vpr and/or Vpx in all primate lentiviruses (Tristem et al, 1990; see Fig. 4A) . Vpx is similarly virion-aεsociated (Cohen et a , 1990a; Yuan et al, 1990; Yu et a , 1988, 1990, 1993) . HIV-2 Vpr is essential for productive infection of human macrophages (Hattori et al, 1990), but like HIV-1 Vpr it is diεpensible for replication in established cell lines (Dedera et al, 1989) . Similarly HIV-2 Vpx is dispenεible in established cell lines (Yu et al, 1988; Guyader et al, 1989; Hu et al , 1989) but is required for infection in fresh macrophages (Guyader et al, 1989; Yu et al, 1991), and augments viral infectivity in peripheral blood lymphocytes (Kappes et al, 1991) . Perhaps most convincing of all, it has been observed that there is a drive in vivo for retention of an intact vpr reading frame and that mutations in vpr lead to a low virus burden in Rhesus monkeys (Lang et al, 1993) . We have cloned the Vpr gene in yeast, and have compared the effect of Vpr protein on haploid yeast cells with the effects of the proteins Vif, Vpu and Nef. We have surprisingly found that the Vpr protein has profound effects on cell growth, while the other proteins tested have no effect. The Vpr protein causes growth arrest, and this appears to be mediated by effects on the cytoskeleton. We have identified the portion of the Vpr protein which is critical for this activity. This critical portion comprises a conserved amino acid sequence motif, H(S/F)RIG, and peptides comprising this portion are active when added extracellularly to mammalian or yeast cells. Since the Vpr protein appears to have an early and possibly critical role in HIV infection, it represents a useful therapeutic target. We have also found that the Vpr protein, and particularly the C-terminal sequence thereof, has a general antiproliferative effect on eukaryotic cells, and therefore is useful in the treatment of conditions mediated by cell proliferation. Summary of the Invention According to one aspect, the invention provides a method of treatment of HIV infection, comprising the step of administering to a subject in need of such treatment an effective amount of an antagonist of Vpr protein, or of a biologically active fragment or analogue thereof comprising at least one motif selected from HFRIG and HSRIG, thereby to prevent HIV infection, to prevent progression of HIV infection to symptomatic AIDS, or to alleviate the symptoms of AIDS. Preferably the Vpr protein comprises at least one sequence selected from the group consisting of HSRIG, HFRIG, HSRIS, HFRAG, HIRAG, HLRAG, RSRKG, RSRIS and RSRIG, In a second aspect, the invention provides an antagonist of the Vpr protein, or of a biologically active fragment or analogue thereof, as defined above, which has the ability to inhibit one or more activities mediated by Vpr, selected from the group consisting of growth arrest, cell replication arrest, cytotoxicity, cytoskeletal disruption, and effects on the endoplasmic reticulum. It is considered that such antagonists will be useful as therapeutic agents for treatment of HIV infection. The invention also encompasses a pharmaceutical composition comprising as active component an antagonist of Vpr as defined above, together with a pharmaceutically- acceptable carrier. The person skilled in the art will recognise that specific antibody, preferably monoclonal antibody, directed against Vpr or a biologically active fragment or analogue thereof, and antisense RNA or triple-stranded DNA which prevents expression of Vpr or of said biologically-active fragment or analogue, provide methods of inhibition of the activity of Vpr, and consequently are within the scope of this invention. Methods for production of monoclonal antibodies against a given peptide sequence, and methods for inducing antisense RNA or triple-stranded DNA production in a target cell are well known in the art. For example, a vpr gene in which the region encoding C-terminal portions of the Vpr protein has been replaced by an inhibitory antisense sequence or by a sequence which encodes an inhibitory peptide could be used for gene therapy of HIV infection or of AIDS. In a third aspect, the invention provides a method of screening compounds suspected of being useful as antagonists of Vpr protein , or of a biologically active fragment or analogue thereof as defined above, comprising the step of measuring the effectiveness of a test compound in inhibiting the activity of Vpr in an assay of a biological activity selected from the group consisting of growth arrest, cell replication arrest, cytotoxicity, cytoskeletal disruption, and effects on the endoplasmic reticulum, as herein described. Our results indicate that the Vpr protein or biologically active fragment or analogue thereof has activities which can be attacked at either the intracellular or extracellular level, and therefore both types of biological activity are within the scope of the invention. According to a fourth aspect, the invention provides a vaccine for prevention of HIV infection or for alleviation of the effects of HIV infection, comprising human immunodeficiency virus-1 or human immunodeficiency virus-2 from which the portion of the HIV genome encoding at least the C-terminal 21 amino acids of the Vpr sequence has been deleted, together with a pharmaceutically- acceptable carrier. Preferably the portion of said genome encoding at least the C-terminal 33 amino acids of the Vpr sequence has been deleted. Even more preferably both a portion of the genome encoding the C-terminal region of the Vpr sequence and the portion of the HIV genome encoding the N-terminal of the Nef gene have been deleted. Relevant portions of the Nef gene are described in our co-pending Patent Application No. PCT/AU94/00254 (WO 94/26776), filed 18 May 1994. According to a fifth aspect, the invention provides a method of treatment of a disease mediated by cell proliferation, comprising administration to a mammal in need of such treatment of an effective amount of Vpr protein, or of a biologically active fragment or analogue thereof comprising at least one sequence of the consensus sequence disclosed herein, thereby to inhibit proliferation of cells mediating the said disease. Diseases mediated by cell proliferation include, but are not limited to, cancer, leukaemias and psoriasis. Proliferating cells are highly calcium dependent. We have found that the anti-proliferative effect of Vpr peptides is blocked by an inhibitor of Ca 2+ -channel transport. Therefore in this aspect of the invention, the Vpr protein or fragment or analogue thereof may optionally be used in conjunction with an enhancer of Ca 2+ -channel transport. According to a sixth aspect, the invention provides a method of treatment of a disease caused by a pathogen, comprising the step of administering to a mammal in need of such treatment of an effective amount of Vpr protein, or of a biologically-active fragment or analogue thereof comprising at least one sequence of the consensus sequence disclosed herein. Diseases caused by bacteria, parasites, yeasts, or fungi are all within the scope of this aspect of the invention. According to a seventh aspect, the invention provides an agent for delivery of a pharmaceutically active substance to a cell membrane or to the interior of a cell, comprising a Vpr peptide or a biologically active fragment or analogue thereof comprising at least one amino acid sequence motif selected from HFRIG and HSRIG, said peptide, fragment or analogue being linked to said pharmaceutically active substance. The invention also provides a method of delivery of a pharmaceutically active substance to a cell membrane or to the interior of a cell, comprising the step of contacting said cell with an agent as defined above. The active substance may be any chemical entity capable of being linked to a peptide, and in particular may be a peptide or a nucleotide. Coupling may be effected by any convenient means, for example chemical coupling using agents such as carbodiimide. Where the active substance is a peptide, the Vpr peptide and the pharmaceutically active peptide may be synthesised together by recombinant means as a fusion protein. The person skilled in the art will be aware of a variety of suitable pharmaceutically active agents which could be delivered in this way, and of methods whereby they may be linked to the Vpr protein. Such a person will also be aware of methods suitable for testing whether the linkage has been effective, and whether the agent retains the desired pharmaceutical activity. In preferred embodiments of the invention, the Vpr protein is a fragment comprising at least the C-terminal 21 amino acids of the Vpr sequence, more preferably at least the C-terminal 33 amino acids of the Vpr sequence. It will be clearly understood that Vpr protein or its biologically active fragments linked to a carrier or fusion protein, such as glutathione-S-transferase (GST) , are within the scope of the invention. It will be further understood that recombinant, synthetic and naturally- derived Vpr protein and fragments and analogues thereof are within the scope of this invention. While the description herein relates specifically to Vpr protein of HIV-1, as described above HIV-2 also possesses a Vpr protein, and it will be clearly understood that the invention is equally applicable to the Vpr protein and the equivalent of the H(S/F)RIG motif derived from HIV-2, and to the Vpx protein of other lentiviruses. Throughout this specification, the single letter code of abbreviations for amino acids is used. S/F indicates that either S or F may be present. Detailed Description of the Invention The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the drawings, in which: Figure 1 illustrates the scheme employed for cloning of the vpr gene for expression in yeast; Figure 2 shows the effect on growth of expression of HIV-1 auxiliary proteins in yeast; Figure 3 shows the morphological changes in yeast cells expressing Vpr. Copper-induced yeast cells were examined by light microscopy. A DY150 [pYEULCBX] control transformant is shown in the top panel (A) and a typical large DY150 [pYEULCBX.Vpr] transformant is shown on the bottom panel (B) . The bar represents 10 μ . Figure 4 shows the results of analysis of induced cells by flow cytometry; Figure 5 shows the identification of the toxic region in Vpr; A series of Vpr constructs is indicated. The region of Vpr produced by the construct is represented by pale blocks, while dark blocks represent GST; GST is not drawn to scale. The position of Baπ-HI (B) , -BcoRI (E) and -Sail (S) sites relative to the protein sequence is indicated. H(S/F)RIG, shown in bold, is encoded by sequences on either site of the Sail site. Growth of the transformed yeast cells producing these proteins is recorded in the right column. Figure 6 shows the relationship between HIV-1 Vpr and proteins from HIV-2, SIV and yeast. A. Alignment of HIV-1 Vpr with Vpr relatives. Vpr and Vpx proteins are aligned in their entirety. Sequences are derived from HIV-1 Vpr NL4-3 (Adachi et al, 1992), HIV-2R0D (Clavel et al, 1992) and SIVmac239 (Regier and Desrosierε, 1990) . Regions with three or more identical amino acids are shaded. B. Alignment of Saclp and HIV-1 Vpr. The entire amino acid sequence of Vpr is aligned with amino acids 77-176 of Saclp (Cleves et al, 1990) . Identical residues are shaded, and the residues in the H(S/F)RIG motif are underlined. Figure 7 illustrates the osmosensitivity of yeast expressing Vpr. Peptide 1 NH 2 -VTRQRRARNGASRS-COOH Peptide 2 NH 2 -CRHSRIGVTRQRRARNGASRS-COOH Peptide 3 NH-,-HFRIGCRHSRIGVTRQRRARNGASRS-COOH Figure 8 illustrates the strategy used for the construction of mutant viruses. Figure 9 shows the replication kinetics of mutant viruses in human PBMC, as measured by reverse transcriptase assay. A. Stimulated cells; B. Unstimulated cells Figure 10 shows the effect of synthetic Vpr peptides on yeast colony formation. A, no peptide; B, Peptide 1; C, Peptide 2; D, Peptide 3. Figure 11 summarises the results of the response of yeast cells to synthetic Vpr peptides. Treatment conditions are as described in Example 11. Figure 12 shows the dose response relationship for Peptide 3. Peptide 3 was added to yeast cells over a concentration range and cells were assayed for colony formation. Figure 13 shows the effect of yeast cell concentration on colony formation in the presence of a Vpr peptide. Various cell concentration were incubated in the presence of 5 μM Peptide 3 and cells were assayed for colony formation. Figure 14 shows the results of flow cytometric analysis of propidium iodide (PI) uptake following incubation of yeast cells with synthetic Vpr peptides for 1 h. Samples are No peptide, Peptide 1, Peptide 2 and Peptide 3. Figure 15 shows the results of flow cytometric analysis of propidium iodide uptake following incubation of yeast cells with Peptide 3 for various times. Figure 16 shows the results of flow cytometric analysis of propidium iodide uptake in RC2a cells electroporated with synthetic Vpr peptides. Figure 17 shows results obtained when mammalian cells were electroporated and analysed by flow cytometry for changes in cell structure as measured by forward and side scatters. Figure 18 shows protection of yeast cells by TMB-8. Figure 19 shows the association of FITC (fluorescein isothiocyanate) -labelled peptides with CD4 + human cells measured by flow cytometry following (A) electroporation, and (B) extracellular addition without electroporation. Figure 20 shows the association of FITC-labelled peptides without electroporation in S. cerevisiae yeast cells measured by flow cytometry. Figure 21 shows the internalised FITC-labelled peptide 3 in human CD4 + cells (A) and in yeast cells (B) . Figure 22 shows the genetic interaction between Vpr and Saclp and actin. Yeasts and bacteria Yeast strains employed in this study were Saccharomyces cerevisiae strain DY150 (iATa ura.3-52 leu2-3, 112 trpl-1 ade2-l his3 -ll canl -100 ) , Candida albicans clinical isolate JRW5, Candida glabrata strain L5 (leu) , Kluyvero yces lactiε strain MW-98-8c ( uraA arg lys ) and Schizosaccharomyces pombe strain SpULA (ade -704 u-ra4-D18 leul-32)h " . Strains were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) . An Bsc-he-richia coli strain TGI Δ(lac-proAB) supE thi hsdA5 F' [traD36 proAB* lacl q lacZAM15] was also employed for toxicity studies and plated onto 2xYT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl ) . Mammalian cells Two CD4+ cell lines, RC2a and Jurkat, were maintained in RPMI-1640, containing 10% heat inactivated foetal calf serum (HIFCS) . Mononuclear cells were isolated from HIV-1 seronegative blood obtained from blood bank volunteers by a standard Ficoll/Hypaque density gradient method. The peripheral mononuclear leukocyte cells (PBMC) were stimulated with phytohaemagglutinin (PHA : 10 μg/10 6 cells) for 48 h at 37°C, and were washed and resuspended in IL-2 medium, containing RPMI-1640 medium with 10% HIFCS, 10% recombinant human Interleukin-2 (Boehringer Mannheim) , 5 mM Hepeε, 0.1% sodium bicarbonate, 25 μg/ml glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml polybrene (Sigma) and 1:1000 anti-interferon (Miles) . Example 1 Molecular Cloning of HIV Genes The HIV-1 genomic clone pNL4-3 was used as the source of HIV-1 genes for amplification by polymerase chain reaction (PCR) . pNL4-3 (Adachi et al, 1986) was obtained from the National Instituteε of Health (NIH) AIDS Research and Reference Reagent Program, National Institutes of Allergy and Infectious Diseases, NIH (Adachi et a , 1986) . The cloning of the vpu and nef genes has been described previously (Macreadie et a , 1992, 1993) . The scheme used for cloning of vpr is described in Figure 1, while the cloning of vif employed PCR and similar known procedures. vpr was amplified from the HIV-1 genomic clone pNL4-3 (Adachi et al, 1986) using PCR and the primers shown. The PCR product was cleaved with BamHI + Smal and cloned into the yeast - E. coli shuttle vector pYEULCBX (Macreadie et al, 1992), and digested with BamHI + -EcoRI (T4 polished) to produce pYEULCBX.Vpr. In like fashion the other HIV-1 genes, nef , vpu and vif were also cloned into pYEULCBX to produce pYEULCBX.Nef27 (Macreadie et al, 1993), pYEULCBX.Vpu (Macreadie et a , 1992) and pYEULCBX.Vif. These plasmids were designed to direct the copper-inducible production of Nef, Vpu and Vif, respectively. vif primers were 5' GCTCCGGATCCATGGAAAACAGATGGCAGG and 5' CGCCCGGGAGCTCTAAAAGCTCTAGTGTCC. The vif PCR product was cloned as a BamHI-Smal fragment. BamHI and Smal cloning sites in the primers (above) are underlined, while sequences in italics represent the vif start and stop codonε. All amplified DNA was sequenced to verify the absence of errors. Cloning of nef , vpr, vpu and vif genes into the yeast expression vector pYEULCBX was designed to direct the copper-inducible production of Nef, Vpu and Vif, respectively. Example 2 Endoq-enouslv-Expreεsed vpr Protein Cauεes Growth Arrest in Yeast In this study we expressed vpr in yeast in order to discern itε functions. At the same time, as part of a general examination of the effects of the HIV-1 regulatory proteins on simple cellular functions, we also produced Vif, Vpu and Nef in haploid yeast and looked for their effects on cell growth. This was achieved by cloning vpr and the genes of other HIV-1 auxiliary proteins into the expression vector pYEULCBX to produce pYEULCBX.Vpr (see Fig. 1), pYEULCBX.Nef27 (Macreadie et al, 1993), pYEULCBX.Vpu (Macreadie et al, 1992), and pYEULCBX.Vif (this study) . Strain DY150 (MATa ura3-52 leu2-3,112 trpl-1 ade.2-1 Jιis3-ll canl-100; Macreadie et al, 1993), obtained from Dr David Stillman at the University of Utah Medical Center, was transformed with the above yeast vectors plus vectors for the copper-inducible production of glutathione S-transferase (GST) and GST fused to Vpr.DY150 was grown on YEPD medium (1% yeast extract, 2% peptone, 2% glucose) . Yeast cells were transformed by the electroporation procedure of Becker and Guarente (1991) and transformantε were grown on minimal selective medium containing 20 μg/ml hiεtidine, adenine and tryptophan and solidified, when required, with 3% Phytagar™ (Gibco) . Expression was induced by the addition of CuS0 4 to the amounts indicated, and growth was asεayed. Tranεformantε were suspended in sterile water and dropped out on to plates for growth at 28°C. The resultε are illustrated in Figure 2, which showε SD plateε (0.67% yeast nitrogen base (Difco) , 2% glucose) containing 0.25 mM CuS0 4 , 20 μg/ml histidine, adenine and tryptophan and solidified with 3% Phytagar™ (Gibco) . The proteins produced by the transformants are indicated. As εhown in Figure 2, profound effects on cell growth were caused by the Vpr protein, while the other HIV-1 proteins tested had no effect on vegetative cell growth. Low levels (0.25 mM) of CuS0 4 caused total growth arrest in cells expressing Vpr (Fig. 2) , while no adverse effectε were cauεed by the other proteinε even with induction levelε as high as 1 mM CuS0 4 . The effect of Vpr was unrelated to copper toxicity, since with no added CuS0 4 , where basal expression from the CUP1 promoter is 5% of the induced level (reviewed in Macreadie et al, 1994), Vpr transfσrmants grew at a slower growth rate than control transformants. The Vpr toxicity was found to be due to growth arrest, not killing, since induced cells, even after 24 hours in the presence of the inducer, formed colonies when plated on media with no added copper. The DY150 [pYEULCBX.Vpr] transformant colonies grown up from the assay were considerably smaller than DY150 [pYEULCBX] transformant colonies. These "small" colonies grew like the parental DY150 [pYEULCBX.Vpr] transformant upon subsequent culture without added copper (data not shown) , indicative of a cell cycle arrest after induction of Vpr synthesis followed by eventual recovery and return to the normal cell cycle. Example 3 Arrested Cells are Greatly Enlarged Examination of cells by light microscopy indicated that induced cells producing Vpr had a grossly altered morphology. As shown in Figure 3, Vpr-producing cells had a diameter of 16 μm, more than twice the diameter of the control DY150 [pYEULCBX] tranεformants grown under the same conditionε. It appeared that moεt of the intracellular space in the large cellε was devoid of structure and occupied by a single large organelle, poεεibly a vacuole. Thiε suggests that the DY150 [pYEULCBX.Vpr] transformantε were arrested in growth before cell division. Example 4 Flow Cytometry Analvsiε Cells were analysed and sorted using a Coulter Epics ® Elite flow cytometry. Illumination was with a 488 nm Argon ion laser, and forward angle light scatter (related to cell size) and εide scatter were recorded. Cells were sorted on the basis of forward angle light scatter. Live cellε were gated by propidium iodide exclusion, indicated by absence of fluoreεcence emission at greater than 600 nm following εtaining with 2 μg/ml propidium iodide. Induced yeaεt cellε were analysed by flow cytometry forward angle light scatter (proportional to cell size) in order to assess the proportion of altered cells. The resultε are shown in Figure 4, and confirmed that in the cell population Vpr transformants exhibited a greater degree of forward light scattering, indicative of their larger εize. Populationε, containing over 50,000 cells, are for DY150 [pYEULCBX] and DY150 [pYEULCBX.Vpr] as indicated. Example 5 Location of Sequences Causing Growth Arrest Includes H(S/F)RIG Repeated Motifs The sequences responεible for cauεing the growth arrest were identified by testing various portions of the Vpr protein for effects on cell growth. Since Vpr fused to glutathione S-transferase (GST) also caused a growth arrest (Fig. 2; construct GST-Vpr, Fig. 5), we also produced a series of GST fusion proteinε in the yeaεt GST-fuεion vector, pYEULCGT (Ward et al, 1994) . Deletion of the laεt 33 amino acidε of Vpr, encoded by an JBcoRI fragment (constructs VprBE and GST-VprBE, Fig. 5) , relieved the growth arrest, while the addition of this portion of Vpr to GST (conεtruct GST-VprEE, Fig. 5) cauεed a growth arrest, indicating that this domain was reεponsible for the growth arrest. A partial growth arrest was also seen with the addition of just the last 21 amino acidε of Vpr to GST (conεtruct GST-VprSE, Fig. 5) . In each case the growth arrest correlated with cell enlargement, as judged by flow cytometry analysis and light microscopy. Significantly, this C-terminal sequence iε the region lacking in many laboratory HIV-1 iεolateε that encode a truncated vpr gene product of 73 amino acidε due to a T inεertion (Yuan et al , 1990; Ogawa et a , 1990; Lavallee et al, 1990) . The Vpr in theεe iεolateε doeε not aεεociate with virionε (Ogawa et al, 1990), preεumably becauεe of the truncation. Our findingε confirm the importance of the same C-terminal region, but for another reason. This growth arrest in yeaεt may be linked to AIDS pathogeneεiε. The region of HIV-1 Vpr that causes cell growth arrest haε been compared with known Vpr relatives, the closest relative being the SIV Vpr followed by HIV-2 Vpr, and then Vpx proteins (Fig. 6A) . The sequence comprises 33% arginine, a much higher arginine content than that found in comparable portions of Vpx proteins. It is notable that there is conservation of a repeated motif, H(S/F)RIG, in Vpr species. The motif is present at amino acids 72-75 (encoded in the -ScoRI-Sall fragment) , and at amino acidε 78-82 (encoded in the Sal l-EcoRl fragment) . The greater toxicity caused by the fragment encoding two copies may indicate a copy number effect or possibly a conformational effect. In a search for a cellular relative to Vpr uεing the program ALIGN, we found that a yeaεt protein, Saclp (Cleveε et al, 1989), haε the most significant sequence similarity of cellular proteinε liεted in the Genbank databaεe (release 82.0). In the alignment of Saclp and Vpr (Figure 6B) it can be seen that Saclp haε 60% identity in the H(S/F)RIG motifε including the terminal Gε, the part of the motif that iε totally conserved in Vpx as well. Over the entire alignment there are 32% identical and 45% similar amino acids. Example 6 Peptide Svnthesiε The peptideε produced were as follows: Peptide 1 NH 2 -VTRQRRARNGASRS-COOH Peptide 2 NH 2 -CRHSRIGVTRQRRARNGASRS-COOH Peptide 3 NH-.-HFRIGCRHSRIGVTRQRRARNGASRS-COOH Peptide 4 NH 2 -HFRIGCRHSRIG-COOH Peptide 5 NH 2 -RHSRIGVTRQRRARNGASRS-COOH Peptide 6 NH 2 -IFRAGTRYFRRG-COOH Peptides were synthesised on an Applied Bioεyεtemε 430A Peptide Synthesizer, uεing the FastMoc solid-phaεe technique in which α-amino groupε were protected by baεe-labile Fmoσ (9-fluorenylmethyloxy- carbonyl) groupε. The εhorteεt sequence waε εyntheεised on to the resin, then approximately one third of the peptide/reεin waε removed from the reaction veεεel. Syntheεiε was then continued on the remainder until the second peptide was aεεembled, at which εtage half of the peptide/reεin waε removed from the reaction veεεel. The third peptide was then asεembled on to the remaining peptide/reεin. The arginine side chains were protected by tert-butyl groupε and glutamic acid by the O-ter -butyl group. Couplingε were achieved by using 2- (lH-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophoεphate activation of amino acidε and N-methylpyrrolidone aε solvent. The peptideε were cleaved from the reεin with trifluoroacetic acid (pluε phenol, ethanedithiol, thioaniεole and water aε εcavengerε) . The peptides were dialysed againεt electroporation buffer (0.213 g/1 Na 2 HP0 4 , 0.068g/l H 2 P0 4 , 93.1 g/1 sucrose) (Wojchowski and Sytkowεki, 1986) before electroporation. For Peptideε 4 to 6, protection waε aε followε: α-amino groupε by base-labile 9-fluorenylmethloxycarbonyl (Fmoc) groupε; arginine side chainε by 2,2,5,7,8- pentamethylchroman-6-εulfonyl (Pmc) ; εerine and threonine by tert-butyl groupε; asparagine, glutamine, histidine and cyεteine by trityl; and glutamic acid by the O-tert-butyl group. Couplings were achieved by using 2-(lH- benzotriazol-1-yl) -1,1,3,3-tetramethyluonium hexafluorophoεphate (HBTU) activation of amino acids and N- methylpyrrolidone (NMP) as solvent. The peptides were cleaved from the reεin with TFA, pluε phenol, ethanedithiol, thioaniεole and water aε εcavengerε. Example 7 H(S/F)RIG Motifε in Synthetic Peptideε Cause Oεmoεenεitivitv We further investigated the function of the H(S/F)RIG motifε uεing the εynthetic peptideε: NH 2 -VTRQRRARNGASRS-COOH NH 2 -CRHSRIGVTRQRRARNGASRS-COOH NH 2 -HFRIGCRHSRIGVTRORRARNGASRS-COOH produced in Example 6, that contain the penultimate 14, 21 and 26 amino acidε, reεpectively, of Vpr. The H(S/F)RIG motif (underlined) is present at zero, one and two copies, reεpectively, within these peptideε. These peptides were electroporated into yeast cells which were then analysed for osmosensitivity. Peptideε, diεεolved in electroporation buffer at 2 mg/ml, were electroporated into yeaεt cellε using a Baekon 2000 (Saratoga, CA) . Conditions for the treatment in the Baekon 2000 were: 2 11 pulses, 8 kV, 0.8 sec burεt time, 100 μεec pulεe time, 10 cycleε, 1 mm gap between εolution and upper electrode. The cuvetteε contained 30 μl of yeaεt εuεpenεion in freεh YEPD growth medium pluε 5 μl of Dulbecco'ε Phoεphate-Buffered Saline and 5 μl of peptide εolution. It waε found necessary to achieve a kill of 60-80% in order to achieve uniform penetration of the surviving cellε. Cells were examined for osmoεenεitivity by plating onto YEPD medium and YEPD medium containing 1.2 M KCl, 1.8 M sorbitol or 0.9 M NaCl, and counting the numbers of colony-forming units, as described by Chowdhury et al (1992) . Osmosenεitivity waε calculated by comparing the relative numbers of colony-forming units on the two media. All viable cellε, including oεmoεenεitive cellε, grew on YEPD, but those that were osmoεenεitive did not grow on high osmotic εtrength media. The results, presented in Figure 7, εhow that cellε treated with the peptide lacking an H(S/F)RIG motif were essentially unperturbed. However, the peptides containing H(S/F)RIG motifs cauεed oεmotic εenεitivity εuch that up to 50% of the cells were killed on high osmotic strength media. The effects were commensurate with the number of copies of H(S/F)RIG motif present, indicating a direct role for this sequence. Example 8 Pathoqenicitv is Aεεociated With the Sequence Containing H(S/F)RIG Motifs The region of the Vpr protein containing H(S/F)RIG motifs may be correlated to the pathogenicity of human and simian immunodeficiency viruseε. A brief compilation of sequences of Vpr and Vpx from human and simian immunodeficiency viruses is εhown in Table 1. There iε almoεt total conservation of the 12 amino acids containing two repeated H(S/F)RIG motifs in HIV-1, a highly pathogenic virus. Seven simian immunodeficiency virus Vpr sequences show high conservation (two changes) of the sequence containing the H(S/F)RIG motifε. However, the two sequences εhown by the aεteriεk have poor conservation of the sequence (8 or 9 changes) . Both the mandrill virus and the Sykeε' monkey viruε εhow poor sequence conservation, and are reported to cause asymptomatic infection (Hirsch et al, 1993; Tsujimoto et al, 1989). In HIV-2 iεolateε there are between two and five changeε from the reference sequence. HIV-2 is leεε pathogenic than HIV-1, and we believe that theεe changeε may be a reaεon for the reduction in pathogenicity. Additionally the preεence of Vpx may reduce pathogenicity. Matεuda et al (1993) εhowed that when Vpx replaced Vpr in HIV-1, the viruε loεt itε infectivity. Thuε we predict that any viruε that produceε Vpx may be expected to be leεε pathogenic than one which produceε Vpr alone. Table 1 H(S/F)RIG Motifε in Vpr Relativeε HIV-1 NL43 HFRIGCRHSRIG HAN HFRIGCRHSRIG MN HFRIGCRHSRIG ELI HFRIGCOHSRIG SC HFRIGCRHSRIG LAI HFRIGCRHSRIG SF2 HFRIGCOHSRIG MAL HFRIGCO . HSRIG OY1 HFRIGCOHSRIG NDK HFRIGCQHSRIS NH52 HFRIGCQHSRMG conεensuε HFRIGCRHSRIG L Q MS SIV SIVmac239 HFRGGCIHSRIG SIVmacl42 HFRSGCSHSRIG SIVmac251 HFRGGCNHSRIG SIVmacMNE HFRGGCTHSRIG SIVmmm H4 HFRSGCAHSRIG SIVmmmPBJ HFRGGCRHSRIG SIV cpz HFRLGCO.HSRIG Table 1 (cont) consensus HFRGGCRHSRIG S I L S T A Q N SIVmndGBl HLAQGCDGTFRE * SIVsykeε HFAAGCPORTRY * HIV-2 ROD HFRAGCGHSRIG D205 HFRAGCGHSRIG ISY HFRAGCGHSRIG NIHZ HFRAGCGHSRIG CAM2 HFRAGCNHSRIG D194 HIRAGCDRSRKG GH1 HLRAGCNRSRIS ST HFRAGCGRSRIG BEN HFRAGCNRSRIG conεensus HFRAGCGHSRIG I DR KS L N Vpx SIVmac239 HCKKGCRCLGEG SIVmacl42 HCKKGCRCLGEG SIVmac251 HCKKGCRCLGEG SIVmacMNE HCKKGCRCLGEG SlVmmm H4 HCKKGCRCLGEE SIVmmmPBJ HCKKGCRCLGGE consensus HCKKGCRCLGEG GE HIV2 ROD HVRKGCTCLGRG HIV2 D205 HYTKGCRCLQEG HIV2 CAM2 HFKRGCTCLGGG HIV2 ISY HFKKGCTCRGEG HIV2 NIHZ HAKRDGTCLGGG HIV2 D194 HFKKGCTCLGRG HIV2 GH1 HFKRGCTCLGGG HIV2 ST HFKRGCTCLGGG HIV2 BEN HFKRGCTCWGED Table 1 (cont.) consensus HFKRGCTCLGGG YRRD R WQRD VT R E A S SIVagml55 HFRCGCRRROPF SIVagm 3 HFRCGCRRRQPF SIVagmTYO HFRCGCRRROPF consensus HFRCGCRRRQPF The overall consensus for the Vpr sequence, excluding those represented by the aεteriεk, is: HFRIGCRHSRIG I L QR MS L G N I s T A Q G D In summary, it appearε that in the sequence HFRIGCRHSRIG, the residues underlined are invariant in Vpr. F can be I or L; the I can be L, G, S or M; the last G can be S. It should also be noted that the C between motifs is invariant. Example 9 Replication Kinetics of Mutant Proviruseε Production and titration of virus culture Half clones of the mutant and wild-type HIV DNA were co-transfected to HeLa cells (5 x 10 6 cells) in T25 flaskε, by the Lipofectamine (GIBCO-BRL) method. PBMC (20 x 10 6 cellε) were added 12 h after tranεfection and the cell free virus production waε measured at regular intervals. The supernatant waε harveεted at maximum production of cell free viruε and used as εtock viruε. Titrationε of viruε εtocks were done in 24 well Linbro plates, and the end point dilution waε scored by both Reverse Transcriptaεe (RT) activity and visible cytopathic effect. RT assay in microtitre plates were performed according to standard methods. Construction of mutant proviruε HIVNL 4.3 molecular clone (Adachi et al, 1986) waε re-cloned aε two half fragmentε into the pKP59 vector for the point mutation of the initiation codons of the nef and vpr geneε. Mutant proviruεes were constructed according to the procedures described in Figure 8, using the mutageneεiε scheme summarized in Table 2. Table 2 Mutagenesis Scheme Mutation Nucleotide Final Clone Gene Oligo position (nt) changes effect Into Nef N7 8788 ATG to AAG no nef pKP3EA N8 8829 ATG to ATC expressed Vpr V2 5559 ATG to GTG no vpr pKP5SE 5565 CAA to TAA expressed Vpr VMM 5770 to 5804 deletion of deletion pKP3EA 36 nucleotideβ of H(S/F)RIG motifs The HIV-1 molecular clone employed was pNL4-3. Because of instability of the full length clone in E. coli , half-clones were constructed in the low copy vector, pKP59, and stably maintained in E. coli . The 5' sequences were introduced as a StuI-ϋteoRI fragment while 3' sequences were introduced as an ficoRI-Avrll fragment. These half-clones could be appropriately digested (Xbal+EcoRl fragment for the 5' clone and -EcoRI+ffaell for the 3' clone) and the cut DNA introduced into mammalian cells where in vivo recombination restored the wild-type viruε. To obtain mutant virus appropriate segments of pNL4-3 were cloned into a phagemid, single-stranded DNA was produced, and second εtrand waε synthesised in the preεence of oligonucleotideε, εhown in Table 3, which were deεigned to introduce εpecific vpr and nef mutations. Table 3 Mutagenic Oligonucleotides Oligonucleotide Code 5'-GGA TTT TGC TAT AAG AAG GGT GGC AAG-3' N7 5'-GTA AGG GAA AGA ATC AGA CGA GCT G-3' N8 5'-CAG AGG ACA GGT GGA ATA AGC CCC CAG AAG-3' V2 5'-CTG CAA CAA CTG CTG TTT ATC *GTT ACT CGACAG AGG-3' VMM * indicateε the εite targetted for deletion in vpr Following purification and verification of sequence changes the DNA waε sub-cloned into the pKP59- half-clone, replacing the wild-type sequence with a mutant sequence. The mutant cloneε do not expreεε Nef, do not express Vpr, or do not express either protein. Infection of PBMC Peripheral blood mononuclear cells were infected at a 0.01 multiplicity of infection (MOI) , and the cell free supernatantε were aεεayed daily for reverse transcriptaεe (RT) production by εtandard techniqueε. In εtimulated PBMC, mutant proviruεes defective for the production of Nef or Vpr produced similar amounts of cell-free virus particles, which were in both cases considerably lesε than in the parent virus strain. The effect of Vpr on virus replication appears to be mediated by the H(S/F)RIG motifs, as shown in Table 4. Table 4 Effect of Deletion of the H(S/F)RIG Motif on the Replication of Virus PBMC, as Measured by Cell-Free RT Activity DAY (P.I) HIVNL 4.3 VPR MOTIF (-) 3 7440 3878 7 269002 174735 10 219986 165945 15 109533 57493 17 93679 42692 A mutant proviruε that waε defective in the production of both Nef and Vpr waε severely repressed in virus production, and showed delayed replication kinetics (Figure 9a) . In unstimulated human primary cells, which closely reεemble the in vivo cell population, both Nef and Vpr are indispensable for cell-free virus production (Figure 9b) . The Vpr " mutant produces smaller amounts of virus, the Nef " mutant exhibits delayed replication kineticε, while the Nef " Vpr ' double mutant shows no virus production. Therefore Nef and Vpr appear to act synergiεtically. Example 10 H(S/F)RIG Motifε in Synthetic Peptideε Cause Growth Arrest in Yeaεt Peptideε were dialyεed againεt PBS and added at a final concentration of 2 μM to yeast cells suspended to a density of 10 6 cells/ml in a final volume of 200 μl water. After incubation for 1 h, 5 x 10 4 cellε were εpread on to εolidified YEPD, and the plateε were examined for colony growth after 40 hours incubation at 28°C. Peptide concentrations were determined by quantitative amino acid analysiε of peptide εolutionε. The addition of Peptide 2 or Peptide 3 caused the cells to completely lose colony forming ability, while the addition of Peptide 1 had no effect (Figure 10) . This compares with Example 5, in which we found a correlation of bioactivity, as assessed by osmosenεitivity, with the intracellular preεence of H(S/F)RIG motifε. Therefore we further inveεtigated peptideε, εuch aε Peptide 4, which contained only the H(S/F)RIG motifε, and which alεo cauεed some osmoεenεitivity, aε εhown in Figure 11. Peptide 4 alεo cauεed complete loεε of colony forming ability. Peptide 5, which iε like Peptide 2 but lackε the cyεteine, alεo cauεed a conεiderable effect, εuggeεting that the cyεteine waε not eεεential for the activity, but that it did increaεe the activity, poεεibly due to a conformational effect. We uεed Peptide 3 to establish a dose responεe relationship. Treatment of cells with a range of concentrations of Peptide 3 indicated that the loweεt peptide concentration which induced complete growth arrest was about 1 μM, aε εhown in Figure 12. Concentrations down to 0.05 μM were partially effective, but below this concentration there was no effect. Example 11 Blockage of the Growth Arrest Effect by Cell Mass There was also an effect cauεed by cell concentration. At high cell concentrationε the effectiveness of the peptides was limited. Figure 13 shows colony formation after treatment of yeast at a range of cell densitieε with 5 μM of Peptide 3. At cell denεities up to 10 5 cells/ml a complete effect can be seen; however, at concentrations above 10 6 cells/ml no effect was observed. Thiε suggeεtε that about 10 6 molecules of peptide per cell are required for inhibition of colony formation. We have also observed that the effect may be abrogated by the presence of medium; for example the effect iε greatly reduced in the presence of YEPD even at concentrations aε low aε 1/10 of normal εtrength. Example 12 Effect of Synthetic Peptideε on Other Microorganisms We have investigated the effectε of theεe peptideε on the growth of several additional microorganiεms, and the results are shown in Table 5. Table 5 Effect of Peptide on Colony Formation of Bacteria, Budding Yeasts and Fiεεion Yeast % "kill" after peptide treatment Peptide S. cerevisiae C. albicans C. glabrata K. lactis Sz . po be E. coli None 0 0 0 0 0 0 1 0 0 0 0 0 0 r 2 100 100 100 100 100 100 e 3 100 100 100 100 100 100 4 100 90 99 100 98 100 5 93 90 99 100 78 61 In E. coli and three budding yeastε, Candida albicans , Candida glabrata and Kluyveromyceε lactis , and in the fiεεion yeaεt Schizosaccharomyces pombe , the reεultε were εimilar to thoεe seen with S. cerevisiae . An effect was also seen on mammalian cells, in which the peptideε inhibit the formation by RC2a cells of a lawn on a culture plate surface. We treated E. coli with Peptideε 1, 2 and 3 in the same way as yeast, except that the treated cells were suspended in 2% glucose/50 mN HEPS. The data in Table 5 εhow that peptideε containing the H(S/F)RIG motif affect the viability of E. coli . However, we found that when E. coli waε εuεpended in PBS and treated with theεe peptideε, no loss of colony-forming ability was observed. It therefore appears that the activity is dependent on the medium used. Example 13 Effect of Peptideε on Yeaεt Cell Permeability We examined cells with Fungolight Live/Dead Stain (Molecular Probes) , and found that cells remained alive, ie. metabolically active, for several hours after the peptide treatments; however, they had lost colony forming ability. Thuε the effect of the peptideε seems to be to produce an irreversible growth arrest. We further examined the peptide-treated cells by staining with propidium iodide, a vital stain, followed by flow cytometry. The results, shown in Figure 14, confirm that after 1 h there is a marked effect caused by Peptides 2 and 3. With Peptide 1 or no treatment, 95% of the cells take up little or no propidium iodide. With Peptides 2 and 3, fewer than half of the cellε take up propidium iodide. Propidium iodide uptake iε uεually indicative of cell death or of membrane damage to the yeaεt. The analyεiε alεo εhows that there iε no significant cell lysiε at thiε time. Kinetic analyεiε of the effect of Peptide 3, shown in Figure 15, indicates that this effect is immediate, with cells taking up propidium iodide within minutes of the peptide being added. Example 14 VPR Peptides Kill CD4 + Cells We have used two CD4 + cell lines, RC2a and Jurkat, to represent promonocytic and T lymphocytic cell lines respectively. Electroporation of peptideε 10 μg of peptide waε added to 1 million CD4 + cellε which were εuεpended in 55 μl Baekon buffer (1.5 mM Na 2 HP0 4 , 0.5 mM KH 2 P0 4 , 0.27 M Sucroεe pH 7.0) and 10 μl of PBS (phoεphate-buffered εaline) . Electroporation conditionε in the Baekon 2000 Advanced Macromolecule Transfer System (San Francisco, CA) were 2 11 pulseε, 8 kV, 0.8 sec burεt time, 62.5 μεec pulse time, 3 cycleε, 85 mm gap between εolution and upper electrode. Peptide- electroporated cellε were reεuspended in 1 ml RPMI-1640 10% HIFCS and incubated in a humidified 5% C0 2 incubator at 37°C. Re-electroporation and pre-treatment of cells Peptide-electroporated cells were harvested at 24 h for re-electroporation of the respective cellε with peptideε or without peptide, and the cells were analysed by flow cytometry after 24 h. One million cells were pretreated either with 0.5 nM TMB-8 hydrochloride ( [8- (diethylamino) -octyl-3,4, 5-trimethoxybenzoate] , HCl) or 0.5 μM proεtaglandin E2 for 30 min before electroporation. Cellε were maintained in the same concentration of the respective reagents after electroporation. Preparation of cells for flow cytometry One million cells were harvested and washed once in PBS at 1600 rpm for 5 min, and the pellet was resuεpended in 200 μl PBS containing 2 μg propidium iodide in preparation for flow cytometry analyεis. Cells were analyεed for a number of parameters 24 and 48 h after electroporation, uεing a Coulter Epicε Elite Flow Cytometer. Forward and εide scatters of a 488 nm argon ion laser were measured. Propidium iodide exclusion waε measured by the absence of fluoreεcence emiεεion at greater than 600 nm. Figure 16 εhows that Peptide 3 kills RC2a cellε to a significant extent, compared to the mock electroporated cellε. Peptide 2 killed a lower number of cellε than did Peptide 3, and the reεultε were comparable in both cell lineε. However, Peptide 1, which lackε the H(S/F)RIG motif, doeε not affect these cell lines. The effect of Peptides 2 and 3 was enhanced by pretreatment with prostaglandin E2, and hence obviateε the need for double electroporation. The effect of the peptide iε modified by pre-treatment with the Ca 2+ -channel blocker TMB-8 HCl. The H(S/F)RIG motif alεo influenceε the cell εtructure aε meaεured by forward and εide scatters, as shown in Figure 17. Peptide 3 has produced a right εhift of both εide and forward εcatterε compared to the mock electroporated and other peptideε electroporated cellε. Thiε εhowε that Peptide 3 induces an increase in both cell size and cellular granularity. Example 15 Blockage of Yeast Cell Growth Arrest by TMB-8 and High Ionic Strength The addition of TMB-8 for 30 min prior to the addition of the peptides abrogates the effect of Peptide 3, as shown in Figure 18. Various concentration of TMB-8 were preincubated with yeaεt cellε for 30 min and then the cells were incubated in the presence or absence of 5 μM Peptide 3. Cellε were then assayed for colony formation. With no TMB-8 there were no colonies formed. Low concentrations of TMB-8 gave protection to about 13% of cells. TMB-8 caused some toxicity at high concentration, but at lower concentrations no toxicity was observed. At theεe lower levelε of TMB-8 Vpr peptideε were not totally effective in inhibiting the colony forming ability of yeaεt cellε. Sodium, calcium, potaεεium or lithium ionε totally abrogated the effect of Vpr peptideε, if added 30 minε before or immediately after the addition of Peptide 3. The oεmotic εupport εorbitol alεo provided partial protection, and total protection waε provided by incubation with 0.1 x YEPD. Theεe reεultε are summarised in Table 6. Thiε apparent protection in the preεence of salts is due to the inability of yeaεt cellε to bind and internaliεe the peptideε containing the H(S/F)RIG motif. However, in the caεe of mammalian cellε the uptake of theεe peptideε waε obεerved in normal serum-containing medium. Table 6 Negation of Effect of Peptide 3 on Yeast Colony Formation Example 15 Viability of Bacterial Cells Producing Vpr Cells were treated with the inducer IPTG and aliquots plated on to 2 x YT + Ampicillin plates after appropriate times, and the number of colonies was counted after overnight incubation of the plateε. The data εhow that the production of GST and GST-Vpr.BE (encoded by the BamHI-.EcoRI fragment of vpr) doeε not kill E. coli cellε, but induction εlowε the growth. The production of GST fuεed to the full-length Vpr protein leadε to εimilar effectε after three hourε induction; however, after 30 hourε in inducer there iε an actual reduction in the number of ampicillin reεiεtant cellε/ml. Thiε iε deεpite the culture reaching a typical optical denεity after overnight incubation, and indicates that the vast majority of cells in the culture have loεt their Amp r determinant (and no longer express vpr) . Furthermore it implicates the C- terminal region of Vpr in the cell death. It is alεo clear that the uninduced cellε have not increased in number, indicating a σytostatic effect in the absence of inducer. However, the cells grew to 10 8 cellε/ml, εuggesting that the toxic effect may be specific to a particular condition, such aε growth in spent medium. Table 7 Protein [Amp* cells/ml] and time after induction produced 30 hours -inducer •-- nducer -inducer +inducer GST 1.1 x 10 8 4 x 10 8 2.2 x 10 8 4.8 x 10 9 4.4 x 10 9 GST-Vpr.BE 6.8 x 10 7 2 x 10 8 4 x 10 7 4.8 x 10 s 2.2 x 10 s GST-Vpr 1.3 x 10 8 4 x 10 8 1.4 x 10 8 1 x 10 8 <2 x 10 6 Example 16 Interaction of Fluorescence-Labelled Peptideε with Cells Figure 19 εhows the asεociation of FITC (fluoreεcein iεothiocyanate) -labelled peptides with CD4 + human cells measured by flow cytometry following (A) electroporation, and (B) extracellular addition without electroporation. Figure 20 εhowε the association of FITC-labelled peptides without electroporation in S . cerevisiae yeast cellε measured by flow cytometry. Peptides 2-4 exhibit high degrees of aεεociation with yeaεt cellε, while there is a considerably lesser asεociation of Peptide 5. Peptide 1, which lackε the H(S/F)RIG motif, exhibitε over one hundred-fold leεε aεεociation with cells than Peptide 2 and 3. By light microscopy we have observed that the FITC- labelled Peptide 3 efficiently targets into yeast and mammalian cells, aε εhown in Figure 21. Peptideε 2 and 4 alεo behave similarly. These data indicate that the H(S/F)RIG motif are εufficient for intracellular targetting and they, or related derivativeε that could alεo be a εubεet of the sequence, will be uεeful carrierε for the delivery of agents into cellε for treatment of diεeaεeε. Figure 21 εhowε the internaliεed FITC-labelled peptide 3 in human CD4 + cellε (A) and in yeaεt cellε (B) . The FITC-labelled human cellε include εome cellε that are εtill intact and others undergoing lyεiε. Example 17 Genetic Interaction Between Vpr and Sacl p and Actin Yeaεt actl and sacl mutants, DBY1195 and DBY1715 respectively, were transformed with pYEULCBX and pYEULCBX.Vpr. Tranεformants were then induced on plates containing 0.5 mM copper sulfate to aεεay for the effectε of the Vpr. DY150 [pYEULCBX] and DY150 [pYEULCBX.Vpr] tranεformantε have been described previously. An example of these results for the sacl -vpr interaction is shown in Figure 22. We have found that Vpr shows structural homology to the yeast protein Saclp. The preciεe function of Saclp in asεembly of the actin cytoεkeleton of yeast cells iε εtill under inveεtigation; however, sacl mutants display profound cytoεkeletal defectε and growth arrest at low temperature (Cleves et al, 1989; Novick et al, 1989; Whitterε et al, 1993) . Production of Vpr in yeaεt possibly causes similar effects to sacl mutantε, due to sequence and functional εimilarity between Saclp and Vpr; the production of Vpr could compete with normal Saclp function and lead to cytoεkeletal defectε, including groεε cell εize and ultimate growth arrest. Indeed, in the many εtudieε of yeaεt with cytoεkeletal defectε, mother cellε are abnormally large and daughter cellε are abnormally εmall (see for example Liu and Bretscher, 1992) . We have found that time-lapse analysiε of newly-induced cellε producing Vpr εhowε the same phenomenon. Osmoεensitivity also indicated possible cytoεkeletal defects induced by Vpr. Large cells producing Vpr were iεolated by flow cytometry and plated onto media containing high osmotic strength and normal media. Only 50% of the cellε capable of growth on normal medium could grow on high osmotic εtrength medium, indicating structural defects in those cells. Vpr appears to produce cytoskeletal defects in mammalian cells as well aε in yeaεt cellε. Work by Levy and colleagues showed that in a rhabdomyosarcoma cell line Vpr produced cell replication arrest and groεε cell enlargement (Levy et al, 1993) . Furthermore, in a CD4 + T-lymphoblastoid cell line it was shown that HIV-1 caused ultras ruetural changeε, including membrane disruption, "ballooning" and vacuoliεation of the endoplaεmic reticulum, during the firεt hour of infection (Fermin and Garry, 1992) . Theεe data are conεiεtent with cytoεkeletal defects, and investigation of the cytoskeleton in those cellε would be of intereεt. What iε the role of Vpr in the HIV-1 life cycle, and iε induced growth arrest relevant to this? For some time there haε been a dilemma regarding the diεtinction between HIV-1 and other retroviruses: retroviruses uεually require cell proliferation for infection, while HIV-1 infects non-proliferating cells such as terminally- differentiated macrophages. Lewis et al (1993) showed that CD4 + cell lines can be productively infected with HIV-1 when they are arrested in G2 growth phase. Non- proliferation of hoεt cellε could therefore be an initial requirement for a productive infection of all or some cell typeε. The function of Vpr may be to bring about growth arrest so that a process like integration may occur. If this were εo, it would account for Vpr (and Vpx counterparts) being virion-aεεociated, εo that early eventε can be initiated. Antibodieε to Vpr have been detected in only 17% of AIDS patientε, but are found in 47% of asymptomatic individualε (Wong-Staal et al, 1987), εuggeεting that the Vpr iε preεent early in infection, and therefore that it iε probably eεεential only at that time. It alεo followε then that inhibitorε of Vpr εhould prevent infection or εlow extracellular spread of the virus. In this εtudy we have εhown that portionε of Vpr containing the εequence HFRIGCRHSRIG or even RHSRIG cauεe cell damage and irreverεible growth arrest when added to yeast cellε. Thiε effect iε related to cell number and peptide concentration, and it appearε that a minimum of 10 6 molecules of peptide per cell iε required to observe the effect. We have alεo εhown that the same sequence was involved in cauεing oεmoεenεitivity and structural defects when peptideε containing thiε εequence were electroporated into cellε. Only intracellular effectε were examined after electroporation, since YEPD in the electroporation medium abrogated the extracellular effect. Peptides containing H(S/F)RIG motifs appear to cause cell damage reεulting in increaεed permeability and cell lyεiε in both mammalian and yeaεt cells. We have observed that Vpr peptides containing the H(S/F)RIG motif are cell aεεociated; fluoreεcence microεcopy using FITC- labelled peptides indicateε that they are internalised within 1 h. We have ascertained that the H(S/F)RIG motif causeε active uptake of the peptide; uptake of FITC- labelled Peptide 1 appeared to be one hundred-fold lower than that of peptideε containing the H(S/F)RIG motif, εuggeεting that the motif doeε promote uptake. In Example 7, peptide uptake waε artificially obtained by electroporation, and the outcome εtudied waε oεmoεenεitivity; the electroporation technique itεelf led to variable degrees of cell death or losε of colony-forming ability. However, among the numerous cells that formed colonies there was a high degree of osmotic sensitivity. The effects observed in Exampleε 10 to 15 are quite different, with total loεε of colony forming ability. Theεe differenceε may be related to the localiεation of the peptide within the cell. Oεmoεenεitivity, rather than loεε of colony forming ability, waε alεo obεerved with the expreεεion of the vpr gene in yeaεt in a more life-like εituation, aε described in Example 2. What AIDS phenomenon then can be correlated with our resultε? Recent εtudieε by Levy et al (1994) εhow that Vpr doeε exiεt in the serum, εuggeεting that it iε releaεed from infected cellε, and indicating that in designing putative antagonists of the protein it is relevant to consider the extracellular effects of Vpr, which may be reεponsible for the killing by HIV of uninfected host cellε, aε well aε itε intracellular effectε. Uεing biologically active fragmentε of Vpr, we have εhown that parts of Vpr, and presumably the entire Vpr protein, irreversibly affect colony-forming ability via the action of the H(S/F)RIG motifs within Vpr. The mode of action of thiε effect may be related to the Ca 2+ ion channel, εince the Ca 2+ ion channel blocker TMB-8 abrogateε the effect, aε εhown in Exampleε 14 and 15. References cited herein are listed on the following pages, and are incorporated herein by this reference. It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodimentε and methodε deεcribed herein may be made without departing from the scope of the inventive concept discloεed in this specification. 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Claims: CLAIMS 1. An antagoniεt of the Vpr protein of human immunodeficiency viruε (HIV) , or of a biologically active fragment or analogue thereof, compriεing at leaεt one amino acid εequence motif εelected from HFRIG and HSRIG, εaid antagoniεt having the ability to inhibit one or more activitieε mediated by Vpr, selected from the group consiεting of growth arrest, cell replication arrest, cytotoxicity, cytoskeletal diεruption, and effectε on the endoplasmic reticulum. 2. An antagonist according to Claim 1, wherein the Vpr protein further comprises at leaεt one sequence εelected from the group conεiεting of HSRIS, HFRAG, HIRAG, HLRAG, RSRKG, RSRIS and RSRIG. 3. An antagoniεt according to Claim 1 or Claim 2, wherein the Vpr protein is a fragment compriεing at least the C-terminal 21 amino acids of the Vpr sequence. 4. An antagonist according to Claim 3, wherein the Vpr protein is a fragment compriεing at leaεt the C-terminal 33 amino acids of the Vpr sequence. 5. An antagonist according to any one of Claims 1 to 4, selected from the group consiεting of an antibody, an anti-εense RNA, and a triple-stranded DNA. 6. An antagonist according to Claim 5, which is an antibody. 7. An antagonist according to Claim 6, which is a monoclonal antibody. 8. An antagonist according to Claim 5, which is an anti-εenεe RNA. 9. An antagoniεt according to Claim 5, which is a triple-stranded DNA. 10. A pharmaceutical composition compriεing as active component an antagonist according to any one of Claims 1 to 9, together with a pharmaceutically-acceptable carrier. 11. A method of screening compounds suspected of being useful as antagoniεts of Vpr protein, or of a biologically active fragment or analogue thereof, compriεing the εtep of meaεuring the effectiveness of a test compound in inhibiting the activity of Vpr in an asεay of a biological activity εelected from the group consisting of growth arrest, cell replication arreεt, cytotoxicity, cytoεkeletal diεruption, and effects on the endoplasmic reticulum. 12. A vaccine for prevention of HIV infection or for alleviation of the effects of HIV infection, comprising human immunodeficiency viruε-1 or human immunodeficiency viruε-2 from which the portion of the HIV genome encoding at least the C-terminal 21 amino acids of the Vpr sequence has been deleted, together with a pharmaceutically- acceptable carrier. 13. A vaccine according to Claim 12, wherein the portion of said genome encoding at least the C-terminal 33 amino acids of the Vpr εequence haε been deleted. 14. A vaccine according to Claim 12 or Claim 13, wherein both a portion of the genome encoding the C- terminal region of the Vpr εequence and the portion of the HIV genome encoding the N-terminal of the Nef gene have been deleted. 15. A vpr gene in which the region encoding C- terminal portionε of the Vpr protein has been replaced by an inhibitory antisense sequence or by a εequence which encodeε an inhibitory peptide. 16. A method of treatment of HIV infection, compriεing the εtep of adminiεtering to a εubject in need of εuch treatment an effective amount of an antagoniεt of Vpr protein, or of a biologically active fragment or analogue thereof, aε defined in any one of Claims 1 to 9, thereby to prevent HIV infection, to prevent progresεion of HIV infection to εymptomatic AIDS, or to alleviate the εymptoms of AIDS. 17. A method of treatment of a disease mediated by cell proliferation, comprising the step of adminiεtration to a mammal in need of εuch treatment of an effective amount of Vpr protein, or of a biologically active fragment or analogue thereof comprising at leaεt one amino acid εequence motif εelected from HFRIG and HSRIG, thereby to inhibit proliferation of cellε mediating the said diseaεe. 18. A method according to Claim 17, wherein the disease mediated by cell proliferation iε a cancer, a leukaemia, or pεoriaεiε. 19. A method according to Claim 17 or Claim 18, wherein the Vpr protein or fragment or analogue thereof is used in conjunction with an enhancer of Ca 2+ -channel tranεport. 20. A method of treatment of a disease cauεed by a pathogen, comprising the step of administering to a mammal in need of such treatment of an effective amount of Vpr protein, or of a biologically active fragment or analogue thereof comprising at least one amino acid εequence motif εelected from HFRIG and HSRIG. 21. A method according to Claim 20, wherein the diεeaεe iε cauεed by a bacterium, a parasite, a yeast or a fungus. 22. An agent for delivery of a pharmaceutically active εubεtance to a cell membrane or to the interior of a cell, compriεing a Vpr peptide or a biologically active fragment or analogue thereof comprising at least one amino acid sequence motif selected from HFRIG and HSRIG, said peptide, fragment or analogue being linked to said pharmaceutically active substance. 23. A method of delivery of a pharmaceutically active substance to a cell membrane or to the interior of a cell, comprising the step of contacting said cell with an agent aε defined in Claim 23. 24. Uεe of Vpr protein, or of a biologically active fragment or analogue thereof comprising at least one amino acid sequence motif εelected from HFRIG and HSRIG in the treatment of a disease mediated by cellular proliferation. 25. Use of Vpr protein, or of a biologically active fragment or analogue thereof compriεing at leaεt one amino acid εequence motif εelected from HFRIG and HSRIG in the treatment of a diseaεe cauεed by a pathogen. 26. An antagoniεt of the Vpx protein of human immunodeficiency viruε-2 (HIV-2) or of a biologically active fragment or analogue thereof, compriεing at leaεt one amino acid εequence motif selected from the group consiεting of HCKKG, CLGEG, CLGEE, CLGGE, CLGRG, HVRKG, HYTKG, HFKRG, HFKKG, HAKRD, CLQEG, CLGGG, CRGEG, CWGED, HFRCG and RRQPF, εaid antagoniεt having the ability to inhibit one or more activities mediated by Vpx, selected from the group conεiεting of group arreεt, cell replication arreεt, cytotoxicity, cytotoεkeletal diεruption and effectε on the endoplasmic reticulum.

Inventors: ARUNAGIRI CHINNIAH, AZAD AHMED A, MACREADIE IAN G, ARUNAGIRI, CHINNIAH, AZAD, AHMED, A., MACREADIE, IAN, G.
Applicants: ARUNAGIRI CHINNIAH, AZAD AHMED A, BIOMOLECULAR RES INST LTD, MACREADIE IAN G, ARUNAGIRI, CHINNIAH, AZAD, AHMED, A., BIOMOLECULAR RESEARCH INSTITUTE LTD., MACREADIE, IAN, G.
Application year: 1995
Main classification: C07K7
IPC codes: A61K35, A61K38, A61K39, A61K48, A61P31, A61P35, A61P37, A61P43, C07H21, C07K7, C07K14, C12N15, C12P21, C12Q1, C12R1