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WO1998035032A9 - Procedes permettant de provoquer une stase de cycle cellulaire - Google Patents

Procedes permettant de provoquer une stase de cycle cellulaire

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Publication number
WO1998035032A9
WO1998035032A9 PCT/US1998/003390 US9803390W WO9835032A9 WO 1998035032 A9 WO1998035032 A9 WO 1998035032A9 US 9803390 W US9803390 W US 9803390W WO 9835032 A9 WO9835032 A9 WO 9835032A9
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WIPO (PCT)
Prior art keywords
protein
vpr
cell
cells
binding
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PCT/US1998/003390
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English (en)
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WO1998035032A2 (fr
WO1998035032A3 (fr
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Priority to AU63335/98A priority Critical patent/AU6333598A/en
Publication of WO1998035032A2 publication Critical patent/WO1998035032A2/fr
Publication of WO1998035032A9 publication Critical patent/WO1998035032A9/fr
Publication of WO1998035032A3 publication Critical patent/WO1998035032A3/fr

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  • the invention relates to methods for inducing cell stasis, blocking cell division and bringing about cell death. More specifically, the present invention provides methods and compositions based on the ability of the Vpr protein, or an analogue of Vpr, to bind to one or more cellular target(s) and block cell division, inducing cell cycle stasis and cell death. Further, the present invention provides methods and compositions based upon the ability of Vpr packaged within both infectious and non- infectious HTV virions to induce cell cycle arrest without de novo production of Vpr.
  • the human immunodeficiency virus type 1 (HTV-1) is the causative agent of acquired immune deficiency syndrome (AIDS).
  • HIV-1 encodes several accessory genes that are important for replication and/or pathogenesis.
  • One ofthese genes, vpr encodes an 14 kDa basic protein that is present in the virion and serves a number of functions.
  • the Vpr gene is non-essential for viral replication in T-cell lines and activated peripheral blood lymphocytes (PBLs) in vitro but is necessary for efficient infection of non-dividing cells such as macrophages.
  • Vpr plays an important role in viral pathogenesis.
  • Functions ascribed to Vpr include transport of the virus core into the nucleus of non-dividing cells, assisting in virus assembly through its association with Gag p6, and a potential role in tr ⁇ /is-activation of the HIV-1 LTR during early stages of transcription.
  • Vpr has also been shown to be capable of inducing cell cycle arrest at the G2 checkpoint in a variety of mammalian cells, including PBLs.
  • Vpr This property of Vpr is proposed to be important for HIV-1 persistence through the inhibition of an effective T-cell expansion during an immune response and potentially contributes to the decline of CD4 + T-cells over time in infected individuals.
  • the cell cycle arrest mediated by Vpr is similar in some respects to the arrest of cells at the G2 phase of the cell cycle induced by damage to DNA.
  • the arrest of cells in G2 by Vpr has been confirmed biochemically by demonstrating that the major regulator of the transition from the G2 to M phase, Cdc2 kinase, is present in Vpr- arrested cells in its inactive hyperphosphorylated form. This is also the predominant form of Cdc2 kinase present in cells arrested by DNA-damaging agents such as nitrogen mustard.
  • Vpr-induced G2 arrest can be alleviated by chemical agents known as methylxanthines, which have previously been shown to alleviate the arrest induced by DNA damage.
  • methylxanthines chemical agents known as methylxanthines
  • the present invention used the yeast two-hybrid system to identify human proteins which are capable of physically associating with Vpr.
  • cDNAs were isolated, including one that encodes a human homologue of the Saccharomyces cerevisiae Rad23 protein, HHR23 A, a protein thought to play a role in DNA repair.
  • the present invention demonstrates that full-length HHR23 A protein when transiently expressed in HeLa cells, interacts physically with bacterially-expressed recombinant GSTVpr. Indirect immunofluorescence and confocal microscopy indicate that the two proteins colocalize within the same subcellular region, principally at or about the nuclear membrane.
  • the Vpr-binding domain in HHR23 A was mapped to the carboxy terminal region of the protein.
  • the present invention is based on the observation of the role that the Vpr protein of lentiviruses plays in arresting cell development in the G2 stage of the cell cycle. Arresting development of the cell by inducing cell cycle stasis ultimately leads to cell death. Based on this observation, the present invention provides compositions and methods for inducing cell cycle stasis and cell death. The present invention further provides methods for identifying compounds that act as an analogue of the Vpr protein to induce cell cycle stasis and cell death. As demonstrated in the Examples, these methods can be used to block abnormal cell growth, such as tumor cell growth and cells involved in autoimmune disorders. The invention further provides a basis for therapeutic methods of controlling cell growth within a patient.
  • compositions and methods for inducing cell cycle arrest and cell death are based on the Vpr protein of lentiviruses, or an analogue of a Vpr protein. These proteins and analogues induce cell cycle arrest when presented to a dividing cell and induce cell death.
  • Further embodiments of the present invention are based on the identification of cellular targets that are bound by the Vpr protein of lentiviruses. Based on these disclosures, the present invention provides methods for screening candidate agents for the ability to function as an analogue of a Vpr protein. In general, these methods are based on binding and/or competitive binding assays that are used to identify agents that bind to the same cellular targets as the Vpr proteins and/or block the binding of the Vpr protein to a cellular target.
  • Two of the herein disclosed protein targets that are bound by the Vpr protein, the B29- 1 and B251 - 1 proteins, are proteins that have not been previously isolated or characterized. Based on the identification ofthese two proteins, further embodiments of the present invention provide the B29-1 and B251-1 proteins in isolated or purified form, nucleic acid molecules that encode the B29-1 and B251-1 proteins, and antibodies that bind either the B29-1 or the B251-1 protein.
  • Another embodiment of the present invention is directed to methods of using virion associated Vpr to mediate arrest of the growth of a cell in the absence of de novo synthesis of Vpr protein. This embodiment provides infection of a host cell with a virion comprising Vpr protein.
  • Such infection involves the initial infection of the host cell but the virion is incapable of establishing a productive infection that results in another round of replicated virions able to infect subsequent new host cells.
  • the growth of the infected host cell is arrested even in the absence of de novo synthesis of Vpr protein by such "non-infectious" virions.
  • Still a further embodiment of the present invention involves the use of such virion associated Vpr protein to arrest the growth of a tumor cell.
  • Cell death usually follows the arrested growth mediated by the Vpr protein.
  • tumor cells may be of any tumor type, including, but not limited to: colon adenocarcinoma, promyelocytic leukemia, bone marrow myelogenous leukemia, bladder carcinoma, osteosarcoma, cervical carcinoma, T-cell lymphoma, acute T-cell leukemia, HTLV-II-cell leukemia, xeroderma pigmentosum (skin cancer), colon carcinoma, and breast adenocarcinoma.
  • Related embodiments of the present invention include compounds useful as pharmaceutical agents for the treatment of cancer comprising a therapeutically effective amount of virion associated Vpr.
  • Another embodiment of the present invention includes methods to restore growth in a cell whose growth has been arrested. Such methods provide for contacting the cell with a compound that reduces the binding of Vpr protein to the cellular target for Vpr protein. Specifically, this embodiment includes restoration of the growth of a cell, whose growth is arrested as a result of the binding of Vpr protein to the cellular target for Vpr protein.
  • Related embodiments contemplated in the present invention include compounds and methods of using said compounds that are effective in restoring growth in a cell whose growth is arrested as a result of the binding of Vpr protein to the cellular target for Vpr protein.
  • Additional embodiments of the present invention include those compounds and pharmaceutical agents and methods of using said compounds and pharmaceutical agents for the treatment of HIV infection comprising a therapeutically effective amount of an agent that reduces the binding of Vpr protein to the cellular target for Vpr protein and specifically includes agents that reduce the binding of virion associated Vpr to the cellular target for the Vpr protein.
  • lane 4 is lysate from HeLa cells cotransfected BSVprXThy and pXCR23A used in the binding assay; lane 5 is lysate from BSV ⁇ rXThy/pXCR23A cotransfected cells incubated with GSTVpr; lane 6 is lysate from BSVprXThy/pXCR23 A cotransfected cells incubated with GST.
  • the arrow indicates the position of the full-length M2 tagged HHR23A protein.
  • Figure 3 Amino acid sequence alignment of HHR23A and the 8 different cDNA clones identified in the 2-hybrid screen for proteins that interact with HIV-1 Vpr.
  • the cDNAs encode proteins with the following lengths: B25-1 45 amino acids (aa), B236-2 46 aa, C3-1 59 aa, C16-1 62 aa, C108-1 112 aa, ClO-1 150 aa, C180-1 174 aa, B213-2 179 aa.
  • the highly conserved internal repeat domain is indicated by the boxed region.
  • GSTVpr binds to the 45 aa C-terminal portion of HHR23A and HHR23B which includes the highly conserved internal repeat domain.
  • A) Ten micrograms of GSTVpr or GST were mixed with fifty micrograms of biotinylated HHR23 A or HHR23B peptide and incubated at 4°C for one hour. GST-containing complexes were selectively recovered by using glutathione sepharose beads. Protein complexes bound to the glutathione beads were subjected to PAGE, transferred to nitrocellulose, and visualized with streptavidin conjugated to horseradish peroxidase using chemiluminescence.
  • Lane 1 is HHR23 A peptide incubated with GSTVpr; lane 2 is HHR23 A peptide incubated with GST; lane 3 is HHR23B peptide incubated with GSTVpr; lane 4 is HHR23B peptide incubated with GST.
  • Ten micrograms of GSTVpr or GST were mixed with fifty micrograms of biotinylated HHR23A or HHR23B peptide and incubated at 4°C for one hour. Increasing amounts of unbiotinylated HHR23A peptide were then added to the binding reactions.
  • Lanes 1-4 show binding reactions containing GSTVpr and HHR23A-biotin, with increasing amounts of unbiotinylated HHR23A peptide added as indicated.
  • Lanes 5-8 show binding reactions containing GST and HHR23 A-biotin, with increasing amounts of unbiotinylated HHR23 A peptide added as indicated.
  • FIG. 5 A Panel A) BS VprThy and pCMV; panel B) BSVprXThy and pCMV; panel C) BS VprThy and pXCB213 (Vpr protein and HHR23AB213 protein); panel D) BSVprXThy and pXCB213 (truncated Vpr protein and HHR23AB213 protein); panel E) mock transfected cells.
  • Figure 5B Panel A) BSVprThy and pCMV; panel B) BSVprXThy and pCMV; panel C) BSVprThy and pXCB213; panel D) BSVprXThy and pXCB213; panel E) BSVprThy and pXCR23 A (Vpr and full-length HHR23 A proteins); panel F)
  • BSVprXThy and pXCR23A (truncated Vpr and full-length HHR23A proteins); panel G) BSVprThy and ⁇ XCR23 Atrunc (Vpr and truncated HHR23A proteins); panel H) BSVprXThy and pXCR23Atrunc (truncated Vpr and HHR23A proteins).
  • FIG. 6 Comparison of the levels of HHR23A and HHR23B RNA expression in Vpr-arrested cells to that detected in the Gl and G2 phases of the cell cycle using an RT-PCR assay.
  • RNA from Vpr-arrested cells HeLa cells were transfected with BSVpr. Forty-eight hours after transfection, total RNA was isolated from transfected cells. At this time point approximately 70 % of the cells were Thyl.2 + (data not shown).
  • RNA was isolated from HeLa cells synchronized by a double thymidine block at hours (Gl) and hours (G2) after release of the block.
  • RNA samples were stained with PI and analyzed by flow cytometry to confirm that the majority of cells was in either the Gl or G2 phase of the cell cycle. Approximately 93 % of the cells harvested at hours after release of the block were in Gl and 97 % of the cells harvested at hours after release of the block were in G2. A series of two-fold dilutions was made of total RNA isolated from the cell populations described above and analyzed by RT-PCR. Panel A shows the RT- PCR products generated using primers that detect HHR23A RNA. Panel B shows the RT-PCR products generated using primers that detect CKShs2 RNA.
  • Figure 7 provides the amino acid sequence identified in the yeast two hybrid system and designated B251-1 and B29-1.
  • Figure 8 panels a-c
  • Figure 9 panels a-c
  • Figure 8 panels a-c
  • Figure 9 panels a-c
  • Figure 10 provides a comparison of Vpr-induced cell-cycle arrest following infection of cells with HIV-1 virus when Vpr was supplied in trans, but not in the absence of Vpr. This demonstrates that virion-associated Vpr is capable of inducing cell cycle arrest from both infectious and non-infectious virions.
  • Figure 11 provides a comparison of Vpr induced cell cycle arrest in the presence or absence of AZT and nevirapine.
  • Figure 12 provides a comparison of Vpr induced cell cycle arrest in the presence or absence of indinivir.
  • Figure 13 provides a ribbon representation of the NMR structure of the C- terminal UBA domain of HHR23A.
  • the UBA domain is an independently folding unit and forms a three helix bundle with a well defined hydrophobic core.
  • Many of the highly conserved amino acids are located in the core of the protein and are clearly important for the overall structure of the domain.
  • the only amino acid sequence differences between the UBA domains of HHR23 A and B are also located in this area. It is likely that this surface area is involved in protein-protein or protein-ligand interaction, i.e., in the interaction with Vpr. Therefore this region is a possible target area for drug design.
  • the present invention is based on the observation that the Vpr protein of lentiviruses can be used to induce cell cycle stasis and cell death in a wide variety of cells, particularly tumor cells. Based on this observation, the present invention provides compositions and methods for arresting cells in the cell cycle, thus inducing cell death. These methods and compositions are based on the observations obtained when the critical role played by the Vpr protein in the arrest of HIV infected cells was observed. In HIV infected cells, the Vpr protein blocks cell development at the G2 stage of the cell cycle.
  • One embodiment of the present invention provides methods for blocking cell division and inducing cell death. These methods comprise the step of contacting dividing cells with a Vpr protein, or an analogue of a Vpr protein.
  • a Vpr protein refers to the 94-amino acid protein encoded by the HIV-1 virus as described by Cohen et al. J Virol (1990) 64:3097, the corresponding protein produced by other HIV strains such as HIV-2, and the corresponding protein produced by other lentiviruses such as the Vpr protein of SIV and the Vpx protein of SIV strain AGM77.
  • the Vpr proteins of the present invention also include minimally modified forms ofthese proteins or fragments of the Vpr protein that retain the ability to arrest cell division. It is well understood that minor modification can be made to the amino acid sequence of proteins without dramatically altering their activity. Preferred modifications include substitution of conservative amino acids for those in the wild-type protein in noncritical regions.
  • the primary amino acid structure may be derivatized to, for example, sugars, lipids, acyl groups, and the like. Modifications to the Vpr proteins that do not interfere with the cell-cycle arresting function of the Vpr protein are also contemplated. Furthermore, the complete amino acid sequence may not be necessary for the requisite activity. Thus, fragments of the Vpr proteins that remain active are also included.
  • Vpr proteins of the present invention will be active in arresting cell division.
  • the test for ascertaining the activity of such proteins is straightforward; the modified Vpr protein need only be tested in comparison to a known active Vpr protein for its ability to arrest cell division and induce cell death when expressed in a mammalian or other eukaryotic cell.
  • an analogue of a Vpr protein refers to agents that possess the cell cycle blocking activity of a Vpr protein by way of binding to the same cellular target(s) as that bound by the Vpr protein.
  • the yeast two hybrid protein system was used to identify several protein targets to which the Vpr protein binds. These targets can be used in competitive binding studies to identify agents that bind to one or more of the Vpr targets so as to function as an analogue of a Vpr protein.
  • the Vpr proteins and the analogues of the Vpr proteins will hereinafter be referred to as the Vpr proteins.
  • the methods and compositions of the present invention can be used to arrest cell division and induce cell death with any cell type from any organism so long as the division of the particular cell type from the particular organism can be blocked using the compositions and methods herein described.
  • the preferred cells are from vertebrate organisms. The most preferred being from mammalian organisms such as humans and commercially important animal such as livestock and pets.
  • the preferred cell types are cells that cause a pathological condition because of uncontrolled or abnormal cell division. In particular, these include all cancer cells and neoplastic conditions as well as cells involved in autoimmune disorders.
  • a Vpr protein is said to be contacted with a cell when the Vpr protein is placed in direct proximity to the cell so that the protein actively binds to its target and arrests cell division.
  • cells whose division is to be controlled using the methods and compositions of the present invention will be found within a mammalian organism that is to be treated.
  • a variety of methods and procedures can be used to systemically or directly administer a Vpr protein such that it will be provided to a dividing cell.
  • a Vpr protein is said to induce cell stasis or block or arrest cell division if the Vpr protein causes the cell to stop dividing when the cell is contacted with the Vpr protein.
  • the Vpr protein will act to block cell division in the G2 phase and inducing cell death.
  • a variety of methods can be employed to determine if cell division is blocked. These typically rely on the use of a marker of cell division, such as nucleotide or amino acid uptake and incorporation, or on the direct assessment of cell numbers. For example, a number of other indices of cell growth can be used, such as labeled thymidine uptake, vital stains such as Alamar blue, trypan blue and the like.
  • cell replication can be measured by the density of culture or with a cell counter or spectrophotometer, by using indicators of growth parameters such as pH of culture medium, by detecting cell byproducts, using kinase assays such as the immunoprecipitation of cdc2 kinase, assaying WEE 1, NIM 1, CAK, and phosphates activity and determining the level of cyclin mRNA expression, and using a microcell physiometer to measure cell metabolism and waste product evolution.
  • certain genes are known to be expressed specifically in particular phases of the cell cycle such as M, Gl and S. Assessment of the level of expression ofthese genes can provide a measure of the status of the cells in culture.
  • the cells may be transfected with promoters associated with these specifically expressed genes, such as CDK or cyclin, wherein the promoter is operably linked to a specific reporter gene such as chloramphenicol acetyl transferase (CAT) or luciferase.
  • promoters associated with these specifically expressed genes such as CDK or cyclin
  • CAT chloramphenicol acetyl transferase
  • luciferase luciferase
  • the agents of the present invention can be administered systemically or directly to the site of action using parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, the route of administration and the nature of the effect desired. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt.
  • the preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt.
  • the most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oil based injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the agents of the present invention can be administered in a systemic or targeted form, or can be directly injected to the site of desired action.
  • a variety of methods have been developed and are being developed to target the delivery of an agent to a particular cell or cell type. Such methods include, but are not limited to, the use of a fusion protein comprising an antibody variable region domain fused to the agent, targeting liposomes and controlled release polymeric matrixes.
  • Such delivery systems can be used to direct the delivery of a Vpr protein to cells whose growth is to be controlled.
  • the agents of the present invention can be provided alone, or in combination with other agents that are used to treat conditions caused by abnormal cell growth.
  • a Vpr protein can be used in conjunction with other chemotherapeutic agents.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • an expression unit that encodes a Vpr protein can be used to transform cells, particularly tumor cells using targeted transformation, in vivo so that the tumor cells are altered to express a Vpr protein. Expression within a tumor cell ensures that the cell will be contacted by the Vpr protein.
  • a variety of techniques are presently available, and others are being developed, for introducing a nucleic acid molecule into a mammalian subject so as to constitutively or inducibly express a protein in a cell within the treated subject. These methods can readily be used to introduce an expression cassette that encodes the Vpr protein, such as those disclosed in US Serial No. 08/322,750, into a target cell or target cell population.
  • Vpr protein can be used to identify targets that bind the Vpr protein.
  • the Vpr protein targets can then be used to rationally design or randomly select an analogue of a Vpr protein and/or can be further used in competitive binding assays.
  • targets that are bound by a Vpr protein can be identified using a yeast two-hybrid system or using a binding-capture assay.
  • yeast two hybrid system an expression unit encoding a fusion protein made up of one subunit of a two subunit transcription factor and the Vpr protein is introduced and expressed in a yeast cell.
  • the cell is further modified to contain 1) an expression unit encoding a detectable marker whose expression requires the two subunit transcription factor for expression and 2) an expression unit that encodes a fusion protein made up of the second subunit of the transcription factor and a cloned segment of DNA.
  • the expression results in the interaction of the Vpr and the encoded protein. This brings the two subunits of the transcription factor into binding proximity, allowing reconstitution of the transcription factor. This results in the expression of the detectable marker.
  • the yeast two hybrid system is particularly useful in screening a library of cDNA encoding segments.
  • a Vpr protein is mixed with an extract of a cell under conditions that allows the association of a binding target with the Vpr protein. After mixing, binding targets that have become associated with the Vpr protein are separated from the mixture. The target that bound the Vpr protein can then be removed and further analyzed. To identify and isolate a binding target, the entire Vpr protein can be used. Alternatively, a fragment of a Vpr protein can be used.
  • a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells whose division can be blocked using the Vpr protein.
  • the cellular extract can be prepared from cells that have been freshly isolated from a subject or from cells or cell lines that have been cultured. A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and the enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the Vpr protein under conditions in which association of the Vpr protein with the binding target can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the Vpr protein with the binding target.
  • the Vpr protein is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to the Vpr protein can be used to immunoprecipitate the Vpr protein and associated binding target. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
  • the binding target can be dissociated from the Vpr protein using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the Vpr protein can be immobilized on a solid support.
  • the Vpr protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the Vpr protein to a solid support aids in separating the protein/binding target pair from other constituents found in the extract.
  • the Vpr protein of HIV-1 was used in a yeast two hybrid system to identify seven proteins that interact with and bind to the Vpr protein. These include: two previously unknown proteins, herein denoted the B29- 1 and B251 - 1 protein ( Figures 8-10); casein kinase II (beta subunit); uracil DNA glycosylase; phosphoglycerate kinase I; ubiquitin conjugating enzyme; and pyruvate kinase.
  • Vpr protein binding targets can be used in methods to identify an analogue of a Vpr protein. Specifically, a Vpr protein and a binding target, such as HHR23 A protein, or a cellular extract containing the Vpr protein and target, are mixed in the presence and absence of an agent to be tested. After mixing under conditions that allow association of the Vpr protein with the target, the two mixtures are analyzed and compared to determine if the tested agent reduced or blocked the association of the Vpr protein with the binding target. Agents that block or reduce the association of the Vpr protein with the binding target will be identified as decreasing the amount of association present in the sample containing the tested agent.
  • a Vpr protein and a binding target such as HHR23 A protein, or a cellular extract containing the Vpr protein and target
  • an agent is said to reduce or block Vpr/binding target association when the presence of the agent decreases the extent to which or prevents the Vpr protein from becoming associated with the binding target.
  • One class of agents will reduce or block the association by binding to the Vpr protein while another class of agents will reduce or block the association by binding to the binding target.
  • An analogue of a Vpr protein will be of the class of agents that bind to the binding target.
  • the Vpr protein and/or binding target used in the above assay can either be an isolated binding partner, such as using a purified Vpr and purified HHR23 A protein, or can be partially purified, such as in the use of a crude cellular extract containing the Vpr protein and an identified but uncharacterized binding target.
  • Vpr protein and binding target have been characterized by an identifiable property, e.g., molecular weight
  • the present assay can be used.
  • either the entire protein can be used or a fragment containing the binding site can be used.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the binding target with the Vpr protein.
  • An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis that may take into account the sequence of the target site and/or its conformation.
  • agents that block Vpr protein/binding target interaction there are two sites of action for agents that block Vpr protein/binding target interaction: the binding target contact site on the Vpr protein and the Vpr protein contact site on the binding target.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the contact sites of the Vpr/target pair.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the binding target contact site on the Vpr protein.
  • an agent will reduce or block the association of the Vpr protein with the binding target by binding to the binding target.
  • analogues of the Vpr protein of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of the Vpr protein, for example, a peptide fragment of the HIV-1 Vpr protein.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • the peptide agents of the invention can be prepared using standard solid phase
  • DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of the Vpr protein or a Vpr binding target.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the Vpr protein intended to be targeted by the antibodies.
  • Critical regions include the contact sites involved in the association of the binding target the Vpr protein.
  • Antibody agents are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptide haptens alone, if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminal with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred.
  • Immortalized cell lines that secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten or is the binding protein itself.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera that contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the Vpr protein or binding target can also be produced in the context of chimeras with multiple species origin. Isolated Protein Binding Targets of the Vpr Protein
  • the yeast two hybrid system was used with the Vpr protein and two previously unidentified proteins were isolated that bind to the Vpr protein.
  • the present invention provides these two proteins, the B29-1 and B251- 1 proteins, as well as allelic variants and conservative amino acid substitutions thereof, in purified or isolated form.
  • the B29-1 protein refers to a protein that has the amino acid sequence depicted in Figure 7 within its amino acid sequence and the B251-1 protein refers to a protein that has the amino acid sequence depicted in Figure 7 within its amino acid sequence.
  • These proteins include the specific fragments of human proteins disclosed herein as well as the actual complete human protein that contains the identified Vpr binding fragments herein denoted as the B29-1 and B251-1 proteins.
  • the B29-1 and B25-1 proteins of the present invention further include naturally occurring allelic variants, proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the requisite ability to associate with a Vpr protein.
  • the B29-1 and B251-1 proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated B29-1 or B251-1 protein.
  • the Vpr binding proteins of the present invention further include conservative variants of the B29-1 and B251-1 proteins herein described. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the ability of the specific protein to bind to a Vpr protein.
  • a substitution, insertion or deletion is said to adversely affect the B29-1 or B251-1 proteins when the altered sequence prevents the protein from associating with a Vpr protein.
  • the overall charge, structure or hydrophobic/hydrophilic properties of B29-1 or B251-1 proteins can be altered without adversely affecting activity of protein.
  • the amino acid sequence of B29-1 or B251-1 proteins can be altered, for example to render the proteins more hydrophobic or hydrophilic, without adversely affecting the ability of the protein to become associated with a Vpr protein.
  • the B29-1 and B251-1 proteins of the present invention further include proteins isolated from organisms other than humans that are structurally similar to the herein exemplified B29-1 and B251-1 proteins and that further bind to a Vpr protein. These proteins can be isolated from any organism or cell that expresses the related B29-1 and B251-1 proteins.
  • the preferred source is other mammalian organisms.
  • allelic variants, the conservative substitution variants of the B29-1 and B251-1 proteins and the conesponding proteins from other organisms will have an amino acid sequence having at least 75% amino acid sequence identity with the B29-1 or B251-1 sequences herein disclosed, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity/homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting identity/homology.
  • the B29-1 and B251-1 proteins of the present invention include molecules having the amino acid sequences disclosed in Figure 8; fragments thereof having a consecutive sequence of at least about 3, 5, 10 or 15 amino acid residues of the B29-1 or B251-1 proteins; amino acid sequence variants of such sequence wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed B29-1 or B251-1 sequences; amino acid sequence variants of the disclosed B29-1 or B251-1 sequence, or their fragments as defined above, that have been substituted by another residue.
  • Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the conesponding B29-1 or B251-1 proteins of other animal species, including but not limited to rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the B29-1 or B251-1 proteins; and derivatives wherein the B29-1 or B251-1 proteins have been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • a detectable moiety such as an enzyme or radioisotope
  • Anti-B29-1 and B251-l Antibodies The present invention further provides antibodies that selectively bind to the
  • B29-1 and B251-l proteins include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complement determining regions.
  • Antibodies are generally prepared by immunizing a suitable mammalian host using a B29-1 or B251-1 protein, or fragment, in isolated or immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
  • Figures 8 and 9 provides a Jameson- Wolf plot of the antigenic index of various regions of B29-1 and B251-1.
  • Such regions provide suitable fragments for use in generating B29-1 and B251-1 specific antibodies.
  • Methods for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known in the art.
  • a carrier such as BSA, KLH, or other carrier proteins
  • direct conjugation using, for example, carbodiimide reagents may be used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
  • B29-1 or B251-1 immunogen is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art.
  • a suitable adjuvant as is generally understood in the art.
  • titers of antibodies can be taken to determine adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, monoclonal antibody preparations are prefened.
  • Immortalized cell lines which secrete a desired monoclonal antibody may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the B29- 1 or B251 - 1 protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of receptor can also be produced in the context of chimeras or CDR grafted antibodies of multiple species origin.
  • the antibodies thus produced are useful not only as modulators of the association of B29-1 or B251-1 with a Vpr protein, but are also useful in immunoassays for detecting B29-1 and B251-1 expression/activity and for the purification of B29-1 and B251-1 proteins and associated binding partners.
  • nucleic acid is defined as RNA or DNA that encodes a peptide as defined above, or is complementary to nucleic acid sequence encoding such peptides, or hybridizes to such nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80%, and more preferably at least 85%, with the peptide sequences.
  • genomic DNA, full length cDNA, mRNA and antisense molecules as well as nucleic acids based on alternative backbone or including alternative bases whether derived from natural sources or synthesized.
  • hybridizing or complementary nucleic acid is defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a Vpr binding protein according to the present invention.
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpynolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • a denaturing agent such as formamide, for example, 50% (vol vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpynolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • Another example is use of 50%) formamide, 5 x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2 x SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
  • nucleic acid molecules of the present invention that encoded either the B29-1 or B251-1 proteins will be preferably in isolated form.
  • a nucleic acid molecule is said to be "isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
  • the present invention further provides fragments of the Vpr binding protein encoding nucleic acid molecule herein disclosed.
  • a fragment of a Vpr binding protein encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode the Vpr contact site of the protein, the fragment will need to be large enough to encode the region of the protein that is contacted by the Vpr protein.
  • the present invention further provides full length cDNAs and genomic DNAs that encode proteins that contain the Vpr binding partners herein identified as the B29-1 and B251-1 proteins.
  • Vpr binding protein encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a variety of such labels are known in the art and can readily be employed with the Vpr binding protein encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled binding target encoding nucleic acid molecule.
  • B29-1 and B251-1 can readily use the amino acid sequence of B29-1 and B251-1 to generate antibody probes to screen expression libraries prepared from cells.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified the B29-1 or B251-1 protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the B29-1 and B251-1 family of proteins.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.
  • Figure 8 and 9 identifies important antigenic and/or putative operative domains found in the B29-1 and B251-1 protein sequence. Such regions are preferred sources of antigenic portions of the B29-1 and B251-1 protein for the production of probe, diagnostic, and therapeutic antibodies.
  • a portion of the B29-1 or B251-1 encoding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the B29- 1 or B251-l family of proteins from any mammalian organisms that contains such a protein.
  • Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
  • pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone a B29-1 or B251-1 -encoding nucleic acid molecule.
  • PCR polymerase chain reaction
  • a PCR denature/anneal extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other B29-1 orB251-l encoding nucleic acid molecules.
  • the _present invention further provides recombinant DNA molecules (rDNAs) that contain a B29-1 or B251-1 encoding sequence.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • a B29-1 or B251-1 encoding DNA sequence is operably linked to expression control sequences and/or vector sequences. The choice of vector and/or expression control sequences to which one of the
  • B29-1 or B251-1 encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the B29-1 or B251-I gene included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a B29-1 or B251-1 encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the B29-1 or B251-1 encoding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.
  • Expression vectors compatible with eukaryotic cells can also be used to form rDNA molecules that contain a B29-1 or B251-1 encoding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV- l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 (Invitrogene), and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a prefened drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphofransferase (neo) gene. Southern et a , JMol Anal Genet ( 1982) 1 :327-341.
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by cotransfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a B29-1 or B251-1 protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a B29- l or B251-l protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the B29-Io ⁇ B251-1 gene product.
  • Prefened eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line, the most prefened being cells that do not naturally express aB29-l orB251-l protein.
  • Any prokaryotic host can be used to express a B29-1 or B251-1 -encoding rDNA molecule.
  • the prefened prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc Natl Acad Sci USA (1972) 69:2110; and Maniatis et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, JMol Biol (1975) 98:503, or Berent et al, Biotech (1985) 3:208 or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a B29-1 or B251- 1 protein that uses one of the B29-1 or B251-1 encoding nucleic acid molecules herein described.
  • the production of a recombinant form of a B29- 1 or B251 - 1 protein typically involves the following steps.
  • a nucleic acid molecule is obtained that encodes a B29- 1 or B251 - 1 protein, such as the nucleic acid molecule depicted in Seq ID No: . If the B29-1 or
  • B251-1 encoding sequence is uninterrupted by introns, it is directly suitable for expression in any host. If not, then a spliced form of the B29-1 or B251-1 encoding nucleic acid molecule can be generated and used or the intron containing nucleic acid molecule can be used in a compatible eukaryotic expression system.
  • the B29-1 or B251-1 encoding nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the B29-1 or B251-1 encoding sequences.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the B29-1 or B251-1 protein.
  • the B29-1 or B251-1 protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with B29- 1 or B251 - 1 encoding sequences to produce a B29- 1 or B251 - 1 protein.
  • B29-1 and B251-1 genes and the B29-1 and B251-1 proteins can also serve as a target for gene therapy in a variety of contexts.
  • B29- 1 or B251 - 1 -deficient non-human animals can be generated using standard knock-out procedures to inactivate a B29-1 or B251-1 gene or, if such animals are non-viable, inducible B29-1 or B251-1 antisense molecules can be used to regulate B29-1 or B251-1 activity/expression.
  • an animal can be altered so as to contain a B29-1 or B251-1 or antisense-B29-l or antisense-B251-l expression unit that directs the expression of B29-1 or B251-1 protein or the antisense molecule in a tissue specific fashion.
  • a non-human mammal for example a mouse or a rat, is generated in which the expression of the B29-1 or B251-1 gene is altered by inactivation or activation. This can be accomplished using a variety of art-known procedures such as targeted recombination.
  • the B29-1 and/or B251- 1 -deficient animal can be used to 1) identify biological and pathological processes mediated by the B29-1 and B251-1 proteins,
  • the present invention further provides the expression control sequences found 5' of the of the newly identified B29-1 and B251-1 genes in a form that can be used in generating expression vectors.
  • the B29-1 and B251-1 promoters identified as being 5' from the ATG start codon in the B29-1 and B251-1 genes, can be used to direct the expression of an operably linked protein encoding DNA sequence.
  • a skilled artisan can readily use the B29-1 and B251-1 promoter in expression vectors using methods known in the art.
  • the dual expression plasmids BSVprThy and BSVprXThy contain either Vpr or, the C-terminal truncation mutant, VprX, and the Thy 1.2 coding sequences which are both expressed from tandem copies of the cytomegalovinis (CMV) immediate-early promoter unit. These vectors also contain simian virus 40 (SV 40) transcription termination sequences and an untranslated intron of the CMV immediate-early promoter.
  • CMV cytomegalovinis
  • HHR23AB213 coding sequences (nt 589 to nt 1155) were amplified by PCR from the HeLa cDNA library plasmid recovered in the 2-hybrid screening.
  • the sequence of the forward or sense primer used for PCR amplification is:
  • This primer contains an Xho I site, a translation initiation site, and the M2 tag (boldface type) at the 5' end.
  • the sequence of the reverse or anti-sense primer used for PCR amplification is:
  • This primer contains an Xho I site, a translation initiation site, and the M2 tag (boldface type) at the 5' end.
  • the sequence of the reverse or anti- sense primer used for PCR amplification is as described for pXCB213.
  • the PCR amplified fragment was inserted into the Xho I and Mlu I sites of pCMV to create pXCHHR23A-l.
  • the DNA sequence of all inserts was confirmed by automated sequencing.
  • HeLa cells were cotransfected with either BSVprThy or BSVprXThy (0.3 ⁇ g) and a 20-fold molar excess of either pCMV (3.2 ⁇ g), pXCB213 (4.0 ⁇ g) or pXCR23A-l (4.0 ⁇ g). Forty eight hours later cells were stained with a monoclonal antibody to the Murine Thy 1.2 cell-surface protein directly conjugated with fluorescein isothiocyanate (FITC) as described (Jowett et al, 1995).
  • FITC fluorescein isothiocyanate
  • HeLa cells were cotransfected with either BSVprThy or BSVprXThy (0.3 ⁇ g) and a 20-fold molar excess of either pCMV (3.2 ⁇ g), pXCB213 (4.0 ⁇ g) or pXCR23A-l (4.0 ⁇ g). Forty eight hours later cells were stained with a monoclonal antibody to the Murine Thy 1.2 cell-surface protein directly conjugated with fluorescein isothiocyanate (FITC) as described (Jowett et al, 1995).
  • FITC fluorescein isothiocyanate
  • a vector was constructed containing Thy 1.2 under control of the CMV immediate early promoter as well as an expression system for the Vpr open reading frame under the control of another copy of the CMV promoter.
  • This vector, BSVprThy would effect expression of both Thy and Vpr in transfected host cells.
  • a control plasmid (BSThy) differs from BSVprThy only in lacking the Vpr open reading frame. The expression plasmids were constructed to contain the Thy 1.2 and the Vpr
  • Thy 1.2 open reading frame was amplified by PCR from a cDNA library.
  • a mouse thymoma cell line cDNA library obtained from Brian Seed, Harvard University, was used as a template for PCR using primers of the following sequences: 5'-CAAGTCGGAACTCGAGGCACCATGAAC-3' (sense) and 5 * -CGCGGTACCACGCGTCACAGAGAAATGAAGTCTAG-3' (antisense) which are complementary to the 5' and 3' ends of the Thy-1 coding sequences and extended to include Xhol site at the 5'-terminal and Mlul and Kpnl sites at the 3'-terminal.
  • the amplified DNA was digested with Xhol and Mlul and ligated into pCMV (Planelles, V. et al. AIDS Res Hum Retroviruses (1991) 7:889) expression vector flanked by the CMV immediate early promoter and the S V40 polyA sequence to provide transcriptional termination sequences and generate pCMV-Thy.
  • the transcriptional unit was then transferred into the Bluescript ® II KS + plasmid (Stratagene La Jolla, CA Cat. No. 212207).
  • the Vpr open reading frame was first cloned into the pCDM8 (Invitrogen, San Diego, CA Cat. No. V308-20) expression vector from pNL4-3.
  • the transcriptional cassette containing the CMV immediate early promoter and the HIV 3 'LTR transcriptional termination sequences were transfened into the above Bluescript vector containing the thy 1.2 expression cassette.
  • a control vector was constructed by subcloning the Thy expression cassette into CDM8 alone (lacking the Vpr open reading frame).
  • an Spel/Xhol fragment of pCDM8 (Invitrogen, San Diego, CA) containing the CMV immediate early promoter was inserted into Spel/Xhol digested NL-Thy (Plannelles et al, 1994) to obtain the plasmid NL-CMV-Thy.
  • NL-Thy contains the open reading frame for Vpr and the Thy 1.2 open reading frame prepared as described above. Digestion of NL-CMV-Thy resulted in a fragment containing the CMV promoter, the Thy 1.2 open reading frame and the 3' LTR sequences from HIV.
  • This fragment was transfened to Pstl digested Bluescript® II KS Plus (Stratagene, La Jolla, CA) to obtain BS-CMV-Thy.
  • Vpr open reading frame was obtained by digesting pNL4-3 (Adachi et al, 1986 (supra)) with Seal and Sad.
  • the Scal/Sacl fragment was cloned into Smal Sacl cleaved plasmid pGEM7Zf(-) (Promega Madison, WI) to obtain pGEM-Vpr.
  • PGEM- Vpr was digested with Xhol and Nsil and the resulting fragment cloned into Xhol/Pstl digested pCDM8 to obtain CDM8-V ⁇ r.
  • a Nrul to BamHI fragment of CDM8-Vpr containing the CMV promoter, Vpr open reading frame and SV40 transcription termination sequences was cloned into BS-CMV-Thy described above digested with Notl, blunt ended by filing in, and with BamHI to obtain BS-Vpr-Thy.
  • the control plasmid BS-Thy was constructed by cloning the NruI/BamHI fragment of pCDM ⁇ containing only the CMV promoter and SV40 transcriptional termination sequences into BS-CMV-Thy digested with Notl, blunt ended by filing in and with BamHI. All cloning steps described followed standard procedures.
  • Plasmid DNA was prepared for transfection by purification on an anion exchange resin (Qiagen Chatsworth, CA Cat. No. 12145) following the manufacturers protocol.
  • HeLa cells human epithelial fibroblast; ATCC CCL 2
  • COS cells African green monkey kidney fibroblast; ATCC CRL 1651
  • 10 ⁇ g of plasmid DNA was added to 5 x 10 6 cells in electroporation media.
  • Electroporation conditions for SupTl cells was as described above for MT2 cells, and for COS and HeLa cells was 250 V at 960 ⁇ F.
  • Thy 1.2 + cells transfected with the Vpr-containing vector was maintained as shown in Table 1.
  • Vpr is able to induce G2 cell anest in cells generally, and this function of the protein is not dependent on HIV infection.
  • the above procedures were repeated using the SW480, HL-60, KG- la, J82, SAOS-2, HeLa, SupTl, Jurkat, MT-2, XP12BE, HCT116 and SKBR3 cell lines.
  • the activity of the Vpr protein in blocking replication in these cell lines is provided in Table 2.
  • the Vpr protein is able to induce G2 cell arrest in cells generally, and this function of the protein is not dependent on the cancer origin of the cell type.
  • the yeast two-hybrid screen was used to identify cDNAs encoding human proteins capable of interacting with HIV-1 Vpr.
  • the complete coding sequence of the HIV- 1 ⁇ 4 . 3 vpr gene was ligated to the yeast Gal4 DNA binding domain (Gal4DB) in a plasmid that directs the expression of a Gal4DB Vpr fusion protein in yeast.
  • the target plasmid was cotransformed into the yeast reporter strain HF7c with a yeast expression library that directs expression of fusion proteins between the Gal4 transcriptional activation domain (Gal4AD) and HeLa cDNA-encoded proteins.
  • the yeast Rad23 protein functions in nucleotide excision repair (NER) in the global genome repair and transcription-coupled DNA repair pathways.
  • HHR23B functions in NER in the global genome repair pathway as part of a complex with the xeroderma pigmentosum complementation group C protein (XPC).
  • HHR23A is also found complexed with XPC in cells (personal communication, F. Hanoaka).
  • HHR23 A and B share extensive overall homology to each other and contain two copies of a highly conserved 50 amino acid (aa) acidic domain that is conserved among all the Rad23 homologues (see Figure 1).
  • This acidic repeat domain shares homology to the C-terminal extension of a bovine ubiquitin conjugating enzyme (UBC), E2(25K) that is thought to promote interaction with its substrate and/or function in cellular localization of the UBC.
  • UBC bovine ubiquitin conjugating enzyme
  • E2(25K) that is thought to promote interaction with its substrate and/or function in cellular localization of the UBC.
  • HHR23 A(B213) encodes the C-terminal portion of
  • HHR23 A which includes the entire C-terminal conserved repeat domain and a portion of the internal conserved repeat domain.
  • the Vpr protein was shown to bind to: two previously unknown human proteins, herein denoted as B251-1 and B29-1 ( Figures 8-10); casein kinase II (beta subunit); uracil DNA glycosylase; phosphoglycerate kinase I; ubiquitin conjugating enzyme; and pyruvate kinase.
  • B251-1 and B29-1 Figures 8-10
  • casein kinase II beta subunit
  • uracil DNA glycosylase phosphoglycerate kinase I
  • ubiquitin conjugating enzyme pyruvate kinase
  • HHR23 A was isolated in a genetic protein interaction screen with Vpr, it seemed likely that HHR23 A and Vpr would interact physically.
  • full-length HHR23 A protein transiently expressed in Hela cells was tested for the ability to bind to a recombinant fusion protein between glutathione-S-transferase (GST) and Vpr (GSTVpr).
  • GSTVpr was purified from bacteria by affinity chromatography on glutathione-sepharose.
  • HHR23 A an expression vector containing the complete coding sequences of HHR23A with an N-terminal FLAG epitope tag was constructed to facilitate detection by Western blotting. Forty eight hours after transfection with the HHR23 A expression plasmid, HeLa cells were lysed and lysates were used in ex vivo binding studies. GSTVpr-associated proteins were selectively recovered by affinity binding to glutathione-sepharose beads. The ability of HHR23A to bind to GSTVpr was determined by Western blot analysis of GSTVpr-associated proteins with a monoclonal antibody directed toward the M2 FLAG epitope ( Figure 2).
  • HHR23A bound to GSTVpr, but no binding was detected with HHR23 A when GST was used in the binding reaction. This result provides evidence for a direct interaction between Vpr and the full-length HHR23 A and confirms and extends the genetic data obtained through the yeast two-hybrid screen.
  • HHR23A To define the intracellular distribution of Vpr and HHR23A, indirect immunofluorescence in HeLa cells expressing either Vpr, HHR23 A, or the truncated form HHR23 A(B213) alone or Vpr and HHR23 A or HHR23 AB213 together was performed and analyzed by confocal microscopy. In contrast to previously reported results, HHR23 A was found to be localized primarily in the perinuclear region. This discrepancy may be accounted for by the differences in the fixation and staining protocols. Also, confocal microscopy allows one to easily distinguish between nuclear and perinuclear localization by determining the staining pattern of proteins within sections throughout the entire cell.
  • HHR23A(B213) was also localized primarily in the perinuclear region.
  • Vpr when expressed alone, localized primarily in the nucleus.
  • a concentration of Vpr in the perinuclear region was also observed.
  • cells expressing both Vpr and HHR23 A or HHR23A(B213) there was a colocalization of Vpr and HHR23A or HHR23A(B213) within the nucleus, and in particular in the perinuclear region. Coexpression of Vpr and HHR23A in cells did not appear to change the subcellular distribution of Vpr.
  • HHR23A required for interaction with Vpr
  • a panel of positive clones identified in the two-hybrid screen were screened to identify Gal4ADHHR23A cDNA fusion proteins encoding shorter fragments of the HHR23 A coding sequences which were still able to bind to Vpr. This was done by hybridizing plasmid DNA isolated from the panel of 173 positive clones identified in the two-hybrid screening with a radio-labeled fragment of HHR23A derived from the Gal4AD/HHR23 A(B213) plasmid.
  • Gal4AD HHR23 A clones encoding fusion proteins of various lengths which maintained interaction with Gal4DB/Vpr in the yeast two-hybrid screen were identified. Sequence analysis ofthese clones identified a common 45 amino acid C-terminal region of HHR23A which was present in all clones ( Figure 3). Interestingly, this minimal domain corresponds to a 50 amino acid domain identified by sequence analysis that shares homology to the C-terminal extension of a bovine ubiquitin conjugating enzyme (UBC), E2(25K) which is thought to promote interaction with its substrate and/or function in cellular localization of the UBC.
  • UBC bovine ubiquitin conjugating enzyme
  • HHR23A The 45 aa C-terminal portion of HHR23A is sufficient for binding to GSTVpr.
  • a chemically synthesized peptide conesponding to the 45 aa C-terminal portion of HHR23A was it for binding to the GSTVpr fusion protein ( Figure 4A). To visualize the peptide following affinity binding with glutathione-sepharose and Western blotting, the peptide was synthesized with a biotin tag on the amino terminal.
  • the 45 aa C-terminal portion of HHR23A comprises most of the internal 50 aa repeat element that is highly conserved between the two human homologues: the corresponding region of HHR23B differs by only 3 aa (see Figure 1).
  • a synthetic peptide derived from the 45 aa C-terminal region of HHR23B was tested for the ability to bind to GSTVpr in the in vitro assay.
  • the HHR23B peptide did specifically bind to GSTVpr but not to GST ( Figure 4A).
  • Figure 4A the yeast two-hybrid screen, it is possible that HHR23B may also interact with Vpr in cells.
  • HHR23A(B213) encoding construct was cotransfected in approximately a 20-fold molar excess over BSVprThy to ensure that the majority of cells which express Vpr and Thyl .2 would also express HHR23A(B213).
  • Cells expressing Vpr and Thyl.2 showed a G1/G2 ratio of 0.15 ( Figure 5 A, panel A).
  • Coexpression of Vpr and HHR23A(B213) resulted in a significant reduction in the degree of cell cycle arrest.
  • the G1/G2 ratio of the Thyl.2 + population was approximately 1.2.( Figure 5 A, panel C). Cells that were mock-transfected or transfected with a plasmid encoding a mutant form of Vpr
  • HHR23A and HHR23B RNA expression are similar in Vpr- arrested cells to that detected in the Gl and G2 phases of the cell cycle.
  • Thy 1.2-positive infected cells
  • Thy 1.2-negative uninfected cells
  • This early event may be mediated by virion-associated Vpr released into cells following infection but prior to provirus formation and de novo expression of Vpr.
  • Vpr is supplied to the virion by co-transfection, resulting in complementation in trans.
  • Virion particles formed in this faction contained Vpr at levels equivalent to wild-type virus (data not shown). Infection of cells with these viruses resulted in cell cycle arrest when Vpr was supplied in trans, but no cell cycle arrest was observed in the absence of Vpr ( Figure 10). This result demonstrates that virion-associated Vpr is capable of inducing cell cycle arrest.
  • Vpr mediated cell cycle stasis in presence of AZT and nevirapine A number of drugs are cunently available for treatment of HIV-1 disease. These drugs block viral infectivity by inhibiting key steps in the viral life cycle. We tested whether infection with viruses blocked at reverse transcription by AZT and nevirapine, nucleoside and non-nucleoside inhibitors, respectively, would still result in cell cycle arrest. Although these agents would block reverse transcription, they would not prevent entry of the virion particle and it is unknown what effect they would have on uncoating and subsequent release of Vpr into the cell. Using similar methodology to that described supra, treatment of uninfected cells with AZT and nevirapine had no effect on the cell cycle.
  • Protease inhibitors are anti-HIV drugs which prevent viral replication by inhibiting cleavage of the Gag precursor, resulting in the production of immature non- infectious particles.
  • virus made in the presence of the protease inhibitor, indinivir could still mediate G2 arrest.
  • HIV-1 expressing wild-type Vpr produced in the presence of indinivir contained reduced amounts of processed Gag proteins yet still contained detectable amounts of Vpr within the virions (data not shown). Infection of HeLa cells with this virus preparation did not yield cells that could be recognized by an anti-HIV- 1 p24 antibody ( Figure 12), indicating the inability of this virus to establish a productive infection.
  • anti- HIV- 1 drugs are highly effective at decreasing viral load in patients yet are less effective at restoring normal T-cell numbers and immune function. It is possible that the production of non-infectious virions containing Vpr may influence the rate at which the immune system regenerates.
  • non-infectious we mean viral particles that may initially infect host cells but are incapable of establishing a productive infection that results in another round of replicated virions able to infect subsequent new host cells.
  • the present results demonstrate that Vpr binds directly to a human cellular protein, HHR23 A.
  • a cDNA which partially encodes one of the human homologs of the yeast Rad23 gene, HHR23A was isolated.
  • the binding of full-length HHR23A from cell lysates with a recombinant GST- Vpr fusion protein was shown.
  • the interaction between Vpr and HHR23 A in vitro was confirmed using recombinant proteins and synthetic peptides.
  • the Vpr-interaction domain was mapped to the C-terminal repeat domain of HHR23A.
  • HHR23A and Vpr Colocalization of HHR23A and Vpr in Hela cells transiently expressing immunotagged HHR23 A and Vpr by indirect immunofluorescence and confocal microscopy. Most significantly, overexpression of HHR23A was shown to lead to a partial alleviation of Vpr-induced G2 arrest. This finding provides functional evidence that Vpr and HHR23 A interact in cells and that this interaction has biological consequences with regards to Vpr- mediated cell cycle arrest.
  • HHR23A is one of two human homologs of the S. cerevisiae Rad23 gene.
  • the Rad23 gene encodes a 42 kDa acidic protein that functions in nucleotide excision repair (NER) in both the global genome repair and transcription-coupled DNA repair pathways in yeast.
  • NER nucleotide excision repair
  • Ex vivo coimmunoprecipitation studies have demonstrated that Rad23 is one component of a higher order protein complex consisting of the multi- subunit transcription factor TFIIH and Rad 14, a zinc metalloprotein that binds specifically to UV-damaged DNA. Rad23 facilitates complex formation between Radl4 and TFIIH via interaction with Radl4 and the Rad25 and TFB1 components of TFUH.
  • HHR23A and its counterpart HHR23B encode acidic proteins of 40 and 43 kDa respectively that share extensive overall homology to each other (57 % identity and 76 % similarity) and with the Rad23 gene of S .cerevisiae (30-34 % identity, 41% similarity).
  • the function of the N-terminal ubiquitin-like domain is unknown.
  • S. cerevisiae it is essential for biological function of Rad23 but does not appear to mediate proteolytic degradation.
  • the 50 aa internal repeat domain shares homology with a C-terminal extension of a bovine ubiquitin conjugating enzyme (E2(25K)) and is fully conserved between the human and murine homologues suggesting a functional role for this region.
  • HHR23A and B do not display significant differences in RNA levels during the mitotic cell cycle.
  • the present results confirm this observation. There was not a significant difference between HHR23 A and HHR23B RNA expression in Vpr-G2- arrested cells and in cells where the predominant population is in the Gl or G2 phase of the cell cycle.
  • HHR23A and B do not exhibit the UV-inducible phenotype of their yeast counterpart where induction of mRNA levels is seen upon UV exposure and during meiotic prophase.
  • the murine Rad23 homologues are expressed in a wide variety of tissues, with increased RNA expression seen in testis tissue suggesting a role in meiotic recombination.
  • the cellular function of HHR23A and HHR23B is less well characterized than that of Rad23.
  • HHR23B was originally identified in association with the XPC protein, the putative homologue of the yeast Rad4 protein. XPC and HHR23B form a protein complex that corrects the genome DNA repair defects of human cells from patients with xeroderma pigmentosum complementation group C (XPC). In vitro reconstitution studies have demonstrated that HHR23B exhibits a stimulatory effect on the conecting activity of XPC. These results indicate that HHR23B functions in one NER pathway known as the global genome repair pathway, the mode of repair that is defective in XPC cells. More recently it has been shown that like HHR23B, HHR23 A also binds to XPC (personal communication, Fumio Hanaoka). Unlike
  • HHR23A Identification of the Vpr interacting domain of HHR23A
  • the eight different HHR23A cDNAs isolated in the two-hybrid screen enabled the localization of the region of HHR23A required for interaction with Vpr.
  • Peptides comprising a region as small as 45 amino acids of the C-terminal are sufficient to bind to GST- Vpr.
  • the corresponding 45 amino acids of HHR23B also binds to GSTVpr.
  • the E2(25K) protein is a class 2 ubiquitin conjugating enzyme (UBC).
  • Class 2 UBCs contain a highly conserved catalytic domain followed by unrelated C-terminal extensions that vary in length and which are thought to promote interaction with the substrate and/or function in cellular localization of the UBC.
  • the other class of UBCs, class I enzymes lack C-terminal extensions and require auxiliary proteins (E3 proteins) for substrate recognition.
  • E3 proteins catalyze the isopeptide bond formation between ubiquitin and the substrate and thus play a key role in the selection of proteins for ubiquination and their subsequent proteolytic degradation.
  • Vpr expression in an infected individual may be the inhibition of clonal expansion of T-cells in response to antigen.
  • antigen presenting cells such as macrophages infected with HIV-1
  • Vpr would contribute to immune dysfunction and serve an important function for the virus by allowing persistence of virally infected cells.
  • Our results suggest that the high proportion of non-infectious HTV-1 particles found in the blood and lymph nodes of infected individuals could contribute to the cell cycle arrest of T-cells and thus immune suppression.

Abstract

La présente invention se fonde sur l'observation selon laquelle la protéine Vpr des lentivirus, en particulier la protéine Vpr du VIH, produit une stase et une mort cellulaires lorsqu'on la met en contact avec une cellule. Se fondant sur ces observations, la présente invention concerne des procédés permettant de provoquer une stase et une mort cellulaires, qui consistent à mettre une cellule en contact avec une protéine Vpr ou un analogue d'une protéine Vpr.
PCT/US1998/003390 1997-02-11 1998-02-11 Procedes permettant de provoquer une stase de cycle cellulaire WO1998035032A2 (fr)

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