WO1997006257A1 - Cellular co-factor for hiv rev and htlv rex - Google Patents

Abstract

DNAs encoding cellular co-factors for the HIV-1 Rev and the HTLV-I Rex post-transcriptional regulator proteins are disclosed. Also disclosed are proteins produced from the DNAs and methods of using the same.

Description

Cellular Co-Factor for HIV Rev and HTLV Rex

Field ofthe Invention

The present invention relates to cellular co¬ factors for the HIV-l Rev and the HTLV-I Rex post- transcriptional regulator proteins, DNAs encoding the same, and methods of use thereof.

Background ofthe Invention

The HIV-l Rev protein is required for the nucleocytoplasmic transport, and hence translation, of a class of incompletely spliced HIV-l mRNAs that encode the viral structural proteins (B. Cullen, Microbiol . Rev . 56, 375-394 (1992)). In the absence of Rev, these late viral RNAs remain sequestered in the nucleus until they are either spliced or degraded (M. Emerman et al. , Cell 57, 1155-1165 (1989); B. Felber et al., Proc . Natl . Acad . Sci . USA 86, 1495-1499 (1989); M. Malim et al., Nature 338, 254-257 (1989)). Rev function is therefore essential for the production of progeny virions by HIV-l infected cells (M. Feinberg et al., Cell 46, 807-817 (1986); J. Sodroski et al., Nature 321, 412-417 (1986)). Rev mediated nuclear RΝA export requires the direct interaction of Rev with a cis-acting, -234 nucleotide (nt) RΝA stem-loop structure termed the Rev Response Element or RRE (T. Daly et al., Wature 342, 816-819 (1989); Mali et al., supra ; M. Malim et al., Cell 60, 675-683 (1990)). It has been proposed that Rev first binds to a high-affinity site within the RRE as a monomer (D. Bartel et al.. Cell 67, 529-536 (1991); L. Tiley et al., Proc . Natl . Acad . Sci . USA 89, 758-762 (1992)) . Subsequently, additional Rev monomers assemble.onto the RRE in an ordered process mediated by both protein-protein and protein-RNA interactions. This multimerization event is critical for Rev function via the RRE (M. Malim and B. Cullen, Cell 65, 241-248 (1991); S. Iwai et al., Nucl. Acids Res . 20, 6465-6472 (1992); D. McDonald et al., J . Virol . 66, 7232-7238 (1992)).

Mutational analysis of the 116 amino acid (aa) Rev transactivator has identified two functional domains. A more amino terminal basic domain acts as both a nuclear/nucleolar localization signal and as a sequence specific RNA binding domain (M. Malim et al., Cell 58, 205-214 (1989A); A. Cochrane et al., J . Virol . 64, 881-885 (1990); J. Kje s et al., EMBO J. 11, 1119-1129 (1992)). This domain is flanked by sequences that are essential for efficient Rev multimerization (Malim and Cullen, supra) . Towards the carboxy-terminus of Rev lies a short, ~10 aa leucine-rich sequence, termed the Rev activation domain, that is dispensable for RRE binding but nevertheless essential for Rev function (M. Malim et al., J . Virol . 65, 4248-4254 (1991) ; L. Venkatesh and G. Chinnadurai, Virology 178, 327-330 (1990)). At least two lines of evidence suggest that the activation domain represents a binding site for an essential cellular Rev co-factor. Most importantly, this short sequence element is the only part of Rev whose integrity is essential for Rev function via a heterologous RNA target site (J. McDonald et al., supra) . Thus, it has been demonstrated that a fusion protein consisting of Rev linked to the MS2 bacteriophage coat protein can induce the nuclear export of an RNA containing multiple copies of the MS2 coat protein RNA binding site. Mutations known to block the activity of the Rev RNA binding and/or multimerization sequence prevent the function of this fusion protein via the RRE yet fail to inhibit function via the MS2 RNA operator. In contrast, mutation of the Rev activation domain abrogates function via either RNA target (J. McDonald et al., supra) . These data therefore imply that the primary role of-the Rev RNA binding/multimerization domain is to facilitate the assembly of a Rev/RRE ribonucleoprotein complex that then recruits the appropriate cellular co-factor to the Rev activation motif.

A second line of evidence indicating that the activation domain is a Rev co-factor binding domain derives from the observation that mutant Rev proteins containing an intact RNA binding/multimerization domain, but lacking a functional activation domain, exhibit a potent dominant negative phenotype in;vivo (Malim et al., supra ; Venkatesh and Chinnadurai, supra) . It has been proposed that these Rev mutants participate with wild-type Rev in the formation of the Rev/RRE ribonucleoprotein complex but then block the function of the wild-type Rev by interfering with the co-operative recruitment of a cellular co-factor. The observation that Rev is functional in cells derived from a wide range of eukaryotes, including primates, mice, birds, frogs, fruit flies and even yeast, implies that this unidentified cellular co-factor must have been evolutionarily conserved (M. Ivey-Hoyle and M. Rosenberg, Mol . Cell . Biol . 10, 6152-6159 (1990); Malim et al., supra ; U. Fischer et al., EMBO J . 13, 4105-4112 (1994); F. Stutz and M. Rosbash, EMBO J . 13, 4096-4104 (1994)).

While HIV-l Rev is the most intensely studied retroviral RNA transport factor, other members of the lentivirinae also encode Rev proteins, while the effectively unrelated T-cell leukemia viruses, including Human T-cell leukemia virus type I (HTLV-I) , encode an equivalent regulatory protein termed Rex (Cullen, supra) . For many of these other Rev and Rex proteins, it has proven possible to mutationally define short protein motifs that can functionally substitute for the activation domain of HIV-l Rev (T. Hope et al., J . Virol . 65, 6001-6007 (1991); L. Tiley et al., J. Virol . 65, 3877-3881 (1991)). In the case of the other primate lentiviruses, as well as the more divergent ovine/caprine lentiviruses Visna Maedi Virus (VMV) and Caprine Arthritis Encephelitis Virus- (CAEV) , the Rev activation domain has been found to be similar in size and composition to that present in HIV-l Rev (Malim et al. , supra ; Tiley et al., supra). However, both the Rev protein encoded by the lentivirus Equine Infectious Anemia Virus (EIAV) and the Rex protein of HTLV-I, share no significant sequence homology with HIV-l Rev and contain larger, divergent activation domains (Hope et al., supra ; I. Weichselbraun et al. J. Virol . 66, 2583-2587 (1992); R. Fridell et al., Rev . J. Virol . 67, 7317-7323 (1993); V. Mancuso et al., J. Virol . 68, 1998-2001 (1994)). In the case of HTLV-I Rex, this -15 amino acid sequence can be at least partially aligned with the relevant HIV-l Rev sequence. In contrast, the -18 aa activation domain of EIAV Rev displays no evident sequence similarity to the -10 aa HIV-l Rev activation motif, even though it can effectively substitute for the latter in mediating HIV-l Rev function.

Summary ofthe Invention

A first aspect of the invention is isolated DNA encoding a cellular co-factor for HIV Rev selected from the group consisting of:

(a) DNA which encodes a protein of SEQ ID NO:2 (e.g., DNA of SEQ ID NO:l);

(b) DNA which hybridizes to DNA of (a) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev; and (c) DNA differing from the DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a cellular co-factor for HIV Rev.

A second aspect of the invention is a recombinant DNA comprising vector DNA and a DNA as given above. A third aspect of the invention is a host cell containing a recombinant DNA as given above.

A fourth aspect of the invention is an oligonucleotide probe capable of selectively binding to a DNA as given above (e.g., a probe that is capable of serving as a PCR extension primer; a probe labelled with a detectable group) .

A fifth aspect of the invention is an isolated protein comprising a cellular co-factor for HIV Rev, which protein is coded for by a DNA as given above.

A sixth aspect of the present invention is an antibody which specifically binds to a cellular co-factor for HIV Rev.

The foregoing and other objects and aspects of the present invention are explained in detail in the drawings herein and the specification set forth below.

BriefDescription ofthe Drawings

Figure 1 shows the primary sequence of the minimal activation domains of selected Rev and Rex proteins. The upper portion of the figure is a schematic representation of the Rev transactivation region. The Rev transactivation region contains an RNA binding domain, indicated by cross-hatching, and an activation domain, which is shown in double cross-hatching. Amino acid sequences of the activation domains of Rev and Rex proteins from several lentiviruses are shown in the lower portion of the figure. All amino acid sequences are shown from the carboxyl to the amino terminus, going from left to right. The numbers on the left and right ends of each sequence indicate the positions of the first and last amino acids, respectively, in the activation domain. Labels on the right hand side of the sequences designate the lentivirus source of the sequences. Going from top to bottom, the displayed sequences correspond to the activation domains of HIV-l Rev, HIV-2 Rev, VMV (Visna Maedi Virus) Rev, CAEV (Caprine Arthritis Encephalitis Virus) Rev, HTLV-I (Human T-cell Leukemia virus type I) Rex and EIAV (Equine Infectious Anemia Virus) Rev. The four leucine residues in the Rev activation domain and their putative homologues in the other lentivirus proteins are indicated by boxes. Figure 2 shows that functioning of Rev activation domain mutants in vivo is closely correlated with their ability to bind proteins of the invention (sometimes referred to herein as "Rab", for the Rev/Rex Activation Domain Binding protein) . The amino acid sequence shown corresponds to amino acids 69 to 90 of the HIV-l Rev protein. Missense mutants are designated by MIO, M15 to M25, M27 and M29. The amino acid substitutions in each missense mutant are indicated by a line above or below the altered amino acids, which is labeled with the name of the mutant. All mutants substituting two amino acid (MIO, M15-M18, M20-M22) have Asp-Leu in place of the wild-type sequence. Single amino acid mutations feature a substituted Asp (M19) or Ala (M27 and M29) residue while three amino acid mutations contain the inserted sequence Glu-Asp-Leu (M23 and M25) or Lys-Asp-Leu (M24) . Below the amino acid sequence is a listing of each Rev mutant fusion protein and the wild-type Rev (WT) (left column) . Rev activity (center column) for each mutant was compared with the WT and is indicated by ++ (>50% WT activity) , + (<50% WT activity) , or - (no activity) . Rab binding of Rev mutants was assessed by expressing each Rev mutant in the yeast indicator strain GGY1::171 as a GAL4 fusion protein and determining the level of 3-gal activity induced by co-expression of the VP16-Rab fusion (right column) . Binding of each Rev mutant to Rab is expressed relative to the wild-type GAL4/Rev fusion protein, which is set at 1.00.

Figure 3 (SEQ ID NO:l) shows the predicted primary amino acid sequence of the human Rab protein. Numbers running down the left side of the figure indicate amino acid position in the sequence. Phenylalanine residues, including the dipeptide motif "FG," as well as runs of serines are indicated by boxes or underlining, respectively.

Figure 4 shows a Western blot analysis of Rab protein expression in several species. The blot shown in the right panel was performed in the presence of soluble Rab protein to assess signal specificity. The source of the protein extract in each lane is designated across the top of the panels. Left panel: lane 1—human, lane 2— mouse, lane 3—quail, lane 4—frog, and lane 5—fly. Right panel: lane 6—human, lane 7—frog, and lane 8—fly. The relative mobility of marker proteins of the indicated size, in kilodaltons, is given to the left of the panels.

Figure 5 shows Rab interaction with the activation domains of multiple Rev and Rex proteins in the mammalian nucleus using a two-hybrid analysis in COS cells. The vertical axis indicates the VP16 fusion proteins tested (HIV Tat, HIV Rev, HLTV Rex, VMV Rev, and EIAV Rev) . For each fusion protein, wild-type (WT) and missense mutants (MIO, M32, and ΔAD) were evaluated. The horizontal axis indicates fold trans-activation of CAT enzyme activity detected in a COS cell culture co-transfected with the indicated VP16 fusion protein expression plasmids above that observed with pBC12/GAL4-Rab and the pG5B/CAT indicator plasmids alone. Data shown are representative of multiple independent transfection experiments that were each internally controlled by co-transfection of a j3-gal expression plasmid.

Figure 6 shows that Rab binds to the Rev:RRE ribonucleoprotein complex in vivo . Panel A is a schematic representation of an in vivo mono-hybrid assay for Rab binding to the Rev:RRE ribonucleoprotein complex. The Tat/Rab fusion protein is indicated by boxes. The Rev protein is shown bound to the SLIIB RNA target sequence. Arrows indicate that Tat will transactivate the HIV-l LTR only if Rab recruits the TAT/Rab fusion protein to the SLIIB-bound Rev protein. Panel B shows activation of the SLIIB/CAT indicator construct in HeLa cells upon co-transfection of the expression plasmids designated on the vertical axis. The horizontal axis indicates the fold transactivation of HIV-l LTR driven pSLIIB/CAT expression when cells were co-transfected with the indicated expression plasmids.

Figure 7 shows the effects of overexpressing Rab on HIV-l Rev and HTLV-I Rex function in COS cells. The vertical axis indicates that COS cells were transfected with the Rev-defective HIV-l provirus expression plasmid HIVΔREV, either alone or in the presence of the HIV-l Rev expression plasmid pcRev or the HTLV-I Rex expression plasmid pcRex. Co-transfections with pcrev and pcrex were done in the presence of an ~20-fold molar excess of a plasmid expressing either the full-length Rab protein or expressing CAT or /3-gal as negative control proteins. The horizontal axis shows the concentrations of HIV-l P24 capsid protein secreted into the culture medium. Data are representative of three independent experiments.

Detailed Description ofthe Invention Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR §1.822 and established usage. See, e.g., Patentin User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C. 20231); U.S. Patent No. 4,871,670 to Hudson et al. at Col. 3 lines 20-43 (applicants specifically intend that the disclosure of this and all other patent references cited herein be incorporated herein by reference) . A. DNAs ENCODING CELLULAR CO-FACTORS OF HIV REV AND HTLV REX.

DNAs of the present invention that code for cellular cofactors of HIV-l REV and/or HTLV-I Rex may be of any species of origin, including mouse, rat, rabbit, cat, and human, but preferably code for proteins of mammalian origin. Thus, DNA sequences which hybridize to a DNA encoding a protein having the sequence given in SEQ ID NO:2 (e.g., a DNA having the nucleotide sequence given in SEQ ID NO:l) and which code for expression of a cellular co-factor of HIV-l REV and/or HTLV-I Rex are also an aspect of this invention. Conditions which will permit other DNA sequences which code for expression of such a protein to hybridize to said sequence can be determined in a routine manner. For example, hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C or even 70°C to DNA encoding the rat serotonin transporter disclosed herein in a standard in situ hybridization assay. See J. Sambrook et al.. Molecular Cloning, A Laboratory Manual , 2d Ed. (Cold Spring Harbor Laboratory 1989) . In general, sequences which code for a protein of the invention and hybridize to the DNA encoding the protein of the invention disclosed herein as SEQ ID NO:2 will be at least 75% homologous, 85% homologous, or even 95% homologous or more with the sequence of the DNA encoding the protein of SEQ ID NO:2. Further, DNA sequences which code for polypeptides coded of the protein given in SEQ ID NO:2, or sequences which hybridize to the DNA encoding the same and code for a protein of the invention, but which differ in codon sequence from these due to the degeneracy of the genetic code, are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Patent No. 4,757,006 to Toole et al. at Col. 2, Table 1. DNAs of the invention may be naturally occuring or synthetic in origin. Those skilled in the art will appreciate that minor alterations can be introduced into the DNAs without departing from the instant invention. Proteins of the invention are, in general, cellular co-factors for HIV-l Rev and/or HTLV-l Rex post-transcriptional regulator proteins, and specifically bind to HIV-l Rev and/or HTLV-l Rex at the same binding site bound by the protein of SEQ ID NO:2, and may have the same biological activity thereof.

B. GENETIC; ENGINEERING TECHNIQUES The production of cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering is well known. See, e.g.. U.S. Patent No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Patent No. 4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Patent No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Patent No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59.

A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding proteins of the invention as given herein and/or to express DNA which encodes proteins of the invention as given herein. An expression vector is a replicable DNA construct in which a DNA sequence encoding a protein of the invention is operably linked to suitable control sequences capable of effecting the expression of the receptor in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. A plificatiQn vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus) , phage, and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination) . The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism. DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

Transformed host cells are cells which have been transformed or transfected with vectors containing a DNA sequence as disclosed herein constructed using recombinant DNA techniques. Transformed host cells ordinarily express the receptor, but host cells transformed for purposes of cloning or amplifying the receptor DNA do not need to express the receptor.

Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are a particularly suitable host for recombinant protein synthesis, and mammalian cells are particularly preferred. Propagation of such cells in cell culture has become a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells- ordinarily include (if necessary) an origin of replication, a promoter located upstream from the DNA encoding the protein of the invention to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence. The transcriptional and translational control sequences in expression vectors to be used in transforming

'vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma,

Adenovirus 2, and Simian Virus 40 (SV40) . See, e.g.. U.S. Patent No. 4,599,308.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV 40 or other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the receptor DNA. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Pat. No. 4,399,216.

Other methods suitable for adaptation to the synthesis of the proteins of the invention in recombinant vertebrate cell culture include those described in M-J. Gething et al.. Nature 293, 620 (1981); N. Mantei et al., Nature 281. 40; A. Levinson et al., EPO Application Nos. 117,060A and 117,058A. Host cells such as insect cells (e.g., cultured

Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out the present invention, as described in U.S. Patents Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.

Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. COli B, E. COU X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using pBR322. Promoters most commonly used in recombinant microbial expression vectors include the beta- lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275. 615 (1978); and Goeddel et al. , Nature 281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8., 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80. 21 (1983)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA encoding the protein of the invention, i.e., they are positioned so as to promote transcription of messenger RNA from the DNA.

Eukaryotic microbes such as yeast cultures may also be transformed with vectors carrying the isolated DNA's disclosed herein. see, e.g.. U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding the receptor as given herein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282. 39 (1979); Kingsman et al., Gene 2, 141 (1979); Tschemper et al., Gene 10. 157 (1980)). Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 1_, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)). Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657. Proteins of the present invention may be isolated and/or purified from natural sources or recombinant sources as given above in accordance with conventional techniques, optionally employing antibodies that specifically bind the proteins as given below.

C. ANTIBODIES AND OLIGONUCLEOTIDE PROBES.

A variety of detectable groups can be employed to label antibodies and probes as disclosed herein, and the term "labelled" is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, β- glucuronidase, alkaline phosphatase, and β-D- galactosidase) , fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., ¹C, ^13lI, ³H, ³²P, and ³⁵S) to the compound being labelled. Techniques for labelling various compounds, including proteins, peptides, and antibodies, are well known. See, e.g., Morrison, Methods in Enzymology 32b, 103 (1974); Syvanen et al., J . Biol . Chem . 284, 3762 (1973); Bolton and Hunter, Biochem . J . 133, 529 (1973). Oligonucleotide probes of the instant invention may be of any suitable length, depending on the specific application thereof. For example, such probes may, in general, be 6, 8 or 12 nucleotides in length to 16, 20, or 30 nucleotides in length or more. Such probes are useful for identifying and making DNAs encoding proteins of the invention.

Antibodies which specifically bind to the proteins of the invention (i.e., antibodies which bind to a single antigenic site or epitope on the transporter) may be polyclonal or monoclonal in origin, but are preferably of monoclonal origin. Such antibodies are useful for the affinity purification of the proteins of the invention, and for the identification; and assay of the proteins in human tissue samples. The antibodies may be of any suitable species, such as rat, rabbit, or horse, but are generally of mammalian origin. The antibodies may be of any suitable immunoglobulin, such as IgG and IgM. Fragments of antibodies which retain the ability to specifically bind the proteins of the invention, such as F(ab')_2/ F(ab'), and Fab fragments, are intended to be encompassed by the term "antibody" herein. The antibodies may be chimeric, as described by M. Walker et al., Molecular Immunol . 26, 403 (1989) . Antibodies may be immobilized on a solid support of the type used as a packing in an affinity chromatography column, such as sepharose, silica, or glass beads, in accordance with known techniques.

Monoclonal antibodies which bind to the proteins of the invention are made by culturing a cell or cell line capable of producing the antibody under conditions suitable for the production of the antibody (e.g., by maintaining the cell line in HAT media) , and then collecting the antibody from the culture (e.g., by precipitation, ion exchange chromatography, affinity chromatography, or the like) . The antibodies may be generated in a hybridoma cell line in the widely used procedure described by G. Kohler and C. Milstein, Nature 256, 495 (1975) , or may be generated with a recombinant vector in a suitable host cell such as Escherichia coli in the manner described by W. Huse et al., Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, Science 246, 1275 (1989).

DNAs of the invention are useful for making proteins of the invention, as described above. Proteins of the invention have a variety of uses. For example, since proteins of the invention specifically bind to HIV-l Rev and HTLV-I Rex (and other Lentivirus and HTLV family Rev's), they are useful in combination therewith as a member of a specific binding pair. Specific binding pairs are useful for a variety of purposes, such as in various immunoassay systems.

In addition, DNAs and proteins of the invention are useful in methods of screening for compounds that affect the binding of a Lentivirus Rev protein or an HTLV family Rev protein with the cellular co-factor thereof. Such compounds are useful in treating or inhibiting the growth of the corresponding virus (e.g., HIV-l, HIV-2, HTLV-I, HTLV-II, bovine leukemia virus (or "BLV") . In general, such methods comprise providing together (e.g., in vitro in an aqueous solution; in vivo in a cell) a first protein and a second protein so that the first and second protein form a complex thereof, wherein the first protein is selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins (e.g., Rev proteins or Rev protein counterparts from HIV-l, HIV-2, HTLV-I, HTLV-II, bovine leukemia virus (or "BLV"), and equine infectious anemia virus) , and wherein the second protein is encoded by a DNA encoding a co-factor of the invention as given above. Then, a test compound is combined with the first and second protein (e.g., by adding it to the aqueous solution or adding to the media containing the cells) and the influence of the test compound on the formation of the complex is detected (for example, by detecting the association or dissociation constant of the complex, the stability of the complex, etc.). Compounds that adversely affect complex formation are candidate compounds for treating or inhibiting the growth of the lentivirus or HTLV family virus. Where the assay is carried out in vivo , any suitable host cell may be employed as described above (e.g., yeast cells; insect cells). Also disclosed herein are recombinant cell useful for carrying out screening methods as given above. In general, such cells are made and used in accordance with the two-hybrid interaction trap method. See, e.g., Fields and Song, Nature 340, 245-246 (1989); Fridell et al., Virology 209, 347-357 (1995)) . Such cells comprise a first DNA that expresses a first fusion protein comprising a DNA binding domain and a first binding partner; a second DNA that expresses a second fusion protein comprising a second binding partner and a transcription activation domain; and a third DNA comprising a binding site operatively associated with a reporter gene, wherein the DNA binding domain specifically binds to the binding site, so that the reporter gene is expressed when the first and second fusion proteins bind to one another (and wherein the reporter gene is not significantly expressed when the first and second binding proteins are not bound to one another, so that the binding of the two may be detected through the activation of the reporter gene) . The first binding partner is either

(a) selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins, as given above, or

(b) a co-factor thereof as given above, and the second binding partner is the other of (a) or (b) . Any suitable host cell as given above may be employed, with yeast cells particularly preferred. Any suitable binding site and corresponding binding domain specifically bound thereby may be employed, with one example being a Gal4 binding site and a Gal4 binding domain. Any suitable transcription activation domain may be employed, including, but not limited to, the Gal4 transcription activation domain, the VP16 transcription activation domain, and the TAT transcription activation domain. Also disclosed are methods of treating or inhibiting the growth (e.g., by interfering- with viral replication) of Lentiviruses or HTLV family viruses (e.g., HIV-l, HIV-2, HTLV-I, HTLV-II, bovine leukemia virus (or "BLV") . Such methods comprise introducing into a cell infected with the virus (e.g., by administering to an animal infected with the virus in need of such treatment) an amount of a compound that inhibits the formation of a complex between a lentivirus Rev protein or an HTLV family Rev protein with the cellular co-factor thereof effective to inhibit the growth of the virus. Such compounds are identified by the methods described above. Animals that may be treated by the method of the invention include both human and animal subjects (e.g., horse, bovine, or any other animal infected by the corresponding virus) . Administration of the compound may be carried out by any suitable means, including parenteral administration (e.g., intraveneous, intraperitoneal, intramuscular, and subcutaneous injection) , topical administration, and oral administration. Where desired, the compounds may optionally be encapsulated into a liposome to facilitate their transport into cells. Dosage of the active compound may be determined by routine experimentation in animal models in accordance with known techniques. The present invention is explained in greater detail in the following non-limiting examples.

EXAMPLES

EXPERIMENTAL PROCEDURES

Yeast two-hvbrid interaction experiments The yeast expression plasmid pGAL4-Rev encodes a fusion protein consisting of the GAL4 DNA binding domain linked to the full-length HIV-l Rev protein (R. Fridell et al.. Virology 209, 347-357 (1995)). A series of 14 missense mutations, in or near the HIV-l Rev activation domain, were generated in the PGAL4-Rev context by replacement of the wild-type Rev CDNA sequence (Xbal to EcoRI) with polymerase chain reaction (PCR) generated CDNA fragments bearing the previously described mutations (M.

Malim et al., J . Virol . 65, 4248-4254 (1991)) listed in

Figure 2. The plasmid PGAL4-Rex was constructed by substituting amino acids 2 to 189 of HTLV-I Rex in place of the Rev sequence present in PGAL4-Rev.

The Y190 yeast indicator strain, the preparation of the oligo-dT primed, pVP16 based CEM CDNA library and the methodology used to screen for interacting proteins have been described (J. Harper et al., Cell 75, 805-816 (1993);

R. Fridell et al., Virology 209, 347-357 (1995)).

Following rescue, library plasmids expressing relevant VP16 fusion proteins were rescreened by transformation into the yeast indicator strain GGY1::171 (G. Gill and M. Ptashne, Cell 51, 121-126 (1987) together with the panel of GAL4 fusion protein expression plasmids described in Figure 2.

After 3 days of selection on culture plates, double transformants were transferred to selective liquid medium.

The next day, equal cell equivalents were analyzed for levels of β-gal expression (R. Fridell et al., Virology

209, 347-357 (1995)).

Where appropriate, molecular clones were sequenced using the dideoxy chain termination method and the sequenase version 2.0 sequencing kit (United States Biochemical) . The Rab CDNA insert was sequenced by the same method using both dGTP and dITP.

RNA and protein expression analysis

Northern analysis of poly(A)+ RNA derived from the human cell lines CEM and HeLa was performed as previously described (M. Malim et al., Cell 60, 675-683 (1990)) using a random primed, α32P-DCTP labeled probe prepared from an internal 853 bp BamHI to Accl Rab CDNA fragment.

The pGEX-4T plasmid was used to express a fusion protein consisting of GST linked to amino acids 101 to 562 of the Rab open reading frame (ORF) (Figure 3) in the BL21

(Ion-) strain of E . coli . Expression was induced by addition of 0.1 Mm IPTG and the resultant GST-Rab fusion protein extracted, purified and used to immunize rabbits. A second fusion protein, consisting of MBP linked to amino acids 101 to 326 of Rab, was expressed in E. coli using the pMAL-C2 expression plasmid, purified using a commercial kit (MBP protein fusion and purification system, New England Biolabs) and then coupled to cyanogen bromide-activated agarose beads (Pierce) . The agarose coupled MBP-Rab fusion protein was used to affinity purify Rab-specific antibodies from the serum of the GST-Rab injected rabbits using buffers and procedures detailed in the Amino Link Immobilization Kit (Pierce) . The resultant Rab-specific rabbit antiserum was! concentrated and dialyzed against phosphate buffered saline (PBS) prior to use.

The tissue culture cell lines used to make protein extracts were HeLa (human) , C127 (mouse) , QC13 (quail) and Schneider 2 (Drosophila) . Frog protein extracts were prepared from Xenopus oocytes. Cells were washed with PBS and then resuspended in 100 11 of PBS. After addition of 100 11 of 2X Laemmli gel loading buffer containing 2-mercaptoethanol, the samples were sonicated, boiled and centrifuged to remove debris. Soluble proteins were separated by 12% SDS-PAGE, transferred to a nitrocellulose filter and then incubated with a 1:20,000 dilution of the affinity purified rabbit Rab antiserum. After vigorous washing, bound antibodies were detected using a horseradish peroxidase-conjugated goat anti-rabbit antiserum and enhanced chemiluminescence (Amersham) .

Immunofluorescence analysis was performed essentially as previously described (M. Malim et al.. Cell 58, 205-214 (1989A)) . CV-1 cells were fixed and permeabilized prior to incubation with a 1:1000 dilution of the rabbit anti-Rab antiserum. After extensive washing, the fixed cells were incubated with a 1:100 dilution of an affinity purified,rhodamine-conjugated goat anti-rabbit antiserum (Boehringer Mannheim) and examined using a Zeiss Axioskop immunofluorescence microscope. GST affinity chromatography Both the GST/Rev and the GST/M10 fusion proteins contain the full-length Rev ORF, in the former case containing an intact activation domain and in the latter the defective, MIO mutant form of the activation domain (M. Malim et al., Cell 58, 205-214 (1989A)). In addition, both Rev fusion proteins also contain the previously described M6 missense mutation of the Rev RNA binding domain (M. Malim et al., Cell 58, 205-214 (1989A) ) . This mutation, which affects amino acids 41 to 44 of Rev, is located well outside the Rev activation domain (Figure 1) . However, inclusion of this mutation markedly increases the yield of full-length Rev protein upon expression in E . coli (data not shown) . Bacterially expressed GST, GST/Rev and GST/M10 proteins were purified as previously described (M. Malim et al., J . Virol . 65, 4248-4254 (1991)). Equivalent levels were then coupled to cyanogen bromide-activated agarose beads (Pierce) and used to prepare columns with a 0.5 ml bed volume.

A DNA fragment encoding the Rab ORF (amino acids 1 to 562, Ncol to Xhol) was cloned into the pGEM3ZF(+) expression plasmid and 35S-labeled Rab protein prepared in a 200 11 rabbit reticulocyte lysate coupled transcription-translation reaction (Promega) using T7 RNA polymerase and 35S-methionine/35S-cysteine. The lysate was then diluted 1:5 with chromatography buffer (CB) (10 Mm Hepes Ph 7.5, 0.1 M NaCl, ImM EDTA, 1 Mm DTT, 10% glycerol, 2mg/ml BSA, 0.5% NP40 and 0.25 Mm PMSF) and equal amounts loaded onto the immobilized GST, GST/Rev and GST/M10 affinity columns. These were then washed with 10 column volumes of CB before bound proteins were eluted with 4 column volumes of 0.1 M glycine, Ph 2.8. Eluted proteins were concentrated and dialyzed against CB prior to analysis by 12% SDS/PAGE. Mammalian two-hybrid assay All mammalian expression plasmids were constructed in the context of plasmid PBC12/CMV (M. Malim et al., J. Virol . 65, 4248-4254 (1991)) with the exception of the pG5B/CAT indicator construct, which has been described (Bogerd and Greene, 1993) . PBC12/GAL4-Rab expresses the GAL4 DNA binding domain (amino acids 1-147) linked to the full-length Rab CDNA sequence. pRev/VP16 and pRex/VP16 have been described (H. Bogerd and W. Greene, J. Virol . 67, 2496-2502 (1993)) and express full-length HIV-l Rev and HTLV-l Rex linked to the VP16 transcription activation domain. Variants of Rev (MIO, M32) and Rex (DAD) bearing defective activation domains were substituted into this same expression plasmid by excision of the wild-type rev gene by cleavage with Sad and Bglll and replacement with a PCR generated Sad; to Bell DNA fragment encoding the appropriate Rev or Rex mutants. Similarly, PCR-generated Sad to Bell DNA fragments encoding wild-type and mutant (DAD) forms of VMV Rev and EIAV Rev were also substituted in place of HIV-l Rev to generate the relevant VP16 fusion protein expression plasmids. All Rev and Rex activation domain mutants have been previously described and characterized (T. Hope et al., J. Virol . 65, 6001-6007 (1991); M. Malim et al., J. Virol . 65, 4248-4254 (1991); L. Tiley et al., J. Virol . 65, 3877-3881 (1991); R. Fridell et al., Rev. J. Virol . 67, 7317-7323 (1993)). The introduced mutations are: HIV Rev M10, LG (78,79) to DL; HIV Rev M32, L 78, 81 and 83 all to A; RexDAD, LSLD (90-93) to GGGG; VMV RevDAD, LE (114, 115) to DL; EIAV RevDAD, L49 and 154 to A.

COS cell cultures (100 mm) were transfected with 1 lg

PG5B/CAT, 500 ng PBC12/GAL4-Rab, 5 lg of a VP16 fusion protein expression plasmid and 500 ng of the internal control indicator plasmid PBC12/CMV/0gal using the DEAE-dextran procedure. The parental PBC12/CMV expression plasmid served as a negative control. At -48 hrs post-transfection, cell extracts were prepared and CAT and 0-gal expression quantified (R. Fridell et al., Virology 209, 347-357 (1995)). Mammalian mono-hybrid assay

The PSLIIB indicator construct and plasmids expressing wild-type (pcrev) and mutant (pM10, pM32) forms of Rev have been described (M. Malim et al., J . Virol . 65, 4248-4254 (1991); L. Tiley et al., Genes Dev . 6, 2077-2087 (1992A) ) . The pcTat/Rab plasmid expresses a fusion protein consisting of the full-length Tat protein linked to the first amino acid of the Rab ORF indicated in Figure 3. HeLa cultures (35 mm) were transfected with 1 lg of the PSLIIB/CAT reporter plasmid, 0.5 lg each of the Tat/Rab fusion protein and Rev protein expression plasmid and 1 lg of carrier DNA using the calcium phosphate procedure. The parental PBC12/CMV expression plasmid was used as a negative control. Cultures were harvested at -48 hrs after transfection and CAT activity quantified (R. Fridell et al., Virology 209, 347-357 (1995)). HIV-l virus rescue HIV-l provirus rescue assays were performed in COS cell cultures essentially as described (M. Malim et al., J. Virol . 65, 4248-4254 (1991)). The Rev- provirus expression plasmid pHIVDRev and the pcrev, pcrex, PBC12/CMV/SEAP, PBC12/CMV/CAT and PBC12/CMV//3gal expression plasmids have been described (L. Rimsky et al., Nature 335, 738-740 (1988); M. Malim et al., J. Virol . 65, 4248-4254 (1991)). The Rab expression plasmid PBC12/CMV/Rab contains the 562 amino acid Rab ORF indicated in Figure 3 (Ncol to Xhol) cloned into the expression plasmid PBC12/CMV. Transfections were performed as described in Figure 7. Secreted p24 levels were quantified using a commercial ELISA kit (DuPont) while SEAP activity was determined as described (J. Berger et al., Gene 66, 1-10 (1988)). RESULTS Using the yeast two-hybrid protein interaction trap (S. Fields and O-K Song, Nature 340, 245-246 (1989); R. Fridell et al., Virology 209, 347-357 (1995)) we sought to identify a human CDNA that encoded a protein able to interact with not only HIV-l Rev, but also HTLV-I Rex, in an activation domain dependent manner. The screened library consisted of the VP16 transcription activation domain fused to CDNA sequences derived from the human CEM T-cell line. This screen led to the identification of 3 clones that encoded VP16 fusion proteins that specifically interacted with the GAL4/Rev bait protein, and 10 clones expressing VP16 fusion proteins that specifically bound GAL4/Rex. None of the three Rev specific clones were able to interact with GAL4/Rex while only one of the ten Rex specific clones could bind to GAL4/Rev. Further experiments demonstrated that this interaction was entirely blocked by the introduction of point mutations into the activation domains of either Rev or Rex (data not shown) . Based on this initial observation, we designated this human protein as the Rev/Rex Activation Domain Binding or Rab protein. Rab binds the Rev activation domain specifically To more fully define the protein sequence specificity of the interaction of Rev with Rab, we took advantage of a previously described (M. Malim et al., J. Virol . 65, 4248-4254 (1991)) set of scanning missense mutations that precisely map the HIV-l Rev activation domain (Figure 2) . These mutations each affect from one to three amino acids either within or adjacent to the activation domain and give rise to wild-type, intermediate or negative phenotypes when assayed for Rev function in mammalian cells. This complete set of Rev mutants was cloned into a yeast GAL4 fusion protein expression plasmid and individually tested for their ability to interact with the VP16/Rab fusion protein in the yeast cell nucleus, as assessed by the level of activation of an integrated lacZ indicator gene. Each of these mutant GAL4/Rev fusion proteins was equivalently stable in yeast, as measured by western blot analysis (data not shown) .

As shown in Figure 2, five Rev mutants that entirely lack effector domain function, including two mutants (M27 and M29) that bear only single amino acid changes in Rev, all proved entirely unable to interact with the Rab fusion protein. In contrast, all of the Rev mutants previously shown (M. Malim et al., J. Virol . 65, 4248-4254 (1991)) to exhibit substantially wild-type Rev activity in vivo also induced substantial levels of j3-gal activity, ranging from a minimum of one quarter to a maximum of -3 times the level seen with wild-type Rev. Strikingly, the three Rev mutants (M18, M20 and M24) previously shown (M. Malim et al., J. Virol . 65, 4248-4254 (1991)) to exhibit only partial (<50%) Rev function in vivo also proved to be significantly attenuated in their ability to interact with Rab (<16% of wild-type) . It is therefore apparent that there is a very close concordance between the ability of Rev activation domain mutants to function in the recruitment of the Rev co-factor in mammaliani cells and the ability of these same Rev activation domain mutants to interact with the Rab protein in the yeast cell nucleus. Sequence of the Rab gene product

The Rab CDNA clone contains a 2584 base pair insert flanked 3' by a stretch of A residues, consistent with priming at the mRNA poly(A) tail. Starting at the first in frame methionine residue, this CDNA contains an open reading frame (ORF) of 562 amino acids which would be predicted to encode a protein of -58 kD (Figure 3) . Computer analysis of available sequence data bases failed to identify any proteins displaying significant homology to the predicted Rab ORF. Similarly, we were also unable to identify any nucleic acid sequences that displayed significant homology to Rab except for two short, unidentified "expressed sequence tags." Rab is therefore a novel human gene. An unusual aspect of the Rab protein sequence is the high concentration of phenylalanine residues, including ten in the form of the dipeptide motif "FG," as well as several runs of serine residues, found concentrated towards the carboxyl-terminus of Rab. The potential significance of these is discussed below. Analysis of Rab MRNA and protein expression The Rab CDNA insert was used to probe a northern blot of MRNA derived from the human T-cell line CEM (the origin of the CDNA clone) and the human cell line HeLa. In both cases, a prominent band of -2.8 kilobases (kb) was observed (data not shown) . Both cell lines also expressed a hybridizing band of -4.6 kb, although this was faint in the CEM cells. Similarly, northern analysis of Rab MRNA expression in a range of human tissues also identified a major 2.8 kb and a minor 4.6 kb Rab transcript in all tissues examined (data not shown) . Based on this analysis, it therefore appeared that this cloned Rab CDNA is close to the full-length of the major species of Rab MRNA expressed in human cell lines and tissues.

An interesting characteristic of both Rev and Rex is that these retroviral regulatory proteins are functional in a wide range of animal cells (M. Ivey-Hoyle and M. Rosenberg, Mol . Cell . Biol . 10, 6152-6159 (1990); M. Malim and B, Cullen, Cell 65, 241-248 (1991); U. Fischer et al., EMBO J. 13, 4105-4112 (1994)). Therefore, it is predicted that the cellular co-factor for Rev and Rex should be conserved across species boundaries. To test this hypothesis, we performed a western analysis on cellular protein extracts of mammalian, avian, amphibian and invertebrate origin using an affinity purified polyclonal rabbit anti-Rab antiserum (Figure 4) . In humans, this procedure identified a predominant band of ~60 Kd (lane 1) and bands of identical mobility were also detected in cellular extracts of mouse, quail and frog origin (lanes 2 to 4) . In the fruit fly, a slightly smaller cross-reactive band was observed (lane 5) . These bands all represented authentic Rab related proteins, in that reactivity could be specifically blocked by addition of a purified, soluble Maltose Binding Protein (MBP)-Rab fusion 3 protein to the western blot during the antibody binding step (lanes 6 to 8) .

Indirect immunofluorescence analysis of the localization of endogenous Rab protein in the primate cell line CVI, using the Rab-specific rabbit antiserum, revealed that Rab is concentrated in the nucleus and nucleolus of these cells in a pattern that is similar to that observed previously with HIV-l Rev and HTLV-I Rex (H. Siomi et al., Cell 55, 197-209 (1988); B. Felber et al., Proc . Natl . Acad . Sci . USA 86, 1495-1499 (1989)). No specific signal was detected when a matched preimmune rabbit serum was used (data not shown) . While Western analysis of nuclear and cytopasmic protein fractions derived from HeLa cells further confirmed the nuclear concentration of Rab, this latter analysis also detected significant levels of cytoplasmic Rab protein (data not shown) . It therefore appears that Rab is primarily, but not exclusively, nuclear in localization.

Although we have been unable to express the full-length Rab protein in a soluble form in bacteria, we are able to express limited amounts of soluble Rab protein in vitro by translation of Rab MRNA in a rabbit reticulocyte lysate. Translation of the Rab ORF indicated in Figure 3 gave rise to a protein that migrated on polyacrylamide gels with the same relative molecular mass as the major Rab-antibody reactive species detected in human cells in vivo (data not shown) . Therefore, the Rab ORF shown in Figure 3 is full-length.

We next used ^3SS-labeled full-length Rab protein generated by in vitro translation to ask whether Rab would bind the Rev activation domain specifically in vitro . For this purpose, we loaded equal levels of the ³⁵S-labeled Rab protein onto columns containing either Glutathione-S-transferase (GST) , or fusion proteins consisting of GST linked to Rev (GST/Rev) or the MIO activation domain Rev mutant (GST/M10) , conjugated to agarose beads. After extensive washing, remaining bound proteins were eluted and subjected to SDS-PAGE. The labeled Rab protein was not retained by the GST or GST/M10 columns but did bind to the GST/Rev column (data not shown) . These data therefore demonstrate that the specific interaction of Rab with the Rev activation domain can be recapitulated in vitro and also strongly suggest that this interaction is direct. Rab binds the activation domains of diverse Rev and Rex proteins in vivo

We next wished to ask whether the interaction of Rab with Rev could be demonstrated in mammalian cells and whether other Rev and Rex proteins, particularly including the highly divergent EIAV Rev (Figure 1) , would also bind to Rab specifically. To address these issues, we performed a mammalian version of the two-hybrid analysis using transient transfection of the primate cell line COS (Figure 5) . The pG5B/CAT indicator plasmid used in this analysis contains 5 GAL4 sites located 5' to a minimal promoter element directing expression of the chloramphenicol acetyl transferase (CAT) gene (H. Bogerd and W. Greene, J. Virol . 67, 2496-2502 (1993)). In this assay, the identities of the bait and prey proteins were reversed such that the Rab protein was now expressed as a GAL4/Rab fusion while the full-length HIV-l, VMV and EIAV Rev proteins and the HTLV-I Rex protein were each expressed as VP16 fusion proteins. For each Rev and Rex protein, plasmids bearing a deleterious missense mutation in the mutationally defined activation domain were also generated (Figure 5) . All VP16 fusion proteins were expressed at comparable levels after transfection into COS cells (data not shown) .

Transient transfection of COS cell cultures with the pG5B/CAT indicator plasmid, along with the PBC12/GAL4-Rab expression plasmid, gave only a low level of CAT enzyme activity. While this level was not significantly enhanced by co-expression of a Tat/VP16 fusion protein, a similar Rev/VP16 fusion protein produced a marked, -18 fold induction in the level of CAT activity (Figure 5) . In contrast, two Rev/VP16 fusion proteins bearing either the M10 or the M32 missense mutation of the Rev activation domain (M. Malim and B, Cullen, Cell 65, 241-248 (199Ϊ)) each failed to enhance expression of the CAT indicator gene.

Similar evidence for an in vivo interaction with Rab was also obtained using VP16 fusion proteins containing either the HTLV-I Rex protein or the VMV Rev or more highly divergent EIAV Rev protein (Figure 5) . In • each case, introduction of a missense mutation (DAD) known to block activation domain function markedly inhibited the in vivo interaction with GAL4/Rab. We therefore conclude that Rab can interact with a range of highly divergent Rev and Rex proteins in the mammalian cell nucleus and that the integrity of the mutationally defined activation motifs is, in each case, critical to this interaction. It should be noted that the endogenously expressed Rab protein (Figure 4) would be predicted to act as an efficient competitive inhibitor of the in vivo interaction between GAL4/Rab and these various Rev or Rex fusion proteins. It is therefore likely that the data presented in Figure 5 significantly underestimate the efficiency of this in vivo interaction. Rab binds to the Rev activation domain when Rev is bound to the RRE

An important predicted property of the cellular co-factor for HIV-l Rev is that it should be able to efficiently interact with the Rev activation domain when Rev is assembled onto the RRE RNA target. To test whether Rab satisfied this prediction, we devised an in vivo genetic assay that uses the HIV-l Tat RNA sequence specific transcriptional activator to provide an indirect measure of the assembly of a protein complex onto an RNA target sequence (Figure 6A) . Normally, Tat activates gene expression from the HIV-l LTR after binding a cis-acting RNA target sequence, termed TAR, that forms the first 59 nt of all HIV-l transcripts (B. Cullen, Microbiol . Rev. 56, 375-394 (1992)). We have previously shown that if the TAR RNA stem-loop is replaced with the RRE-derived stem-loop IIB (SLIIB) minimal RNA target sequence for HIV-l Rev, then trans-activation of the HIV-l LTR can only be observed if Tat is fused to an HIV-l Rev protein bearing an intact RNA binding domain (L. Tiley et al., Proc . Natl . Acad . Sci . USA 89, 758-762 (1992B) ) . The assay delinea.ted in Fig. 6A takes this approach one step further. Here, the TAR element is again replaced by RRE SLIIB in an indicator construct in which the HIV-l LTR is linked to CAT (PSLIIB/CAT) . However, in this case Rev is expressed in its wild-type form while Tat is expressed as a Rab fusion protein. Only if Rab can induce the efficient recruitment of this Tat/Rab fusion protein to the RRE-bound Rev protein will Tat be brought to the HIV-l LTR promoter element and, hence, be able to activate HIV-l LTR driven CAT expression (Figure 6A) .

Results obtained using this mono-hybrid assay on the homologous RRE RNA target are presented in Figure 6B. As can be readily seen, neither HIV-l Rev alone, nor the Tat/Rab fusion protein alone, was able to induce a level of CAT expression that exceeded the basal level seen in HeLa cells transfected with the PSLIIB/CAT indicator construct alone. However, the simultaneous expression of both Rev and Tat/Rab in the nuclei of these human cells induced a readily detectable -19 fold induction in CAT protein expression. Assembly of Rab onto the Rev:RRE ribonucleoprotein complex was, as predicted, fully dependent on the integrity of the Rev activation domain as shown by the lack of activity of the Rev MIO and M32 activation domain mutants in this assay (Figure 6B) . Importantly, these two mutations have no effect on the ability of Rev to bind the RRE in vivo (L. Tiley et al., Genes Dev. 6, 2077-2087 (1992A) ) .

Overexpression of Rab can enhance Rev and Rex function in vivo The data presented in Figure 6 show that Rab can bind to the Rev activation domain when Rev is part of the Rev:RRE ribonucleoprotein complex. Therefore, Rab can effectively compete with the endogenous Rev co-factor for binding to the Rev activation domain. This finding suggested that an in vivo assay for Rev function performed under conditions where the level of Rev expression was sub-optimal should be able to uncover a phenotype for Rab even in the face of a significant endogenous level of Rab expression. In particular, we hypothesized that', if Rab is not the authentic Rev co-factor, then overexpression of Rab should competitively inhibit Rev function. In contrast, if Rab is the authentic Rev co-factor, then overexpression of Rab might promote the recruitment of Rab to the RRE RNA target by sub-optimal levels of Rev protein, leading to an increase in Rev activity. Similarly, the HTLV-I Rex protein, which can also act via the HIV-l RRE element (L. Rimsky et al., Nature 335, 738-740 (1988)), might also display enhanced activity in the presence of Rab if this protein is indeed the authentic Rev/Rex co-factor.

To test this hypothesis, we first introduced a premature termination codon into the rev gene present in a full-length HIV-l proviral clone. We then transfected the resultant HIVDRev provirus into COS cells along with different levels of the HIV-l Rev expression plasmid pcrev or the HTLV-I Rex expression plasmid pcrex (L. Rimsky et al., Nature 335, 738-740 (1988)) and determined the level of each of these plasmids that induced -10% of the level of P24 capsid protein production that was seen with a saturating level of Rev. We next transfected COS cells with the HIVDRev proviral clone, along with this limiting level of either pcrev or pcrex, together with an -20 fold molar excess of a PBC12/CMV based plasmid (M. Malim et al., J. Virol . 65, 4248-4254 (1991)) expressing the full-length Rab protein or expressing either CAT or β-gal as negative control proteins. Based on immunoprecipitation analysis, we estimate that cells transfected with the Rab expression plasmid express 5 to 10 fold higher levels of Rab than normal (data not shown) . Transfection efficiency was monitored by co-transfecting a constant level of an expression plasmid encoding the secreted alkaline phosphatase (SEAP) indicator gene (J. Berger et al.. Gene 66, 1-10 (1988)). After -88 hours, the supernatant media were removed from the transfected cultures and levels of P24 capsid protein and SEAP activity determined. As shown in Figure 7, we consistently observed a 3-6 fold increase in the level of HIV-l P24 capsid protein secreted by the culture overexpressing the Rab protein when compared to the cultures expressing either CAT or β-gal. In contrast, Rab overexpression had no significant effect on the level of supernatant SEAP activity. These data therefore strongly indicate that Rab is an important human cellular co-factor for HIV-l Rev and HTLV-I Rex.

The foregoing examples are intended to be illustrative of the present invention, and are not to be taken as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Cullen, Bryan R. Bogerd, Hal P.

(ii) TITLE OF INVENTION: Cellular Co-Factor for HIV Rev and HTLV Rex

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Bell, Seltzer, Park and Gibson

(B) STREET: Post office Drawer 34009

(C) CITY: Charlotte

(D) STATE: North Carolina

(E) COUNTRY: USA

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(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Sibley, Kenneth D.

(B) REGISTRATION NUMBER: 31,665

(C) REFERENCE/DOCKET NUMBER: 5405-118

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 919-420-2200

(B) TELEFAX: 919-881-3175

(C) TELEX: 575102

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2583 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 244..1929

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TGGCGGCGGC GGCGGCGGTT GTCCCGGCTG TGCCGGTTGG TGTGGCCCGT CAGCCCGCGT 60

ACCACAGCGC CCGGGCCGCG TCGAGCCCAG TACAGCCAAG CCGCTGCGGC CGGGTCCGGC 120

GCGGGCGGCG CGCGCAGACG GAGGGCGGCG GCCGCGGCCA GGGCGGCCCG TGGGACCGCG 180

GGCCCCCGGC GCAGCGCTGC CCGGCTCCCG GCCCTGCCGG CCTCCTCCCT TGGCGCCGCG 240 GCC ATG GCG GCC AGC GCG AAG CGG AAG CAG GAG GAG AAG CAC CTG AAG 288 Met Ala Ala Ser Ala Lys ArgtLys Gin Glu Glu Lys His Leu Lys 1 5 10 15

ATG CTG CGG GAC ATG ACC GGC CTC CCG CAC AAC CGA AAG TGC TTC GAC 336 Met Leu Arg Asp Met Thr Gly Leu Pro His Asn Arg Lys Cys Phe Asp 20 25 30

TGC GAC CAG CGC GGC CCC ACC TAC GTT AAC ATG ACG GTC GGC TCC TTC 384 Cys Asp Gin Arg Gly Pro Thr Tyr Val Asn Met Thr Val Gly Ser Phe 35 40 45

GTG TGT ACC TCC TGC TCC GGC AGC CTG CGA GGA TTA AAT CCA CCA CAC 432 Val Cys Thr Ser Cys Ser Gly Ser Leu Arg Gly Leu Asn Pro Pro His 50 55 60

AGG GTG AAA TCT ATC TCC ATG ACA ACA TTC ACA CAA CAG GAA ATT GAA 480 Arg Val Lys Ser Ile Ser Met Thr Thr Phe Thr Gin Gin Glu Ile Glu 65 70 75

TTC TTA CAA AAA CAT GGA AAT GAA JGTC TGT AAA CAG ATT TGG CTA GGA 528 Phe Leu Gin Lys His Gly Asn Glu Val Cys Lys Gin Ile Trp Leu Gly 80 85 90 95

TTA TTT GAT GAT AGA TCT TCA GCA ATT CCA GAC TTC AGG GAT CCA CAA 576 Leu Phe Asp Asp Arg Ser Ser Ala Ile Pro Asp Phe Arg Asp Pro Gin 100 105 110

AAA GTG AAA GAG TTT CTA CAA GAA AAG TAT GAA AAG AAA AGA TGG TAT 624 Lys Val Lys Glu Phe Leu Gin Glu Lys Tyr Glu Lys Lys Arg Trp Tyr 115 120 125

GTC CCG CCA GAA CAA GCC AAA GTC GTG GCA TCA GTT CAT GCA TCT ATT 672 Val Pro Pro Glu Gin Ala Lys Val Val Ala Ser Val His Ala Ser Ile 130 135 140

TCA GGG TCC TCT GCC AGT AGC ACA AGC AGC ACA CCT GAG GTC AAA CCA 720 Ser Gly Ser Ser Ala Ser Ser Thr Ser Ser Thr Pro Glu Val Lys Pro 145 150 155

CTG AAA TCT CTT TTA GGG GAT TCT GCA CCA ACA CTG CAC TTA AAT AAG 768 Leu Lys Ser Leu Leu Gly Asp Ser Ala Pro Thr Leu His Leu Asn Lys 160 165 170 175

GGC ACA CCT AGT CAG TCC CCA GTT GTA GGT CGT TCT CAA GGG CAG CAG 816 Gly Thr Pro Ser Gin Ser Pro Val al Gly Arg Ser Gin Gly Gin Gin 180 185 190

CAG GAG AAG AAG CAA TTT GAC CTT TTA AGT GAT CTC GGC TCA GAC ATC 864 Gin Glu Lys Lys Gin Phe Asp Leu Leu Ser Asp Leu Gly Ser Asp Ile 195 200 205

TTT GCT GCT CCA GCT CCT CAG TCA ACA GCT ACA GCC AAT TTT GCT AAC 912 Phe Ala Ala Pro Ala Pro Gin Ser Thr Ala Thr Ala Asn Phe Ala Asn 210 215 220

TTT GCA CAT TTC AAC AGT CAT GCA GCT CAG AAT TCT GCA AAT GCA GAT 960 Phe Ala His Phe Asn Ser His Ala Ala Gin Asn Ser Ala Asn Ala Asp 225 230 235

TTT GCA AAC TTT GAT GCA TTT GGA CAG TCT AGT GGT TCG AGT AAT TTT 1008 Phe Ala Asn Phe Asp Ala Phe Gly Gin Ser Ser Gly Ser Ser Asn Phe 240 245 250 255

GGA GGT TTC CCC ACA GCA AGT CAC TCT CCT TTT CAG CCC CAA ACT ACA 1056 Gly Gly Phe Pro Thr Ala Ser His Ser Pro Phe Gin Pro Gin Thr Thr 260 265 270

GGT GGA AGT GCT GCA TCA GTA AAT GCT AAT TTT GCT CAT TTT GAT AAC 1104 Gly Gly Ser Ala Ala Ser Val Asn Ala Asn Phe Ala His Phe Asp Asn 275 280 285

TTC CCC AAA TCC TCC AGT GCT GAT TTT GGA ACC TTC AAT ACT TCC CAG 1152 Phe Pro Lys Ser Ser Ser Ala Asp Phe Gly Thr Phe Asn Thr Ser Gin 290 295 300

AGT CAT CAA ACA GCA TCA GCT GTT AGT AAA GTT TCA ACG AAC AAA GCT 1200 Ser His Gin Thr Ala Ser Ala Val Ser Lys Val Ser Thr Asn Lys Ala 305 310 315

GGT TTA CAG ACT GCA GAC AAA TAT GCA GCA CTT GCT AAT TTA GAC AAT 1248 Gly Leu Gin Thr Ala Asp Lys Tyr Ala Ala Leu Ala Asn Leu Asp Asn 320 325 330 335

ATC TTC AGT GCC GGG CAA GGT GGT GAT CAG GGA AGT GGC TTT GGG ACC 1296 Ile Phe Ser Ala Gly Gin Gly Gly Asp Gin Gly Ser Gly Phe Gly Thr 340 345 350

ACA GGT AAA GCT CCT GTT GGT TCT GTG GTT TCA GTT CCC AGT CAG TCA 1344 Thr Gly Lys Ala Pro Val Gly Ser Val Val Ser Val Pro Ser Gin Ser 355 360 365

AGT GCA TCT TCA GAC AAG TAT GCA GCT CTG GCA GAA CTA GAC AGC GTT 1392 Ser Ala Ser Ser Asp Lys Tyr Ala Ala Leu Ala Glu Leu Asp Ser Val 370 375 380

TTC AGT TCT GCA GCC ACC TCC AGT AAT GCG TAT ACT TCC ACA AGT AAT 1440 Phe Ser Ser Ala Ala Thr Ser Ser Asn Ala Tyr Thr Ser Thr Ser Asn 385 390 395

GCT AGC AGC AAT GTT TTT GGA ACA GTG CCA GTG GTT GCT TCT GCA CAG 1488 Ala Ser Ser Asn Val Phe Gly Thr Val Pro Val Val Ala Ser Ala Gin 400 405 410 415

ACA CAG CCT GCT TCA TCA AGT GTG CCT GCT CCA TTT GGA GCT ACG CCT 1536 Thr Gin Pro Ala Ser Ser Ser Val Pro Ala Pro Phe Gly Ala Thr Pro 420 425 430

TCC ACA AAT CCA TTT GTT GCT GCT GCT GGT CCT TCT GTG GCA TCT TCT 1584 Ser Thr Asn Pro Phe Val Ala Ala Ala Gly Pro Ser Val Ala Ser Ser 435 440 445 ACA AAC CCA TTT CAG ACC AAT GCC AGA GGA GCA ACA GCG GCA ACC TTT 1632 Thr Asn Pro Phe Gin Thr Asn Ala Arg Gly Ala Thr Ala Ala Thr Phe 450 455 460

GGC ACT GCA TCC ATG AGC ATG CCC ACG GGA TTC GGC ACT CCT GCT CCC 1680 Gly Thr Ala Ser Met Ser Met Pro Thr Gly Phe Gly Thr Pro Ala Pro 465 470 475

TAC AGT CTT CCC ACC AGC TTT AGT GGC AGC TTT CAG CAG CCT GCC TTT 1728 Tyr Ser Leu Pro Thr Ser Phe Ser Gly Ser Phe Gin Gin Pro Ala Phe 480 485 490 495

CCA GCC CAA GCA GCT TTC CCT CAA CAG ACA GCT TTT TCT CAA CAG CCC 1776 Pro Ala Gin Ala Ala Phe Pro Gin Gin Thr Ala Phe Ser Gin Gin Pro 500 505 510

AAT GGT GCA GGT TTT GCA GCA TTT GGA CAA ACA AAG CCA GTA GTA ACC 1824 Asn Gly Ala Gly Phe Ala Ala Phe Gly Gin Thr Lys Pro Val Val Thr 515 520 525

CCT TTT GGT CAA GTT GCA GCT GCT iGGA GTA TCT AGT AAT CCT TTT ATG 1872 Pro Phe Gly Gin Val Ala Ala Ala Gly Val Ser Ser Asn Pro Phe Met 530 535 540

ACT GGT GCA CCA ACA GGA CAA TTT CCA ACA GGA AGC TCA TCA ACC AAT 1920 Thr Gly Ala Pro Thr Gly Gin Phe Pro Thr Gly Ser Ser Ser Thr Asn 545 550 555

CCT TTC TTA TAGCCTTATA TAGACAATTT ACTGGAACGA ACTTTTATGT 1969

Pro Phe Leu

560

GGTCACATTA CATCTCTCCA CCTCTTGCAC TGTTGTCTTG TTTCACTGAT CTTAGCTTTA 2029

AACACAAGAG AAGTCTTTAA AAAGCCTGCA TTGTGTATTA AACACCAGGT AATATGTGCA 2089

AAACAGAGGG CTCCAGTAAC ACCTTCTAAC CTGTGAATTG GCAGAAAAGG GTAGCGGTAT 2149

CATGTATATT AAAATTGGCT AATATTAAGT TATTGCAGAT ACCACATTCA TTATGCTGCA 2209

GTACTGTACA TATTTTTCTT AGAAATTAGC TATTTGTGCA TATCAGTATT TGTAACTTTA 2269

ACACATTGTT ATGTGAGAAA TGTTACTGGG GAAATAGATC AGCCACTTTT AAGGTGCTGT 2329

CATATATCTT TGGAATGAAT GACCTAAAAT CATTTTAACC ATGCTACTGG AAAGTAACAG 2389

AGTCAAAATT GGAAGGTTTT ATTCATTCTT GAATTTTTCC TTTCTAAAGA GCTCTTCTAT 2449

TTATACATGC CTAAATTCTT TTAAAATGTA GAGGGATACC TGTCTGCATA ATAAAGCTGA 2509

TCATGTTTTG CTACAGTTTG CAGGTGAAAA AAAATAAATA TTATAAAATA AAAAAAAAAA 2569

AAAAAAAAAA AAAA 2583

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 562 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Ala Ser Ala Lys Arg Lys Gin Glu Glu Lys His Leu Lys Met 1 5 10 15

Leu Arg Asp Met Thr Gly Leu Pro His Asn Arg Lys Cys Phe Asp Cys 20 25 30

Asp Gin Arg Gly Pro Thr Tyr Val Asn Met Thr Val Gly Ser Phe Val 35 40 45

Cys Thr Ser Cys Ser Gly Ser Leu Arg Gly Leu Asn Pro Pro His Arg 50 55 60

Val Lys Ser Ile Ser Met Thr Thr Phe Thr Gin Gin Glu Ile Glu Phe 65 70 75 80

Leu Gin Lys His Gly Asn Glu Val Cys Lys Gin Ile Trp Leu Gly Leu 85 90 95

Phe Asp Asp Arg Ser Ser Ala Ile Pro Asp Phe Arg Asp Pro Gin Lys 100 105 110

Val Lys Glu Phe Leu Gin Glu Lys JTyr Glu Lys Lys Arg Trp Tyr Val 115 120 125

Pro Pro Glu Gin Ala Lys Val Val Ala Ser Val His Ala Ser Ile Ser 130 135 140

Gly Ser Ser Ala Ser Ser Thr Ser Ser Thr Pro Glu Val Lys Pro Leu 145 150 155 160

Lys Ser Leu Leu Gly Asp Ser Ala Pro Thr Leu His Leu Asn Lys Gly 165 170 175

Thr Pro Ser Gin Ser Pro Val Val Gly Arg Ser Gin Gly Gin Gin Gin 180 185 190

Glu Lys Lys Gin Phe Asp Leu Leu Ser Asp Leu Gly Ser Asp Ile Phe 195 200 205

Ala Ala Pro Ala Pro Gin Ser Thr Ala Thr Ala Aβn Phe Ala Asn Phe 210 215 220

Ala His Phe Asn Ser His Ala Ala Gin Asn Ser Ala Asn Ala Asp Phe 225 230 235 240

Ala Asn Phe Asp Ala Phe Gly Gin Ser Ser Gly Ser Ser Asn Phe Gly 245 250 255

Gly Phe Pro Thr Ala Ser His Ser Pro Phe Gin Pro Gin Thr Thr Gly 260 265 270

Gly Ser Ala Ala Ser Val Asn Ala Asn Phe Ala His Phe Asp Asn Phe 275 280 285

Pro Lys Ser Ser Ser Ala Asp Phe Gly Thr Phe Asn Thr Ser Gin Ser 290 295 300

His Gin Thr Ala Ser Ala Val Ser Lys Val Ser Thr Asn Lys Ala Gly 305 310 315 320

Leu Gin Thr Ala Asp Lys Tyr Ala Ala Leu Ala Asn Leu Asp Aβn Ile 325 330 335

Phe Ser Ala Gly Gin Gly Gly Asp Gin Gly Ser Gly Phe Gly Thr Thr 340 345 350

Gly Lys Ala Pro Val Gly Ser Val Val Ser Val Pro Ser Gin Ser Ser 355 360 365

Ala Ser Ser Asp Lys Tyr Ala Ala Leu Ala Glu Leu Asp Ser Val Phe 370 375 380

Ser Ser Ala Ala Thr Ser Ser Asn Ala Tyr Thr Ser Thr Ser Asn Ala 385 390 395 400

Ser Ser Asn Val Phe Gly Thr Val Pro Val Val Ala Ser Ala Gin Thr 405 410 415

Gin Pro Ala Ser Ser Ser Val Pro Ala Pro Phe Gly Ala Thr Pro Ser 420 425 430

Thr Asn Pro Phe Val Ala Ala Ala Gly Pro Ser Val Ala Ser Ser Thr 435 440 445

Asn Pro Phe Gin Thr Asn Ala Arg Gly Ala Thr Ala Ala Thr Phe Gly 450 455 460

Thr Ala Ser Met Ser Met Pro Thr Gly Phe Gly Thr Pro Ala Pro Tyr 465 470 475 480

Ser Leu Pro Thr Ser Phe Ser Gly Ser Phe Gin Gin Pro Ala Phe Pro 485 490 495

Ala Gin Ala Ala Phe Pro Gin Gin Thr Ala Phe Ser Gin Gin Pro Asn 500 505 510

Gly Ala Gly Phe Ala Ala Phe Gly Gin Thr Lys Pro Val Val Thr Pro 515 520 525

Phe Gly Gin Val Ala Ala Ala Gly Val Ser Ser Asn Pro Phe Met Thr 530 535 540

Gly Ala Pro Thr Gly Gin Phe Pro Thr Gly Ser Ser Ser Thr Asn Pro 545 550 555 560

Phe Leu

Claims

THAT WHICH IS CLAIMED IS:

1. Isolated DNA encoding a cellular co-factor for HIV Rev selected from the group consisting of:

(a) DNA which encodes a protein of SEQ ID NO:2;

(b) DNA which hybridizes to DNA of (a) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev; and

(c) DNA differing from the DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a cellular co-factor for HIV Rev.

2. Isolated DNA according to claim 1 which encodes a protein of SEQ ID NO:2.

3. Isolated DNA according to claim 1 having the nucleotide sequence of SEQ ID NO:l.

4. A recombinant DNA comprising vector DNA and a DNA according to claim 1.

5. A recombinant DNA according to claim 4, wherein said vector DNA comprises a plasmid.

6. A recombinant DNA according to claim 4, wherein said vector DNA comprises a virus.

7. A recombinant DNA according to claim 4, wherein said vector DNA comprises a baculovirus.

8. A host cell containing a recombinant DNA of claim 4.

9. A host cell containing a recombinant DNA of claim 4 and capable of expressing the encoded cellular co¬ factor for HIV Rev.

10. A host cell according to claim 9, wherein said host cell is a mammalian cell.

11. A host cell according to claim 9, wherein said host cell is an insect cell.

12. An oligonucleotide probe capable of selectively binding to a DNA of claim 1.

13. An oligonucleotide probe according to claim 12, which probe is capable of serving as a PCR extension primer.

14. An oligonucleotide probe according to claim 12, which probe is labelled with a detectable group.

15. An oligonucleotide probe according to claim 14, which detectable group is a radioactive atom.

16. An isolated protein comprising a cellular co-factor for HIV Rev, which protein is coded for by a DNA according to claim 1.

17. An antibody which specifically binds to a protein of claim 16.

18. An antibody according to claim 17 which is labelled with a detectable group.

19. An antibody according to claim 18, which antibody comprises a monoclonal antibody.

20. A method of screening for compounds that affect the binding of a Lentivirus Rev protein or an HTLV family Rev protein with the cellular co-factor thereof, comprising: providing together a first protein and a second protein so that said first and second protein form a complex thereof, wherein said first protein is selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins, and wherein said second protein is encoded by a DNA selected from the group consisting of (a) DNA which encodes a protein of SEQ ID NO:2; and (b) DNA which hybridizes to DNA of (a) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev; then combining a test compound with said first and second protein; and then detecting the influence of said test compound on the formation of said complex.

21. A method according to claim 20, wherein said method is carried out in vitro .

22. A method according to claim 20, wherein said method is carried out in vivo .

23. A method according to claim 20, wherein said method is carried out in vivo in a yeast cell.

24. A method according to claim 20, wherein said method is carried out in vivo in an insect cell.

25. A recombinant cell useful for screening for compounds that affect the binding of a Lentivirus Rev protein or an HTLV family Rev protein to the cellular co¬ factor thereof, comprising: a first DNA that expresses a first fusion protein comprising a DNA binding domain and a first binding partner; a second DNA that expresses a second fusion protein comprising a second binding partner and a transcription activation domain; and a third DNA comprising a binding site operatively associated with a reporter gene, wherein said DNA binding domain specifically binds to said binding site, so that said reporter gene is expressed when said first and second fusion proteins bind to one another; wherein said first binding partner is either (a) selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins, or (b) encoded by a DNA selected from the group consisting of (i) DNA which encodes a protein of SEQ ID NO:2 and (ii) DNA which hybridizes to DNA of (i) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev; and said second binding partner is the other of (a) or (b) above.

26. A recombinant cell according to claim 25, wherein said cell is a yeast cell.

27. A recombinant cell according to claim 25, wherein said binding site is a Gal4 binding site and said binding domain is a Gal4 binding domain.

28. A recombinant cell according to claim 25, wherein said transcription activation domain is selected from the group consisting of the Gal4 transcription activation domain, the VP16 transcription activation domain, and the TAT transcription activation domain.

29. A recombinant cell according to claim 25, wherein said first binding partner is selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins, and said second binding partner is encoded by a DNA selected from the group consisting of (i) DNA which encodes a protein of SEQ ID NO:2 and (ii) DNA which hybridizes to DNA of (i) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev.

30. A recombinant cell according to claim 25, wherein said first binding partner is encoded by a DNA selected from the group consisting of (i) DNA which encodes a protein of SEQ ID NO:2 and (ii) DNA which hybridizes to DNA of (i) above under stringent conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03 M sodium citrate, 0.1% SDS at 60°C, and which encodes a cellular co-factor for HIV Rev; and wherein said second binding partner is selected from the group consisting of Lentivirus Rev proteins and HTLV family proteins.

31. A recombinant cell according to claim 25, wherein said binding partner is selected from the group consisting of Lentivirus Rev proteins and HTLV family Rev proteins is HIV-l Rev or HTLV-I Rex.

32. A method of treating a lentivirus or HTLV family viruses, comprising introducing into a cell infected with the virus a compound that inhibits the formation of a complex between a lentivirus Rev protein or an HTLV family Rev protein with the cellular co-factor thereof.