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WO1997006257A9 - Cofacteur cellulaire pour vih rev et htlv rex - Google Patents

Cofacteur cellulaire pour vih rev et htlv rex

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WO1997006257A9
WO1997006257A9 PCT/US1996/012986 US9612986W WO9706257A9 WO 1997006257 A9 WO1997006257 A9 WO 1997006257A9 US 9612986 W US9612986 W US 9612986W WO 9706257 A9 WO9706257 A9 WO 9706257A9
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rev
dna
protein
proteins
hiv
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  • 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 .
  • 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)) .
  • 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 .
  • 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 R ⁇ A export requires the direct interaction of Rev with a cis-acting, -234 nucleotide (nt) R ⁇ A stem-loop structure termed the Rev Response Element or RRE (T. Daly et al . , Nature 342, 816-819 (1989) ; Malim et al . , supra ; M. Malim et al .
  • 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 . Na tl . 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-R ⁇ A interactions. This multimerization event is critical for Rev function via the RRE (M. Malim and B. Cullen, Cell 65, 241-248 (1991) ; S.
  • 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. Kjems 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) .
  • Rev activation domain a short, -10 aa leucine-rich sequence, termed the Rev activation domain, that is dispensable for RRE binding but nevertheless essential for Rev function
  • the activation domain represents a binding site for an essential cellular Rev co-factor.
  • 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) .
  • 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.
  • 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)) .
  • HIV-l Rev is the most intensely studied retroviral RNA transport factor
  • other members of the lentivirinae also encode Rev proteins
  • the effectively unrelated T-cell leukemia viruses including Human T-cell leukemia virus type I (HTLV-I)
  • Rex an equivalent regulatory protein termed Rex (Cullen, supra)
  • Rex an equivalent regulatory protein termed Rex (Cullen, supra)
  • Rex an equivalent regulatory protein termed Rex
  • 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) .
  • 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 .
  • DNA which encodes a protein of SEQ ID NO:2 (e . g. , DNA of SEQ ID NO:l) ;
  • 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.
  • 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
  • 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 M10, 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 (M10, 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 -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 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
  • 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 3-gal expression plasmid.
  • FIG. 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 HIV ⁇ REV, 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 jS-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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Proteins of the invention are, in general, cellular co-factors for HIV-l Rev and/or HTLV-1 Rex post-transcriptional regulator proteins, and specifically bind to HIV-l Rev and/or HTLV-1 Rex at the same binding site bound by the protein of SEQ ID NO:2, and may have the same biological activity thereof.
  • 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.
  • suitable control sequences capable of effecting the expression of the receptor in a suitable host.
  • 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.
  • Amplification 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)) .
  • 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 .
  • 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
  • a vector e.g. Polyoma, Adenovirus, VSV, or BPV
  • the host cell chromosomal replication mechanism 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.
  • suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Pat. No. 4,399,216.
  • baculovirus expression vector e.g., vectors derived from Autograp ⁇ a californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
  • baculovirus expression vector e.g., vectors derived from Autograp ⁇ a californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
  • 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. coli 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 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 .
  • 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.
  • 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.
  • 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, ⁇ - glucuronidase, alkaline phosphatase, and ⁇ -O- galactosidase) , fluorescent labels (e.g., fluorescein, luciferase) , and radiolabels (e.g., 14 C, 131 I, 3 H, 32 P, and 35 S) to the compound being labelled.
  • enzymes e.g., horseradish peroxidase, ⁇ - glucuronidase, alkaline phosphatase, and ⁇ -O- galactosidase
  • fluorescent labels e.g., fluorescein, luciferase
  • radiolabels e.g., 14 C, 131 I, 3 H, 32 P, and 35
  • Oligonucleotide probes of the instant invention may be of any suitable length, depending on the specific application thereof.
  • 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 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.
  • antibody 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) .
  • D ⁇ As 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 imrnunoassay systems.
  • 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") .
  • viruses e.g., HIV-l, HIV-2, HTLV-I, HTLV-II, bovine leukemia virus (or "BLV"
  • BLV bovine leukemia virus
  • methods comprise providing together (e . g.
  • 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)
  • the second protein is encoded by a DNA encoding a co-factor of the invention as given above.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • parenteral administration e.g., intraveneous , intraperitoneal, intramuscular, and subcutaneous injection
  • topical administration e.g., topical administration
  • oral administration e.g., 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.
  • 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) ) .
  • 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.
  • RNA and protein expression analysis were performed 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.
  • 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) .
  • MBP protein fusion and purification system New England Biolabs
  • 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.
  • PBS phosphate buffered saline
  • 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) .
  • 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, M10 mutant form of the activation domain (M. Malim et al. , Cell 58, 205-214 (1989A) ) .
  • 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) .
  • 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 .
  • CB chromatography buffer
  • the introduced mutations are: HIV Rev M10, LG (78,79) to DL; HIV Rev M32, 0 L 78, 81 and 83 all to A; RexDAD, LSLD (90-93) to GGGG; VMV
  • 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
  • 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//?gal expression plasmids have been described (L. Rimsky et al . , Nature 335, 738-740
  • the Rab expression plasmid PBC12/CMV/Rab contains the 562 amino acid Rab ORF indicated in Figure 3 ( ⁇ col 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
  • yeast two-hybrid protein interaction trap S. Fields and O-K Song, Na ture 340, 245-246 (1989) ; R. Fridell et al . , Virology 209, 347-357 (1995)
  • the screened library consisted of the VP16 transcription activation domain fused to CD ⁇ A sequences derived from the human CEM T-cell line.
  • 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) .
  • 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.
  • 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 -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 .
  • 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.
  • Rab protein sequence 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.
  • Rev and Rex 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.
  • 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) ) .
  • the assay delineated in Fig. 6A takes this approach one step further.
  • 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) .
  • 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) .
  • Rab is the authentic Rev co-factor
  • 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.
  • the HTLV-I Rex protein which can also act via the HIV-l RRE element (L. 5 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.

Abstract

La présente invention décrit des cofacteurs cellulaires qui encodent les ADN pour les protéines régulatrices post-transcriptionnelles VIH-1 Rev et HTLV-I Rex. Des protéines produites à partir des ADN ainsi que les procédés pour utiliser celles-ci sont également décrits.
PCT/US1996/012986 1995-08-10 1996-08-09 Cofacteur cellulaire pour vih rev et htlv rex Ceased WO1997006257A1 (fr)

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