A NOVEL INTEGRIN SPECIFICITY FOR THE HIV Tat PROTEIN

Abstract: The present invention relates to HIV Tat proteins and integrin cell surface receptor that specifically recognizes the HIV Tat protein. The integrin is a heterodimer containing an alpha v subunit and one or more beta subunits, such as alpha v beta 5 derived from human cells and alpha v beta 8 derived from rat cells. The present invention further relates to reactive fragments having sequences corresponding to the binding regions of the HIV Tat protein and the Tat binding integrins. Methods of controlling the binding of the HIV Tat protein to cells expressing such integrins are also provided.

Description: A NOVEL INTEGRIN SPECIFICITY FOR THE HIV Tat PROTEIN This invention was made in part with Government support under Grant Nos. CA 42507, CA 28896, CA 08686, CA 52412 and CA 30199 from the National Cancer Institute. The United States Government may have certain rights in this invention. BACKGROUND OF THE INVENTION The present invention generally relates to the HIV Tat protein and more particularly to the integrin cell surface receptor capable of binding to the HIV Tat protein. Human immunodeficiency virus (HIV-1) encodes a regulatory protein, termed "Tat", that transactivates genes expressed from the long terminal repeat of the virus. Tat exists as either a 72 or 86 amino acid protein, depending on whether it is expressed from one or both coding exons through differential splicing. Both forms are functional transactivators and contain an acidic domain at the amino terminus, a basic region (six arginines and two lysines out of nine contiguous residues) and a cysteine-rich region (seven out of sixteen residues) . The basic domain has been shown to be responsible for nuclear/nucleolar targeting of the Tat protein as well as its capacity to bind to its cognate RNA sequence, TAR. Although the cysteine-rich domain is also critical for transactivation, its function is less defined but has been proposed to mediate metal-linked dimerization of the protein. The 14-amino acid sequence encoded by the second exon has the tripeptide arginine-glycine-aspartic acid (RGD) as its most notable feature. An RGD sequence is required for integrin mediated cell adhesion to extracellular matrix proteins such as fibronectin, vitronectin, and fibrinogen as reported in Pierschbacher and Ruoslahti, Nature 309:30-33, (1984) and Ruoslahti and Pierschbacher, Science 238:491-497, (1987). It has recently been demonstrated that Tat may function as an exogenous factor. Extracellular Tat is internalized by cells and transported to the nucleus, where it retains the ability to transactivate the HIV promoter as discussed in Frankel and Pabo, Cell 55:1189- 1193 (1988) . In addition, Tat can be released from HIV-1 acutely infected cells or Tat-transfected cells. Similar processes of release and uptake have been observed with another retroviral transactivator protein, namely, Tax of HTLV-l. Furthermore, Tat has been shown to modulate cell proliferation, both in the suppression of proliferation of antigen-activated T-cells and in the specific stimulation of proliferation of Kaposi's sarcoma (KS) derived cells as reported in Ensoli et al., Nature 345:84-86 (1990). Thus, Tat may contribute to the pathogenesis of HIV via mechanisms that go beyond the activation of virus replication. However, the details of these mechanisms are not well understood. For example, it is not known how extracellular Tat stimulates KS cell growth or how it is internalized. One mechanism underlying the interactions of cells with one another, with extracellular matrices and with soluble proteins, such as the Tat protein, is binding of cell surface receptors to a cognate ligand on the surface of cells or in the extracellular matrix. In many cases, such a receptor belongs to a class of proteins known as integrins. The integrins are a large family of cell surface glycoproteins that mediate cell-to-cell and cell- to-matrix adhesion as described, for example, in Ruoslahti and Pierschbacher, supra (1987) . All known members of this family of adhesion receptors are heterodimers consisting of an α and a β subunit noncovalently bound to each other. Over the past few years, the primary structures of six integrin β subunits from mammalian cells and one from Drosophila have been deduced from cDNA. Eleven distinct α subunits have thus far been described. The adhesion of cells to extracellular matrices is mediated in many cases by the binding of a cell surface receptor to an RGD containing sequence in the matrix protein, as reviewed in Ruoslahti and Pierschbacher, supra (1987) . The RGD sequence is a cell attachment site at least in fibronectin, vitronectin, fibrinogen, von Willebrand factor, thrombospondin, osteopontin, and possibly various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by their interactions with specific receptors. Because of the importance of the Tat protein in the maintenance and propagation of HIV infection, a need exists to control the effects of this protein on normal and HIV infected cells. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION The present invention relates to methods of inhibiting HIV Tat protein binding to a cell expressing an HIV Tat binding integrin cell surface receptor ("integrin") . The methods are accomplished by blocking the binding of the HIV Tat protein to the integrin by binding the HIV Tat protein with a reagent having reactivity with the integrin binding site of the HIV Tat protein or by binding the integrin with a reagent having reactivity with the HIV Tat binding site of the integrin. The integrin can be, for example, human ^ 5 or rat cXγB> 8 . The The reagent can be an antibody or active fragments of either the integrin or the HIV Tat protein. The present invention further provides fragments of the HIV Tat protein that can be used in the claimed methods. Such fragments containing an integrin binding site include, for example, the basic domain of the HIV Tat protein. A secondary binding site for cell adhesion may be an RGD-containing region of the Tat protein. Thus, the fragments of the present invention can also include an RGD-containing domain of the HIV Tat protein. Isolated nucleic acids encoding the HIV Tat protein, a Tat binding integrin or reactive fragments of either are also provided. The invention further relates to vectors having a nucleic acid encoding such polypeptides or fragments and to host cells containing these vectors. Methods of detecting ligands of the Tat binding integrins in a sample are also provided. Such methods include contacting a Tat binding integrin with the sample and determining the binding of the integrin to the sample. Binding of the integrin to the sample indicates the presence of an integrin-reactive ligand. The methods can be used to detect or purify HIV Tat proteins, active fragments of such proteins or HIV Tat peptide mimetics. The present invention additionally relates to methods of increasing the binding of HIV Tat proteins to cells expressing the integrins of the present invention by overexpressing the integrins. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the structure of the Tat protein and Tat-derived peptides. Shown are the cysteine rich domain and the basic domain in exon 1 and the RGD sequence in exon 2. Figure 2 shows the results of cell adhesion to Tat peptides. Figure 2A shows the results of rat L8 cells; Figure 2B shows the results of human SK-LMS cells. Wells of microtiter dishes were coated with various concentrations of Tat 1-86 (□) , Tat 45-86 (f) , or Tat 57- 86 (A) . L8 cells (~10 cells per well) were added to each well and incubated at 37"C for one hour. The attached cells were fixed and stained with crystal violet. The dye was eluted and the absorbance at 600 nm measured in an ELISA reader. SK-LMS cells were tested for attachment to the same peptides, including a 12 amino acid peptide consisting of the basic domain, Tat 45-57 (Δ) . Figure 3 shows the results of studies relating to the inhibition of cell attachment to Tat with anti-α v β 3 VNR antibodies. Wells of microtiter dishes were coated with either fibronectin, vitronectin or Tat peptide 45-86 at 5 μg/ml. SK-LMS or L8 cells (~10 cells per well) were added to each well in the presence of anti-α v β 3 (VNR) or anti-α 5 B 1 (FNR) integrin antibodies. The attached cells were fixed and stained with crystal violet. The dye was eluted and the absorbance was measured in an ELISA reader. Figure 4 shows the affinity chromatography on Tat peptide. An extract of surface iodinated L8 cells was fractionated on Tat peptide 45-86 coupled to Sepharose. After washing, the column was eluted with either 1 mg/ l of the Tat peptide or 200 μg/ml of the full-length Tat protein. The fractions were analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non- reducing conditions. Figure 5 shows the results of immunoprecipitation of material affinity-purified on Tat peptide. An extract of surface-iodinated SK-LMS cells was fractionated on Tat 45-88 coupled to Sepharose as in Figure 4. After sequential elution with GRGDSP, Tat 57- 86 and Tat 45-57 (basic 12-mer) , peak fractions of each eluate were immunoprecipitated with antibodies to the α v , β u β j or β 5 subunit cytoplasmic domain, and analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non- reducing conditions. Shown are the immunoprecipitations of the flow-through (unbound) , GRGDSP eluate and Tat 45- 57 eluate (basic 12-mer) . Figure 6 shows the results of immunoprecipitation of material affinity-purified on GRGDSPK-Sepharose. An extract of surface-iodinated SK- LMS cells was fractionated on GRGDSPK-Sepharose. After elution with GRGDSP (1 mg/ml) , the flow-through (unbound) and eluate (bound) was immunoprecipitated with antibodies to the α v , B 1# B 3 or β 5 subunit cytoplasmic domains, and analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non-reducing conditions. Figure 7 shows the elution of integrin from Tat column with EDTA or NaCl. Extracts of surface-iodinated L8 or SK-LMS cells were fractionated on Tat 45-86 coupled to Sepharose. Sequential elution of the columns with 10 mM EDTA (in 100 mM NaCl; lanes 2,6), 250 mM NaCl (lanes 3,7) and Tat 45-86 (1 mg/ml; lanes 4,8), followed by immunoprecipiTation with a polyclonal anti-vitronectin receptor antibody and analysis by SDS-polyacrylamide gel (7.5%) electrophoresis under, non-reducing conditions are shown. Lanes 1 and 5 are immunoprecipitates of the flow- through material. Figure 8 shows the results of inhibition of SK- LMS cell binding to vitronectin and Tat peptide. SK-LMS cells were added to microtiter wells coated with Tat 45- 86 or vitronectin (VN) in the presence or absence of inhibitory antibodies. P3G2 is a monoclonal antibody shown previously to inhibit the interaction of a v B 5 with vitronectin. LM 609 inhibits the interaction of α y β 3 with vitronectin. Anti-VNR is a polyclonal antibody raised against the α^ integrin purified from a placental extract on a GRGDSPK-Sepharose column. Figure 9 shows the effect of antibodies and peptide on Tat transactivation. LTR-CAT was transfected into L8 cells and transactivated by exogenous GST-Tat fusion protein in the presence of anti-α v B 3 polyclonal antibody (anti-VNR) , rabbit anti-α 5 β 1 (anti-FNR) , normal rabbit serum, or Tat basic peptide. Lane 1, LTR-CAT alone; lane 2, LTR-CAT plus GST-Tat fusion protein; lane 3, LTR-CAT plus GST protein; lanes 4-8, LTR-CAT plus GST- Tat fusion protein; and lane 4, 300 μg basic peptide; lane 5, 80 μl anti-α v B 3 ; lane 6, 8 μl anti-α v B 3 ; lane 7, 80 μl anti-α g β, serum; lane 8, normal rabbit serum. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to integrin cell surface receptors, referred to herein as "integrins," that bind to the HIV Tat protein. In human cells, such integrins are the known α y β 5 , while rat cells may contain a previously unidentified β subunit, designated herein as β g , and the known subunit α v . The α v subunit is the most versatile of the integrin a subunits. It has been known to combine with three, possibly four, different β subunits. Thus, the rat β 8 subunit of the present invention may add at least one additional β subunit to this group. The human β 5 subunit was previously known to combine with α v , although the Tat binding activity of human ^B 5 was not previously known. The present invention particularly relates to a novel interaction between the HIV Tat protein and the integrins to which the Tat protein bind. Although previous reports suggest an RGD-dependent mechanism in the binding of Tat to rat L8 cells, as described in Brake et al., J. Cell Biol. 111:1275-1281 (1990), it has now been discovered from cell adhesion and affinity chromatography data that the basic region of the Tat protein mediates this interaction. Immunoprecipitation of the protein purified on the Tat columns identified the Tat binding proteins as components of the integrin 0:^ 5 . This result was consistent with data showing that cell adhesion to Tat could be blocked by polyclonal antibodies to the ct y β j integrin. Antibodies to this integrin would be expected to bind to the α v subunit shared by α y β 5 and α^ and inhibit the function of both heterodimers. The C- y β g integrin has been shown to bind to vitronectin through the RGD sequence. Although the Tat protein contains an RGD sequence, it has been shown in the studies relating to the present invention that the integrin recognition sequence is the basic domain of Tat. This conclusion is based on the complete correlation found between the ability of Tat-derived peptides to support cell attachment and bind the ^ integrin in affinity chromatography with the presence of the basic sequence in the peptide. In contrast, the presence of absence of the RGD sequence had no influence in either type of assay. Apparently the RGD sequence is present in a context not suitable for integrin binding because even the α v B 3 integrin, which has the greatest binding activity with short RGD-containing peptides as shown in Figure 6 failed to bind appreciably to the RGD-containing Tat peptides. Most integrins require divalent cations for their activity and are dissociated from their ligands in the presence of EDTA. In addition to utilizing the basic domain, the interaction between α y β 5 and Tat has the unusual feature that it is stable in the presence of 10 mM EDTA and is therefore not divalent cation-dependent. The interaction of the o y β 5 integrin with Tat was, however, inhibited by high NaCl concentrations. Although this salt sensitivity is unusual among integrins, it is not unprecedented. For example, the α 3 β, integrin also demonstrates a salt-labile, RGD-independent binding to collagen and laminin as discussed in Elices et al., J. Cell Biol. 112:169-181 (1991). It could be that α 3 β. also binds to a basic region within these proteins. Interestingly, Q-JB., also has been found to bind to fibronectin in an RGD-dependent manner, suggesting that CT j β, integrin may have two functionally distinct ligand binding sites. The α 4 β, integrin has two distinct ligand binding sites, one for the endothelial cell ligand, V-CAM, and the other for an alternatively spliced segment of fibronectin as reported in Elices et al., Cell 60:577-584 (1990). The inability to inhibit the binding of L8 and SK-LMS cells to Tat with a monoclonal antibody that inhibits their interaction with vitronectin suggests that c- y β g may have a second ligand binding site for a basic extracellular protein yet to be identified. The integrin Ilb/IIIa may also share some of the basic peptide binding properties of α γ β 5 . Peptides containing both an RGD and a basic segment bind more avidly to Ilb/IIIa than peptides containing RGD alone as reported in Savage et al., J. Biol. Chem. 265:11766-11772 (1990). Both rat L8 and human SK-LMS cells remained round when plated on a surface coated with Tat and Tat- derived peptides containing the basic domain. This behavior is consistent with the finding that cells expressing a^E> 5 attach to vitronectin, but do not spread. According to reports in Wayner et al. , J. Cell Biol. 113:919-929 (1991), spreading on vitronectin is dependent on the presence of α^. Based on early studies with rat L8 cells, the Tat was found to bind an integrin containing a β subunit designated herein as B 8 and the known α v . In further studies with human cells, the Tat protein was found to bind the known human integrin, a v B 5 . However, the rat integrin may also be G- y β g because it behaved identically to the human a^ integrin in the affinity chromatography experiments and migrated similarly in SDS-PAGE. The antibodies prepared against the cytoplasmic peptide of the human β 5 subunit were weakly reactive against a number of rat cell lines. Poor reactivity of the antibodies with the rat B 5 subunit may therefore explain the lack of immunoprecipitation of the L8 integrin observed. It is also possible that the β subunit of the Tat binding integrin from the L8 cells may be an alternatively spliced β 5 variant or a completely different β subunit altogether. The rat B 8 subunit is distinguishable from known β subunits. First, direct comparisons show that the apparent molecular weight of the β 8 subunit is lower than many of the previously characterized β subunit. In addition, although the α v subunit has been shown to associate with the B 1# β 3 , or β 5 subunits and may also combine with B 6 , antibodies to the cytoplasmic tails of each of these known subunits failed to immunoprecipitate the Tat binding receptor. The possibility that the β subunit differs from a previously characterized β subunit by alternative splicing or proteolytic processing cannot be excluded. However, it is clear that the L8 cells have a β j subunit that is functional, but does not bind to Tat. The α v subunit is the most versatile of the integrin subunits in that it is known to combine with three, possibly four, different β subunits. It now appears that at least one additional β subunit can be added to this group. The rat α^ integrin is likewise distinguishable from the known integrins in its binding specificity. For example, the α 4 β, integrin binds to sequences that do not contain the RGD sequences, yet the Tat protein does not contain any sequences significantly homologous to the α 4 B 1 target sequence. An important characteristic of the β subunit is that in combination with the α v subunit it forms the integrins of the present invention that binds to the HIV Tat protein. The specificity of the integrins of the present invention is such that it can bind the intact Tat protein or a truncated form lacking the RGD-containing region and, therefore, can be used to control the activities of the Tat protein. The β component of the Tat binding integrins gives a double band in a non-reduced SDS-gel electrophoresis in the molecular weight range of about 75-95 kD, representing two forms of the same β subunit or two different subunits. The present invention is contemplated to include both. The full length Tat protein contai s the following amino acid sequence: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRR AHQNSQTHQASLSKQPTSQSRGDPTGPKE. A basic domain of the Tat protein has been found to be the dominant binding site for the integrin of the present invention. This basic region contains nine amino acid residues of which six are arginine and two are lysine. The amino acid sequence of the basic region is RKKRRQRRR in the HIV strain HXB2. In studies to determine the role of RGD in binding the Tat protein, it was found that a variant peptide in which RGD was replaced with KGE was still capable of binding to the Tat protein with only a small reduction in the binding activity. Whereas Brake et al., supra (1990) found that the cell attachment to the Tat protein was inhibited by short RGD-containing peptides, such peptides did not have a significant effect on the binding of the Tat protein to the integrin in affinity chromatography. This result was supported by later studies in which RGD was replaced by KGE. Thus, it appears that the RGD-containing region is a secondary binding site of the Tat protein. This secondary binding region contains the amino acid sequence: PTSQSRGDPTGPKE in the HIV strain HXB2. Other ligands which bind to the Tat binding integrins can contain both a basic domain and a nearby RGD sequence. While the basic region appears to be the primary binding site of these integrins than the RGD- containing region, at least some of the RGD-directed integrins recognize basic regions in addition to the RGD- containing sequences. This conclusion is supported in part by studies in which peptides that contain both an RGD and a basic segment bound more avidly to the α πb /β 3 integrin than peptides containing RGD alone as described in Savage et al., J. Biol. Chem. 265:11766-11772 (1990). Accordingly, the present invention also provides isolated fragments specific to the Tat binding integrins, particularly to the β subunmits such as, for example, human β 5 and rat β g . Such fragments can be regions obtained from the native Tat protein or synthetic polypeptides having amino acid sequences of the Tat protein regions. These fragments are necessarily of sufficient length to be distinguishable from fragments of other known βs and, therefore, are "specific to" or "unique to" the β subunits of the Tat binding integrins, particularly human B 5 and rat β 8 , for example. Such fragments specific to B 8 can be determined using methods disclosed herein and known in the art. These fragments also retain the Tat binding function and can therefore be used, for example, as inhibitors of the binding of the Tat binding integrins to the Tat protein, or as an indicator to detect the Tat binding integrins of the present invention. One skilled in the art can determine other uses for such fragments. The terms "substantially purified" or "isolated" mean that the material, either polypeptide or nucleic acid, is substantially free of contaminants normally associated with the native or natural environment. A reactive fragment of the HIV Tat protein refers to a polypeptide having substantially the amino acid sequence of a portion of the HIV Tat protein that retains the integrin binding site so as to remain substantially reactive with the Tat binding integrins. Similarly, a reactive fragment of a Tat binding integrin refers to a polypeptide having substantially the amino acid sequence of a portion of the integrin that retains the Tat binding function. Thus, modifications of the amino acid sequence that do not substantially destroy the functions and that retain the essential sequences of rat B 8 or human B 5 are included within the definition of these β subunits. Amino acid sequences, such as those for β 1 , β 2 and B 3 , having less than 50% homology with the sequence of β or β 5 are not substantially the same sequence and, therefore, do not fall within the definition of these β subunits. Given the amino acid sequences set forth herein, additions, deletions or substitutions can be made and tested to determine their effect on the function of the β subunits. In addition, one skilled in the art would recognize that certain amino acids can be modified to alter integrin binding function. The invention also provides reagents having specificity for the integrins of the present invention and particularly the rat B 8 and human β 5 subunits. One skilled in the art could readily make reagents, such as antibodies, that are specifically reactive with the β subunits of the present invention. Such reagents can be used to immunologically distinguish these B subunits from other molecules. Various methods of raising such antibodies are well established and are described, for example, in Antibodies, A Laboratory Manual. E. Harlow and D. Lane, pp. 139-283 (Cold Spring Harbor Laboratory, 1988) , incorporated herein by reference. The invention further provides isolated nucleic acids that encode the Tat protein or reactive fragments thereof having binding sites or regions recognized by the β-containing integrins of the present invention. Similarly, isolated nucleic acids encoding the Tat binding integrins or reactive fragments thereof are also provided. Following standard methods as described, for example, in Maniatis et al., Molecular Cloning, Cold Spring Harbor (1989) , these nucleic acid sequences can be identified, isolated and then cloned into an appropriate expression vector. The vector can then be inserted into a host, which will then be capable of expressing the Tat recombinant proteins, Tat binding integrins or reactive fragments of each. Thus, the invention also relates to vectors containing nucleic acids encoding such sequences and to hosts containing these vectors. The present invention further relates to nucleic acids that can be used as probes for diagnostic purposes. Such nucleic acid probes can hybridize with a nucleic acid having a nucleotide sequence specific to the Tat binding integrins or to the Tat protein but do not hybridize with nucleic acids encoding non-Tat binding integrins or Tat proteins, particularly other cell surface receptors or non-Tat proteins, respectively. Nucleic acids are also provided which specifically hybridize to either the coding or non-coding DNA of the Tat binding integrins or the Tat protein. Such nucleic acids can be identified and prepared by methods known in the art, such as a standard nucleic acid synthesizer. In the methods of inhibiting the binding of Tat proteins to the Tat binding integrins provided by the present invention, the binding of the Tat binding integrins, such as α v B 8 or α y β 5 , to the Tat protein can be blocked by various means. For example, the binding of a β 8 - or β 5 -containing integrin can be blocked by a reagent that binds the β subunit or the β-containing integrin. Examples of such reagents include, for example, peptides and polypeptides containing the basic region of the Tat protein or antibodies specifically reactive with the β subunit or a β-containing integrin. Thus, the binding of the Tat protein to the Tat binding integrins can be blocked by binding the integrins with Tat-derived peptides having an amino acid sequence that recognizes the Tat binding site of the integrins. Alternatively, blocking can be carried out by binding the Tat protein or a reactive fragment thereof with a reagent specific for the Tat protein at a site that inhibits the Tat protein from binding with the integrin, such as an anti-Tat antibody or a reactive fragment of the integrin. The ability to block the binding of the Tat protein by the Tat-binding integrins can be used to control HIV mediated conditions. Thus, the activities of the Tat protein as a growth factor for Kaposi's sarcoma cells and as a transactivator of the HIV promoter when internalized by cells can be inhibited or otherwise controlled. Since the binding of the Tat binding integrins to the Tat protein can mediate cell adhesion, preventing this binding can prevent the adhesion of cells to the endogenous ligand of the integrin. Other activities of the integrin can be similarly inhibited by the use of the Tat mimicking compounds of the present invention. Alternatively, cell adhesion can be promoted by increasing the expression of these integrins by a cell. The activities of the endogenous ligand or ligands can be mimicked with the compounds of this invention. As a method of controlling the binding of Tat proteins to cells expressing the Tat binding integrins, the binding can be enhanced by methods in which the Tat binding integrin is overexpressed in such cells. Methods of enhancing the overexpression of such integrins can be accomplished, for example, by introducing a nucleic acid encoding for the integrin into the genome of a cell by methods known to those skilled in the art. The present invention further provides methods of detecting ligands that bind the Tat binding integrins. The methods include contacting the integrin or binding fragment thereof with a solution containing ligands known to or suspected of binding the Tat binding integrins. Ligands that bind such integrins are then detected. Assays useful to carry out these methods are well known in the art and are described, for example, in Hautanen et al., J. Biol. Chem. 264:1437-1442 (1989) and Smith et al., J. Biol. Chem. 265: 11008-11013 (1990), both of which are incorporated herein by reference. These methods can be used to identify or screen for additional compounds that are bound by the Tat binding site on the integrins of the present invention. Such compounds can be naturally occurring ligands, such as derivatives of the Tat binding site or other compounds capable of being bound by the integrin Tat binding site. Also included are synthetic compounds designed to mimic the desired binding activity of the Tat protein or other Tat binding integrin binding ligands. Such compounds having the desired binding function can be peptides, peptide derivatives or mimetics, or other compounds, so long as they are capable of being bound by the Tat protein's integrin binding site. The following examples are intended to illustrate but not limit the invention. EXAMPLE I Peptide Synthesis The intact Tat protein was synthesized using t- butoxycarbonyl (BOC)-protected amino acids for stepwise synthesis on an Applied Biosystems 431A (Foster City, CA) solid phase automated peptide synthesizer. Amino acids were added as hydroxybenzotriazole (HOBt) esters using n- methylpyrrolidone as the coupling solvent. The synthesis was accomplished starting with 0.5 mmol of Boc-Glu(OBzl)- O-phenylacetamidomethyl resin (0.69 g substituted at 0.72 mmol) with a minimum of two couplings for each amino acid. The average repetitive coupling efficiency was 99.32% as determined by a qualitative ninhydrin assay. The cysteine sulfhydryls were protected with a p- methylbenzyl group to yield a fully reduced form after low/high HF cleavage using suitable scavengers. All other peptides used were synthesized with an Applied Biosystems Model 430A synthesizer using similar chemistry. EXAMPLE II Isolation of Tat-bindinσ Proteins To identify the integrins or other cell surface molecules capable of binding to Tat, affinity chromatography according to Example VII was performed on the 86-amino acid Tat protein and the Tat derived peptides. L8 rat myoblast cells (ATCC # CRL-1769) were chosen for this experiment since they had previously been shown to bind to Tat and vitronectin but not to laminin, collagen, or fibronectin as described in Brake et al., supra. (1990) . In SDS-PAGE under non-reducing conditions, a band of 150 kD and a doublet of 75-95 kD were identified that bound to a full length Tat protein coupled to a Sepharose column and eluted with 1 mg/ml of a Tat peptide (amino acid residues 45-86) containing the basic region and the RGD region but lacking the cysteine rich region of the protein. A second peptide representing the cell adhesion site of fibronectin, GRGDSP, was not effective at eluting the Tat binding proteins. In another study, the peptide covering residues 45-86 coupled to Sepharose bound the same proteins. Identical bands were eluted with the truncated peptide (residues 45-86) or the full length Tat protein, demonstrating that the peptide and protein had similar binding sites. An additional band of approximately 120 kD was eluted from the full length Tat protein column, but not from the shorter Tat peptides. This band has not been further identified, but may represent a subunit of a second binding integrin. To investigate the role of the RGD sequence further, the peptide that contained residues 45-86 was synthesized with the KGE sequence substituted for RGD. The same proteins were bound to the KGE variant peptide and were eluted from the Tat column with this variant peptide indicating that the role of RGD in binding Tat is not a primary one. To determine whether these cells contained RGD binding proteins that did not bind to Tat, affinity chromatography on GRGDSP-Sepharose was performed with the unbound material from the Tat peptide column. A band with a similar electrophoretic mobility to the 150 kD Tat binding protein together with a band distinct from the 85 and 95 kD bands obtained from the Tat column were eluted from the GRGDSP-Sepharose column. These bands were identified as the subunits of the α y β 3 integrin. To identify the integrins or other cell surface molecules capable of binding to Tat, affinity chromatography was performed using the 86-amino acid Tat protein and the Tat peptides. As in the cell attachment experiments shown in Figure 2, all of the peptides that contained the basic domain of Tat gave similar results with rat L8 and human leiomyosarcoma SK-LMS (ATCC No. HTB 88) cells. Shown in Figure 4 are the proteins from an iodinated L8 cell extract bound to the Tat 45-86 peptide and eluted with this peptide or with full-length Tat. In all experiments with peptides containing the Tat basic region, bands of 150 kD and a doublet at approximately 90 kD were eluted from the column. Changing the RGD sequence to KGE or deleting the second exon entirely had no discernible effect on the identity of the proteins eluted from the column. Even the 12 amino acids comprising the basic domain were sufficient to bind and elute these bands (see Figure 5) . However, peptides lacking the basic domain did not bind or elute significant amounts of iodinated cell surface proteins. Figure 5 shows an immunoprecipitation of proteins eluted from the Tat 45-86 peptide column. The column was eluted sequentially with the peptide GRGDSP followed by the Tat 57-86 peptide and finally the 12 amino acid peptide containing the basic domain of Tat. Shown is the material eluted from the column with the indicated peptide after immunoprecipitation with antibodies to the specified integrin subunits. A small amount of material eluted with the peptide GRGDSP could be immunoprecipitated with β, and β 3 antibodies. However, the majority of the β, and β 3 subunit-associated material did not bind to the column and was detected in the unbound fraction. This suggests that the peptide GRGDSP and other peptides lacking the basic domain were relatively ineffective at eluting the proteins from the Tat peptide columns. In contrast, the majority of the material eluted with the basic peptide was detectable with the anti-β 5 subunit antibody (Figure 5) . The heterogeneity of the β 5 subunit may have been due to partial proteolysis resulting from harvesting the cells for the chromatography with trypsin. No labeled material could be immunoprecipitated from the fractions eluted with the Tat 57-86 peptide (not shown) . These results, together with the inhibition of cell attachment by the anti-VNR serum (Figure 3) , indicate that the α y β 5 integrin binds to the Tat protein and that this binding occurs at the basic domain of Tat. In contrast to the results obtained with the basic peptide, affinity chromatography with the peptide GRGDSPK coupled to Sepharose revealed a strikingly different pattern (Figure 6) . Whereas the predominant integrin binding to Tat-Sepharose was a^, the α^ integrin was the only integrin substantially enriched in the GRGDSPK-bound fraction (Figure 6) . Although some α y β 5 was observed in the bound fraction, the majority of this integrin was in the unbound fraction. This is in agreement with reports that demonstrated a relatively weak affinity of a^> s for the peptide GRGDSPK as described in Freed et al., EMBO J. 8:2955-2965 (1989). Together these data suggest that although these cells contain a functional α^ integrin that is capable of binding the peptide GRGDSPK, the RGD sequence in Tat is present in a context unfavorable to integrin binding. Since the a y B 5 integrin binds to the basic domain of Tat and not the RGD sequence, it appears to be an unusual interaction of an integrin with its ligand. To test this further, the EDTA-sensitivity of the integrin association with Tat was determined. It is known that integrins typically require divalent cations to bind their ligands and can be eluted from ligand " affinity columns with EDTA. The interaction between α y β 5 and Tat was, however, insensitive to elution with 10 mM EDTA in affinity chromatography experiments (Figure 7) . The NaCl concentration in the solution containing 10 mM EDTA was lowered from 150 mM to 100 mM to insure elution would result from divalent cation chelation and not an overall increase in the salt concentration of the solution. Immunoprecipitations of the peak fractions eluted with 10 mM EDTA, 250 mM NaCl, or the Tat 46-86 peptide revealed that the receptor was eluted from the Tat column with high salt or with the peptide, but chelating the divalent cations which are normally required for integrin function was not effective in disrupting this unusual integrin-ligand interaction. Since the interaction between Tat and α y β 5 was not a previously defined integrin-ligand binding, experiments were undertaken to determine if Tat bound to the same site on α y β 5 as vitronectin, the principal α y β 5 ligand. The monoclonal antibody P3G2 (Bristol Myers- Squibb) had previously been shown to bind to α y β 5 and to inhibit its interaction with vitronectin. This result was reproduced using SK-LMS cells as shown in Figure 8. However, the monoclonal antibody did not inhibit the binding of the cells to Tat. As shown in Figure 3, a polyclonal anti-a y B 3 antibody did inhibit the interaction of both Tat and vitronectin with the SK-LMS cells. A monoclonal antibody recognizing α y β 3 (LM 609) did not inhibit the binding of the cells to either substrate. The results suggest that α y β 5 mediates the attachment of SK-LMS cells to both vitronectin and Tat and that distinct regions of the receptor may be utilized for the binding to each ligand. EXAMPLE III Identification of the Tat-bindin Proteins To determine the identity of the Tat binding proteins in L8 cells, immunoprecipitations were performed with polyclonal antibodies to the cytoplasmic domains of α y . , β g , and β 6 . Antibodies against the α v cytoplasmic domain precipiTated all three bands eluted from the Tat peptide column. However none of the reagents against various integrin B subunits known or suspected to be associated with the α y subunit, β,, β 3 , β 5 , and B 6 , precipitated the complex. Control experiments with rodent cell lines and tissues demonstrated that the reagents were capable of reacting with the relevant integrins. These results indicate that the protein eluted from the Tat columns is an integrin comprised of the c- y subunit and possibly one or two previously unknown β subunits. The unbound fraction from the Tat column was rechromatographed on a GRGDSPK-Sepharose column. The material purified on the GRGDSP-Sepharose column was immunoprecipitated by antibodies to both the α y and β 3 subunits. These cells synthesize a functional vitronectin receptor (o_ y β 3 ) , that did not bind to the Tat column. EXAMPLE IV Cell Adhesion Assays Cell attachment assays were performed essentially as described in Ruoslahti et al., Meth. Enzvmol. 82 Pt A:803-831 (1982), incorporated herein by reference. Microtiter plates (96 wells) were coated with the full length peptide (Tat-86) , a shorter Tat peptide of residues 45-86 (Tat-41) or a second truncated Tat peptide of residues 56-86 (Tat-30) as the substrate for one hour in the presence of 0.25% glutaraldehyde. The plates were washed and then treated with 1 M ethanolamine and 2.5 mg/ml bovine serum albumin. L8 rat cells were detached from their substrate with 0.5 mg/ml trypsin as described in Brake et al., supra. washed three times with 0.5 mg/ml soybean trypsin inhibitor and resuspended in DMEM at 10 cells/ml. 100 μl of cell suspension was added to each well in the presence or absence of inhibitory antibody. After a one hour incubation, the attached cells were fixed in 3% paraformaldehyde and stained with 0.5% crystal violet. The dye was eluted from the stained cells with 100 μl of 50% ethanol containing 100 mM sodium citrate (pH 4.2). Attachment was quantified by reading the absorbance at 600 nm. The approximate O.D. values obtained in this study are summarized in Table 1. Table 1 Peptide O.D. (600 nm) Tat-86 0.92 Tat-41 0.93 Tat-30 0.06 To confirm that the cell adhesion to Tat was mediated by an integrin, various antibodies were used to inhibit cell adhesion to Tat. The above method was generally followed with 100 μl of either vitronectin or Tat peptide of residues 45-86 (Tat-41) as the substrate. In addition, approximately 10 L8 cells per well were added to each well in the presence of anti-vitronectin receptor (anti-VNR) or anti-fibronectin receptor (anti- FNR) antibodies. No antibodies were used in the controls. The attached cells were fixed and stained with crystal violet. The dye was eluted and the O.D. was measured in an ELISA reader. The approximate O.D. values obtained in this study are summarized in Table 2. The anti-VNR polyclonal antibodies against the vitronectin receptor inhibited the adhesion of these cells to both vitronectin and to the Tat peptide. Anti- FNR antibodies were not effective in preventing the adhesion of these cells to either substrate. These results indicate that a receptor related to the vitronectin receptor is responsible for the adhesion of the cells to Tat. The cell attachment assays with a variety of Tat derived peptides (Figure 1) were used to investigate the mechanism of cell interaction with the Tat protein. L8 rat skeletal muscle cells were chosen for these experiments because they had been shown to bind to Tat in a previous study described in Brake et al., supra. In agreement with this earlier study, L8 cells readily attached to the Tat protein (Figure 2A) . L8 cells also attached to the Tat 45-86 peptide that contained the RGD sequence and the basic domain, but not the cysteine rich domain. An unexpected result was that the cells did not attach to a shorter peptide in which the basic regions was deleted (residues 57-86) with residue 57 changed from arginine to lysine) , even though this peptide contained the cell attachment sequence RGD (Figure 2A) . Similar results were obtained with the human leiomyosarcoma cell line, SK-LMS (Figure 2B) . The cells bound only to those peptides that contained the basic region. Peptides with the RGD sequence changed to KGE or deleted entirely could still support cell attachment, provided the basic region was retained (Figure 2B) . In fact, a peptide containing only the basic domain and three flanking amino acids (residues 45-57) supported cell attachment as well as full-length Tat, on a molar basis. These results, together with the result that the attachment of cells to Tat was inhibited by heparin, suggest that the basic region of Tat, in addition to its function in transactivation, is required for cell adhesion, and that the RGD containing region of Tat by itself is incapable of supporting cell adhesion. EXAMPLE V Antibodies Against Various α and β Subunits Polyclonal antibodies against the cytoplasmic tails of v , β 3 , and β 5 were raised in rabbits by immunization with synthetic peptides coupled to keyhole limpet hemocyanin. The peptides used were KRVRPPQEEQEREQLQPHENGEGNSET from the C-terminus of α v as described in Suzuki et al., Proc. Natl. Acad. Sci. USA 83:8614-8618 (1986), KFEEERARAKWDTANNPLYKEATSTFTNITYRGT from the C-terminus of B 3 as described in Fitzgerald, J. Biol. Chem. 262:3936-3939 (1987), and KKPISTHTVDFTFNKSYNGTVD from the C-terminus of B 5 as described in Suzuki et al. , Proc. Natl. Acad. Sci. USA 87:5354-5358 (1990), all incorporated herein by reference. All peptides were synthesized with an Applied Biosystems Model 43OA (Foster City, CA) . Additional details on the preparation of some of these antibodies are described in Freed et al., EMBO J. 8:2955-2965 (1989) . The anti-β 1 subunit antiserum has also been described in Giancotti and Ruoslahti, Cell 60:849-859 (1990) . Each of the antibodies was shown to bind to the appropriate integrin subunit in immunoblotting and to immunoprecipitate integrins containing these subunits from various surface labeled cell lines. Polyclonal antibodies prepared against the α y β 3 and ct g β., integrins have been described in Argraves et al. , J. Cell Biol. 105:1183-1190 (1987) and Suzuki et al., Proc. Natl. Acad. Sci. USA 83:8614-8618 (1986) . Monoclonal antibodies (LM 609) were a gift of Dr. David Cheresh and are described in Cheresh and Spiro, J. Biol. Chem. 262:17703-17711 (1987) or Bristol Myers-Squibb Pharmaceutical Research Institute (P3G2) which is described in Wayner et al., J. Cell Biol. 113:919-929 (1991) . To determine whether adhesion to Tat was mediated by an integrin, various antibodies were used to inhibit cell adhesion to Tat. Polyclonal antibodies against the vitronectin receptor (α: y β 3 integrin) inhibited the adhesion of the L8 and SK-LMS cells to both vitronectin and to the Tat 45-86 peptide (Figure 3) . Anti-fibronectin receptor (ct g β, integrin) antibodies did not prevent the adhesion of these cells to either substrate. Control experiments showed that the anti-α y β 3 antiserum did not inhibit the attachment of cells to fibronectin, whereas the anti-α g β, antiserum did (Figure 3) . A receptor related to the α y β 3 integrin, therefore, appeared to be responsible for the adhesion of the cells to Tat. EXAMPLE VI Immunoprecipitation Assays Immunoprecipitations were performed by incubating material in the presence of 5 μl of immunized rabbit serum and 50 μl protein A-Sepharose for 1 hour. The receptor-antibody-protein A complex was spun down and washed three times with 0.5% Triton X-100, 150 μM NaCl, 50 μM Tris, pH 7.4. The complex was then boiled in electrophoresis sample buffer and loaded on 7.5% SDS- polyacrylamide gels. EXAMPLE VII Affinity Chromatography The Tat-binding proteins were isolated from surface-iodinated cells essentially as described in Pytela et al., PNAS fUSA. 82:5766-5770 (1985) and Pytela et al., Cell 40:191-198 (1985), both incorporated herein by reference. Cells were detached from culture plates in 100 μg/ml trypsin (Sigma) as in the cell adhesion assays, and washed three times in 500 μg/ml soybean trypsin inhibitor (Sigma) . Cells were surface iodinated and extracted with a buffer containing 150 μm octyl glucoside, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 μg/ml aprotinin, 1 μg/ml leupeptin, 0.4 μg/ml pepsTatin, 150 mM NaCl and 50 mM Tris, pH 7.4. The extracts were clarified at 15,000 x g and passed over a column containing various peptides coupled to cyanogen bromide activated Sepharose 4B (Pharmacia). After an incubation of 2 hours, the column was washed with several volumes of extraction buffer containing 50 mM octyl glucoside. The bound receptor was then eluted with the appropriate peptide at a concentration of 1 mg/ml in the buffer used to wash the column. Aliquots were boiled in electrophoresis sample buffer and run on 7.5% SDS-polyacrylamide gels or used for immunoprecipitation. EXAMPLE VIII Production of Polyclonal Antibodies Polyclonal antibodies can be prepared by any method known in the art using as the immunogen an appropriate protein or a synthetic peptide derived therefrom. In addition, the integrin of the present invention, the Tat protein or equivalent compounds can be used as the immunogen. Such methods as described, for example, in Argraves et al., J. Cell Biol. 105:1163-1173 can be used. For example, a synthetic peptide containing at least one lysine residue is coupled to Keyhole Limpet Hemocyanin (KLH) as follows: by stirring 5.6 ml KLH (10 mg/ml in phosphate buffered saline) with 0.5 ml m- maleimidobenzoyl-N-hydroxysuccinimide ester (25 mg/ml in dimethyl formamide) for 30 minutes at room temperature. The mixture is filtered and 25 g of the peptide is added followed by three hours of stirring at room temperature. After dialysis against phosphate buffered saline, the mixture containing the coupled peptide is emulsified in Freund's complete adjuvant, mixed and injected into a New Zealand White female rabbit. After one month, the animal is reinjected with the mixture emulsified in incomplete adjuvant. The animal is bled approximately two weeks after the second injection. The blood is allowed to clot and the resulting serum is used as a source of polyclonal antibodies. EXAMPLE IX Inhibition of Transactivation Assay Cells were seeded in 10 cm dishes (10 cells/dish) . The following day, cells were transfected with 5 μg LTR-CAT and/or 5 μg LTR-Tat plasmids by DEAE dextran precipitation as described in David et al. , Basic Methods in Molecular Biology, p. 388 (Elsevier 1986) , incorporated herein by reference. Two days posttransfection, cells were incubated with 80 μg or 8 μl rabbit anti-human vitronectin receptor (anti-α y β 3 ) , anti- fibronectin receptor (anti-α- g β,,) or normal rabbit serum in 3 ml of complete DMEM for 30 minutes before the addition of 0.5 μl of E. coli extract containing glutathione transferase-Tat fusion protein. Basic peptide (300 μg) of Tat protein was also included for the inhibition assay of Tat transactivation. Sixteen hours later, cells were harvested and disrupted by 3 cycles of freezing and thawing to harvest the cell lysate, which was then used for CAT assay as described in Gorman et al. , Mol. Cell. Biol. 2:1044-1051 (1982), incorporated herein by reference. To determine whether α y β 5 serves as the receptor for Tat internalization, the effects of basic Tat peptides and antibodies to α y β 3 on transactivation by extracellular Tat were studied. Antibodies to α 5 β 1 were used as a control. As shown in Figure 9 (lanes 1-3) , a recombinant GST-Tat fusion protein added to L8 cells was able to efficiently and specifically transactivate an LTR reporter gene transfected into these same cells. However, CAT activity was not diminished in the presence of anti-α y β 3 antibodies at concentrations sufficient to inhibit cell adhesion (lane 5) nor was it inhibited in the presence of 300 μg of basic peptide (lane 4) . These results suggest that the entry of functional Tat protein into L8 cells is not dependent on its interaction with the C- y β g integrin, consistent with an earlier suggestion that Tat internalization was not receptor-mediated as described in Mann and Frankel, EMBO J. 10:1733-1739 (1991) . Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims: We claim: 1. A method of inhibiting HIV Tat protein binding to a cell expressing an HIV Tat binding integrin, comprising blocking the binding of said HIV Tat protein to said integrin, wherein said blocking is at a binding site comprising a non-RGD binding region. 2. The method of claim 1, wherein the binding of said HIV Tat protein to said integrin is blocked by binding the HIV Tat protein with a reagent having reactivity with an integrin binding site of said HIV Tat protein. 3. The method of claim 2, wherein said reagent is an antibody. 4. The method of claim 2, wherein said reagent is a reactive fragment of said integrin. 5. The method of claim 1, wherein the binding of said HIV Tat protein to said integrin is blocked by binding said integrin with a reagent having specificity for said integrin at a HIV Tat protein binding site. 6. The method of claim 5, wherein the reagent is a reactive fragment of said HIV Tat protein. 7. The method of claim 6, wherein said reagent has substantially an amino acid sequence of the HIV Tat protein residues 45-86 or a reactive fragment thereof. 8. The method of claim 7, wherein said reactive fragment comprises a basic region of said HIV Tat protein. 9. The method of claim 8, wherein said basic region has substantially an amino acid sequence RKKRRQRRR. 10. An isolated fragment of HIV Tat protein having binding reactivity with a HIV Tat binding integrin, wherein said fragment comprises a non-RGD binding region. 11. The isolated fragment of claim 10, wherein said fragment contains a basic domain of said HIV Tat protein. 12. The isolated fragment of claim 11, wherein said basic domain comprises substantially the amino acid sequence RKKRRQRRR. 13. The isolated fragment of claim 11, wherein said fragment further comprises an RGD-containing domain of said HIV Tat protein. 14. The isolated fragment of claim 13, wherein said fragment comprises substantially the amino acid sequence RKKRRQRRRXPTSQSRGDPTGPKE, wherein X is from zero to 15 amino acids. 15. The isolated fragment of claim 10, wherein said integrin is a y B 5 . 16. An isolated nucleic acid encoding a reactive fragment of HIV Tat protein having a basic domain. 17. The isolated nucleic acid of claim 16, wherein said basic domain has substantially the amino acid sequence RKKRRQRRR. 18. An isolated nucleic acid encoding a Tat binding integrin subunit or a reactive fragment thereof. 19. The isolated nucleic acid of claim 18, wherein said integrin subunit is a β subunit. 20. The isolated nucleic acid of claim 19, wherein said β subunit is β 5 . 21. A vector comprising the nucleic acid of claim 19. 22. A host containing the vector of claim 21. 23. A reagent having specific reactivity with a Tat binding integrin or fragment thereof at a binding site that inhibits the binding of Tat protein with said integrin. 24. The reagent of claim 23, wherein said reagent is an antibody. 25. The reagent of claim 23, wherein said Tat binding integrin is α. y β 5 . 26. The reagent of claim 23, wherein said fragment is a β subunit of said integrin. 27. The reagent of claim 26, wherein said B subunit is β 5 . 28. A nucleic acid probe that specifically hybridizes with the nucleic acid encoding a HIV Tat binding integrin without hybridizing to a nucleic acid encoding a non-HIV Tat binding integrin or a reactive fragment thereof. 29. A method of detecting in a sample a ligand that binds to a HIV Tat binding integrin, comprising: (a) contacting said integrin with said sample; and (b) determining the binding of said integrin to said sample, wherein binding indicates the presence of said ligand. 30. The method of claim 29, wherein said ligand is a HIV Tat protein or a reactive fragment thereof. 31. The method of claim 29, wherein said ligand is a peptide mimetic. 32. A method of increasing the binding of HIV Tat protein to a cell expressing a HIV Tat binding integrin, comprising overexpressing said integrin in said cell.

Inventors: RUOSLAHTI ERKKI I, VOGEL BRUCE E, WONG-STAAL FLOSSIE Y, RUOSLAHTI, ERKKI, I., VOGEL, BRUCE, E., WONG-STAAL, FLOSSIE, Y.
Applicants: JOLLA CANCER RES FOUND, UNIV CALIFORNIA, LA JOLLA CANCER RESEARCH FOUNDATION, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1992
Main classification: C07K7
IPC codes: A61K38, A61K39, A61P31, C07K7, C07K14, C07K16, C12N15, C12Q1, G01N33