Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90-kDa cell surface binding protein.

The HIV-1 transactivator protein Tat is essential for viral gene expression and replication. Tat is taken up by cells and transactivates the HIV-LTR promoter in the cell nucleus. The present studies show that cells adhere to both synthetic and recombinant Tat, and, using synthetic peptides, we localize the binding site to a region spanning amino acid residues 49-57 (peptide Tat49-57). Tat49-57 also inhibited cell attachment to solid phase full-length Tat peptide and to recombinant Tat protein. Using Tat peptide affinity chromatography, we identified a 90-kDa cell surface protein that binds to Tat. The 90-kDa protein could be eluted from the Tat column using the Tat49-57 peptide. A 90-kDa cell surface Tat binding protein was also identified by coprecipitation with Tat after incubation with radiolabeled cell membrane preparations. Co-precipitation of the 90-kDa protein was inhibited by competition with a Tat49-65 peptide, but not with Tat55-86. Our findings suggest that cellular attachment to Tat is mediated through a 90-kDa cell surface protein that binds to a Tat domain between amino acids 49 and 57.

( 1 Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 The HIV-1 transactivator protein Tat is essential for viral gene expression and replication. Tat is taken up by cells and transactivates the HIV-LTR promoter in the cell nucleus. The present studies show that cells adhere to both synthetic and recombinant Tat, and, using synthetic peptides, we localize the binding site to a region spanning amino acid residues 49-57 (peptide ). Tat49"Z7 also inhibited cell attachment to solid phase full-length Tat peptide and to recombinant Tat protein. Using Tat peptide affinity chromatography, we identified a 90-kDa cell surface protein that binds to Tat. The 90-kDa protein could be eluted from the Tat column using the Tat49-67 peptide. A 90-kDa cell surface Tat binding protein was also identified by coprecipitation with Tat after incubation with radiolabeled cell membrane preparations. Co-precipitation of the 90-kDa protein was inhibited by competition with a Tat49"'6 peptide, but not with Tat6S-S6. Our findings suggest that cellular attachment to Tat is mediated through a DO-kDa cell surface protein that binds to a Tat domain between amino acids 4 9 and 67.

Tat49-6?
The HIV-1 Tat protein is a strong transactivator of the HIV-LTR promoter (Green and Loewenstein, 1988;Frankel and Pabo, 1988) and may also regulate cellular genes and cell behavior (Brake et al., 1990;Roy et al., 1990;Ensoli et al., 1990). Tat transactivaton of the HIV-LTR promoter is essential for both viral gene expression and virus replication (for reviews see Green (1991) and Sharp and Marciniak (1990)). T a t can be rapidly taken up by HeLa cells and localizes in the nucleus (Green and Loewenstein, 1988;Frankel and Pabo, 1988). Tat has been found to stimulate the growth of cultured Kaposi's sarcoma cells (Ensoli et al., 1989). Recently, Tat was shown to promote lymphocytic and skeletal muscle cell attachment (Brake et al., 1990). The ability of Tat to be taken up and affect cell behavior suggests a cellular receptor for Tat. In this regard, T a t contains an amino acid sequence, Arg-Gly-Asp (RGD), at residues 72-74, which is a well known integrin receptor recognition sequence (reviewed in Akiyama * This work was supported in part by National Institutes of Health Grant A128201 and by Research Career Award 5 KO6 A104739 from the National Institute of Allergy and Infectious Diseases (to M. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Tat, Peptides, and Antibodies-HIV-1 (BRU isolate) Tatffi, Tat"", thesized by the solid-phase method and purified by HPLC' as previously described (Green and Loewenstein, 1988). A scrambled peptide containing the sequence RRKQRRKRR was custom synthesized by Research Genetics using the same method and also purified by HPLC. Antibody to Tatffi peptide was prepared in rabbits by methods previously described (Green et al., 1983). Anti-TateG antibody was affinity purified on Tatffi-Sepharose columns. Recombinant Tat was purchased from Advanced Biotechnologies. Antibodies to 81, p2, 84, aV, and avo3 integrins were purchased from Telios. Antibodies to p3 integrin were purchased from AMAC Inc., and control IgG was purchased from Kirkegaard-Perry.
Cell Attachment Assays-Cell attachment was determined using a modification of a previously described method (Klebe et al., 1974). Various amounts of synthetic Tat, Tat deletion peptides or recombinant Tat protein diluted in 200 pl of water were added to 16-mm diameter wells in 24-well cell culture plates (Costar) and air dried overnight at 4 "C. After drying, each well was treated with 3% bovine serum albumin for 1 h at 37 "C. The bovine serum albumin was then removed by aspiration. Next, PC12 and NG108-15 cells were detached by agitation, and H9 cells were collected in suspension. Cells were then washed three times in phosphate-buffered saline, pH 7.4 (PBS), and resuspended in DMEM (PC12 and NG108-15 cells) or RPMI (H9 cells) containing 100 pg/ml transferrin, 100 PM putrescine, 20 n M progesterone, 30 nM NaSe03, and 5 rg/ml insulin. 1 X lo4 cells were seeded in each well. Cells were incubated for 60 min at 37 "C in Tat37 serum-free medium. The unattached cells were then removed by washing the wells twice with 0.5 ml of 0.02 M sodium phosphate containing 0.15 M NaCl (PBS). The attached cells were fixed with methanol and stained with Diff Quick (Baxter Scientific Products). The number of attached cells was determined by counting the center 2-mm2 field in each of triplicate wells. The 2-mm2 field represents 1.6% (1/64) of the cells which remained adherent so the average value of the triplicates was multiplied by 64 to represent the total number of adherent cells per well. The number of adherent cells was then divided by the total number of cells added to the well and multiplied by 100 to determine the percent of adherent cells.
The peptide competition attachment assays were performed in two ways. First, the amount of Tat substrate was varied and a constant amount 10 pg of soluble peptide was added. Second, attachment of cells to 5 pg of Tat substrate was challenged with increasing amounts of soluble peptide. Competition to Tat was performed with a scrambled and polylysine each dissolved in DMEM at a concentration of 1 mg/ml. Competative peptides were added at the time of cell seeding.
For the antibody inhibition of attachment studies, Telios and AMAC monoclonal antibodies were tested at a final 1/10 dilution on wells coated with 20 pg of recombinant Tat. Using a 1 mg/ml stock, 50 p1 of control mouse IgG were added to the 450-pl assay well at the time of H9 cell seeding. Telios polyclonal rabbit antiserum to avo3 integrin and heat inactivated normal rabbit serum were tested at a final 1/25 dilution. Attachment was determined as described above.
Cell Surface Iodination-Cells were iodinated by the lactoperoxidase method as previously described (Lew et al., 1986). Briefly, cells (3-5 X lo7) were washed in PBS three times and resuspended in 100 11 of PBS. Two mCi of ['251]iodine (Amersham Corp.), 60 pl of 2 mg/ ml lactoperoxidase (Sigma) in PBS, and 20 p1 of 0.0125% H202 were added and the cell suspension was gently agitated for 5 min. Next, 30 pl of 0.05% H202 were added, and cells were incubated for an additional 10 min, followed by the addition of 40 p1 of 0.05% H202 and a 15-min incubation. Cells were washed three times with PBS and either used for (i) TataG incubation and immunoprecipitation analysis or (ii) frozen on dry ice and stored at -20 "C until used for affinity chromatography.
Affinity Chromatography-Peptide affinity columns were prepared at 4 "C by incubating peptide (1 mg/ml) with CNBr-activated Sepharose 4B according to the manufacturer's instructions. As control columns, both an unrelated 20-amino acid peptide from the laminin A chain or bovine serum albumin were coupled to Sepharose by the same procedure. Cell surface-labeled cells (see above) were lysed and sonicated in binding buffer (100 mM Tris-HCI, pH 7.4, containing 25 mM n-octyl 0-D-ghcopyranoside, 150 mM NaC1, 2 mM phenylmethylsulfonyl fluoride, and 1 mM MnC12). The lysate was clarified by centrifugation, applied to the column, and circulated overnight. In some experiments the bound protein fractions were eluted with 20 mM EDTA. In other experiments elution of the 90-kDa protein off the Tat column was carried out using the Tat49-57 peptide. For elution with the peptide, columns were prepared as described above and then 1 mg of Tat49-57 was added to 5 ml of binding buffer and circulated overnight. A parallel column was tested using the scrambled 49-57 peptide to try to elute the 90-kDa protein. In all experiments, eluates were dialyzed against water at 4 "C, lyophilized, and resuspended in SDS sample buffer for resolution on SDS-polyacrylamide gels and autoradiography, or resuspended in PBS containing 1% Triton X-100 for immunoprecipitation analysis.
Immunoprecipitation of Tat column fractions from ['4C]leucinelabeled H9 cell lysates was preformed as previously described (Roberts et al., 1988) using antibodies to pl, p4, and aV at a final 1/25 dilution.
Briefly, Tat column fractions were incubated with specific antibodies for one hour at 4 "C and then incubated with a final 10% suspension of Protein A-Sepharose for one h at 4 "C. The beads were then washed, and the bound proteins were dissociated by both heat and SDS and analyzed by 7.5% polyacrylamide gel electrophoresis and autoradiography.
Co-immunoprecipitation Analysis of T a p Protein Complexes-To analyze Tat interactions with cell surface proteins of intact cells, Molt3 cells were surface-labeled with lz5I as described above and incubated in 500 pl of PBS containing 400 ng of Tat=/ml PBS or PBS alone for 60 min at 37 "C with shaking. Cells were washed twice with PBS and lysed in 1 ml of PBS containing 0.5% Nonidet P-40, 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride at 4 "C. After 15 min, nuclei were removed by centrifugation and 200-pl aliquots of the supernate were immunoprecipitated with anti-TaP antibody as follows. Samples were made up to 1.5 ml with immunoprecipitation buffer containing a final concentration of 150 mM NaC1, 50 mM Tris-HC1, pH 7.4, 0.1% BSA, 0.05% Nonidet P-40, and, in initial studies, 10 mM EDTA. In order to determine if the Tat-90-kDa protein interaction could occur in the absence of EDTA, EDTA was eliminated from the immunoprecipitation buffer in later studies and the Tat-90-kDa protein interaction was unaffected. Samples were incubated in the immunoprecipitation buffer overnight at 4 "C with 50 pl of affinity purified anti-Tats6 antibody. For the deletion peptide competition of the co-immunoprecipitation, either 1 mg of the TataG, Tat6*-%, or Tat4'-% peptides were added. Washed Protein A Sepharose beads (30 +I, 7 mg) were added and the mixture was rotated 1 h at Tat86 Tat49-57 Tat55-86 Poly-lyslne gel. Gels were stained with (homassie Hlue, dried, and exposed to xray film with an intensifying screen at -70 "C. To analyze 'Tat interactions with cell memhrane-associated proteins, Molt:] cells were washed twice with RPMI lacking cysteine and methionine and were resuspended at 2 X 10" cells per ml in the same medium. After incuhatinn at 37 "C for 1 h, 10 ml of cells were metabolically Iaheled hv addition of 980 pCi of Tran""S-lahel (ICN, 1.179 Ci/mmol, 1 5 7 Cvs, H5"ij Met) and further incubated for 4.5 h. Cells were washed twice with cold I'BS, resuspended in I'HS contnining 80 mM sucrose, and disrupted with four strokes of a Dounre homogenizer fitted with a 13 pestle. Nuclei were removed hv centrifugation at 5,000 X p for 5 min. EDTA and KC1 were added to 10 mM a n d 50 mM final concentration, respectively, to the supernant fluid followed hv rentrifugation at 100,000 X for 1 h. The memhrane pellet was rinsed sequentially with 100 mM NnCl and distilled water. Tat peptide (Tat8') ( Fig. 1). All cell lines attached to Tatw' and t o polylysine. Spreading and process formation was ohserved with the neuronal cells on Tat, but not on polylysine (Fig. 2 ) . For hoth neuronal cell lines, approximately 40-50'; attachment was reproducibly ohserved at : i p g with 80-9Or; nf cells attached a t 30 pg. The H9 cell line was less adherent compared t o the neuronal cells, with approximately 2.5' ; o f cells adhering a t 5 p g and less than 50% attachment at 30 pg.
The active hinding region within Tat"' was localized using T a t deletion peptides with overlapping amino acid seqrlences. PC12 cells hound Tatw, Tat:" ' :, and Tat'" '", hut did not hind within a hasic %amino acid sequence encoded in rc~sidrrt.~ Tat"q T h e Tat"' ' I' peptide also promoted cell spreading in the neuronal cells (Fig. 2).
In the presence of 10 pg of soluble Tat"" '" peptick. attachment to hoth recombinant and synthetic Tat was competit ively inhihited hy approximately 5 0 5 a t varin~rs suhst rate levels (Fig. S A ) . With a constant suhstrate level o f 5 p g , inhibition of binding to hoth recornhinant Tat antl Tat"' was not ohserved with the addition of 1 p g o r less o f soluhle Tat4" ''7. Inhihition was ohsenred with addit inn of soluhle Tat4" " peptide and reached SOr; with the addition of 5 pg o f soluhle peptide (Fig. 314). With hoth varying and constant suhstrate levels, polylysine and a scramhled Tat"' '" did not inhibit cell binding to Tat. These data demonstrate that the HIV-1 Tat protein contains a cell attachment site in the 49-57-amino acid sequence.

Identification of a Cell Surface Binding Protein for Tat of 90 kDa in Various Cell Lines by HIV-1 Tat Peptide Affinity
Chromatography-The data suggest that Tat can interact with a cell surface receptor protein. Since integrins comprise a large family of adhesion receptor molecules, we first tested whether antibodies to various integrin subunits could block cell adhesion to recombinant Tat. Monoclonal antibodies to Pl, p2, p3, and aV did not inhibit attachment to Tat (Table   11). Rabbit antisera to aVp3 inhibited attachment by approximately 50%, but control normal rabbit sera also inhibited attachment. A monoclonal antibody to p4 integrin inhibited attachment to Tat by over 90% without inhibition by the control mouse IgG (Table 11). These data suggest involvement of the p4 integrin subunit in cell attachment to Tat.
Affinity chromatography of cell surface labeled proteins

C.
PC12 was carried out using Tat86 immobilized on Sepharose 4R to identify a cellular binding protein.
A major protein hand of 90 kDa from PC12 cells was eluted from the Tat peptide column with 20 mM EDTA and could be visualized by Coomassie Blue staining (Fig.  4.4). Additional 90-kDa protein could be detected eluting from the column with higher concentrations of EDTA (not shown). Tat binding proteins which stained with Coomassie Rlue, hut were not cell surface associated, were also ohserved. A similar cell surface iodinatable 90-kDa Tat hinding protein was found in Kaposi's sarcoma cells (Fig. 4 8 ) . Furthermore, the presence of a protein with the same electrophoretic mobility that bound Tat'" was detected in HI) cells by Coomassie Blue staining (Fig.  4C). In addition t.o the 90-kDa protein, a protein band of'  kDa was ohserved in some of the preparations in variahlc and The 90-kDa protein could also he eluted from the T a t column using the Tat"' '" peptide (Fig. 5). The scrambled Tat4!' "' peptide did not elute the 90-kI)a protein.
Tat affinity column unhonnd flow-through and PO m%I EDTA-eluted fractions from l'~-lahelcd H9 cclls were immunoprecipitated using anti-Pl, d t , and (rV antibodies t o identify the 90-kDa band. While the Tat untmlnd frnctions contained immunoprecipitahle proteins. the Tat hintlinE proteins did not immunoprecipitate with these antihotlies (data not shown). These data suggest that the Tat hinding proteins isolated hy our column chromatography are not J l , A . or WV integrins and suggests the possihilitv of multiple cell recognition sites on Tat and multiple Tat binding proteins.
Detection of a Complex between Tat and a 90-kDa Protein by Co-immunoprecipitation with Anti-Tat Antibody-To provide additional direct evidence that Tat can associate with a 90-kDa cell surface protein in uitro, cells were metabolically labeled with [35S]Met/[35S]Cys, and the membrane fraction was isolated, lysed with detergent, and incubated with Tatffi. Immunoprecipitation with anti-Tats6 antibody (Fig. 6B, lane  3 ) but not with normal serum (Fig. 6B, lane 1 ) precipitated a major band of 90 kDa. The 90-kDa protein was not immunoprecipitated in the absence of Tatffi (Fig. 6B, lane 2 ) .
We also determined in co-immunoprecipitation studies that Tat can associate with the 90-kDa cell surface protein using cells surface labeled with 1251 and incubated with Tats6. The cells were then lysed with detergent, and the extract was immunoprecipitated with anti-Tats6 antibody. A major labeled band of 90 kDa was found in the immunoprecipitate (Fig. 6A,  lane 3 ) , indicating that Tat added to intact cells can form a complex with the 90-kDa cell surface protein. The 90-kDa protein was not detected when normal rabbit serum was used instead of anti-Tatffi antibody (Fig. 6A, lane I ) or in the absence of added Tat (Fig. 6A, lane 2 ) . In addition to the 90-kDa protein, a protein band of 180-200 kDa was observed by co-immunoprecipitation analysis in Molt3 cells (Fig. 6).
That the 90-kDa protein binds Tat within the 49-65 sequence was demonstrated by competing co-immunoprecipitation of the iodinated 90-kDa cell surface protein with Tat deletion peptides. In the absence of Tatffi, no immunoprecipitable complex was formed (Fig. 7, lane 5 ) . Tatffi formed an immunoprecipitable complex with a 90-kDa protein (Fig. 7, lanes I and 6). Tat5bffi did not inhibit Tat interaction with the 90-kDa protein (Fig. 7, lane 3 ) , while Tat49-65 almost completely blocked Tat interaction with the 90-kDa protein (Fig. 7, lanes 4 and 8). These data demonstrate that Tat binds in solution to the 90-kDa cell surface protein via residues 49-65 on Tat.

DISCUSSION
We have demonstrated that the HIV-1 transactivator protein, Tat, is active for cell attachment with a variety of cell lines, and using synthetic Tat peptides we mapped the cell attachment site to amino acid residues 49-57. A synthetic Tat peptide comprised of residues 49-57 (Tat49-57) promoted cell attachment and spreading and competitively inhibited PC12 cell attachment to recombinant Tat protein and to a fulllength synthetic Tat% peptide. Using Tatffi peptide affinity chromatography and co-immunoprecipitation studies, we identified a novel 90-kDa cell surface protein which binds TatM. The specificity of the Tat-90-kDa binding protein binding was confirmed in co-immunoprecipitation studies where These data demonstrate that Tat recognizes a 90-kDa cell surface binding protein on various cell types and that the cell attachment site is within amino acid residues 49-65.
Although integrins are members of a related family of cell adhesion receptors and one of the tested antibodies against a member of the integrins reduced attachment to Tat, it is unlikely, for several reasons, that a known integrin is involved in Tat-mediated cell adhesion. First, antibodies to D l , p2, p3, CUV, and aVp3 did not inhibit cell attachment to Tat. Second, while anti-p4 inhibited attachment to Tat, antibodies to p4 (and 01 and aV) integrin did not immunoprecipitate any Tat49-65 , but not Tat5s-86 blocked co-immunoprecipitation. protein from the 20 mM EDTA Tat affinity column fraction which contains the 90-kDa Tat binding protein. Third, it is known that integrins change their mobility after reduction in SDS gels (Tamura et al., 1990) and the mobility of the 90-kDa protein was unaffected by reduction.
Tat contains an RGD sequence at residues 72-74. The RGD sequence is found in many adhesive proteins including fibronectin, laminin, thrombospondin, and fibrinogen (for reviews, see Ruoslahti and Pierschbacher (1986), Hynes (1987), and Yamada (1990). Here, we localized by two methods a Tat binding domain within a 9-amino acid sequence encoded in residues 49-57. TatM and Tat49-57 promoted cell attachment,  whereas and Tat"45 peptides did not promote cell attachment. Attachment to Tat was inhibited using the protein in co-immunoprecipitation experiments. Tat5&% did not disrupt Tat-90-kDa protein co-immunoprecipitation. Our observations may appear at odds with a recent report supporting the role of the Tat RGD (residues 72-74) sequence in cell attachment (Brake et al., 1990). In the latter study, however, multiple cell recognition sequences on Tat are implicated as only partial reduction in attachment was observed (42-58%) in T lymphocyte cell lines HUT-78 and MOLT-4 and monocyte line THP-1 when the RGD site in Tat was modified to RGE. Similar evidence of multiple cell binding sites in Tat is present in our studies since only partial inhibition (50%) of cell binding to Tat was observed by addition of soluble Tat49-57 peptide. Taken together, these data imply the existence of multiple active cell binding sites on Tat.
AIDS patients suffer from a variety of syndromes which may not be related directly to immunodeficiency. These syndromes include dementia and Kaposi's sarcoma. While the exact pathogenesis of these syndromes remains unclear, a more direct role for the Tat viral protein has been suggested (Brake et al., 1990;Roy et al., 1990;Ensoli et al., 1990). Cell adhesion to Tat, and possibly Tat activity within host cells, may play a role in AIDS pathogenesis. Here we show that Tat interacts with a cell surface binding protein of 90 kDa which may be facilitate Tat binding and possibly entry into cells.