ViROl.OtiY485,

451-454

(1991)

Tat Protein of Human Immunodeficiency

Virus Type 1 Is a Monomer ANDREW

Division of Molecular

P.

RICE'

AND

When Expre

CMIS

FRED CHAN

Virology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030 Received June 24, 1991; accepted August 1, 199 1

Human immunodefkkncy virus transcription dirsotsd by the HIV-o-plaxes. We havs blots to ddpeet Tat, in CCYSc&l exlracts. Thess rasults agree with other studies which indicate that Tat is a monomsrk Academic

Press, Inc.

Human immunodeficiency virus type 1 (HIV-l) is a complex retrovirus (I), encoding six genes in addition to the gag, PO/, and env genes found in all replicationcompetent retroviruses. Two of these additional genes, rev and tat, are essential for viral replication and encode trans-acting proteins that regulate HIV-l gene expression, The Rev protein controls the cytoplasmic accumulation of spliced versus unspliced HIV-1 mRNAs (reviewed in 2). The Tat protein stimulates the transcription rate directed by the HIV-1 long terminal repeat sequences (LTR), acting at the levels of transcriptional initiation and elongation (reviewed in 3). Tat is a potent transactivator, able to stimulate HIV-1 LTRdirected transcription several hundredfold in some experimental systems (4, 5). The genetic element within the HIV-1 LTR that responds to Tat is known as TAR. Both genetic and biochemical experiments have demonstrated that Tat binds directly to TAR RNA rather than to TAR DNA (3). The TAR RNA element, which consists of nucleotides + 14 to +45 (with + 1 defined as the site of transcriptional initiation), forms a stable stem and loop structure at the 5’ends of all HIV-l transcripts (6). Tat makes direct contact with a 3-nt bulge and 5 surrounding base pairs in the TAR stem (7). In addition, a 68-kDa cellular protein binds specifically to the TAR loop and may be involved with Tat in formation of a protein-RNA ternary complex (8). The Tat-TAR RNA complex forms on the 5’ end of nascent transcripts (9) and thereby stimulates the activity of transcription complexes by an unknown mechanism. Tat has also been shown to regulate TAR-mediated post-transcriptional processes in some experimental systems, most

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notably in Xenopus oocytes (see 70 and references therein). The HIV-l Tat protein is encoded in Tao exens and ranges in size from 86 to 101 a pending on the viral isolate (11). ant in length in all isoletes and e the second exon encodes 72 residues of the first ex tion activity (72). RNA spiictng called Tnv or Tev, which contains of Tat fused to 39 residues of the 90 residues from the second exon o (73, 14). Tnv possesses full Tat activii but Me Rev activity. In the course of HIV-l i re, three proteins with Tat activity are a 72-residue protein containing only the f&t Tat exon, a full-length Tat protein ranging in size from W to 101 residues, and a 201 -residue Tnv pro&.&t. The functional transactivation domain of ail three pn&eins appears to be the amino terminal 72 residues from the first Tat exon. The Tat protein has two regions w&h striking sequence features, a basic region and a c rich region. The basic region, whioh con&&s 8 and arginines be%veen residues 49 and 57, E%F@S8s the nuclear localization signal for Tat (75, I@, In adzWon, the basic region directly contacts RNA in the Tat protein-TAR RNA complex (7, 17, 78). The eysteine-rich region contains 7 cysteines between rains 22 and 37. The function of the cysteine residues b ~~k~~wn, but they have been proposed to be irrvb In b&d&g metal ions and protein-protein interaMen (tg. The cysteine residues are crucial for Tat funr%on, ae substitution of single cysteine residues in m abolishes transactivation activity; an exception to this

be addressed. 451

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is the cysteine at residue 31 where mutation reduces but does not abolish activity (20-23). To date, information about the structure of Tat has come from studies using Tat expressed and purified from Escherichia co/i (19, 24, 25) or Tat expressed in a wheat germ cell-free translation system (26). E. coli-expressed Tat was originally reported to bind either Zn*+ or Cd’+ and form a homodimer (19). An alternative purification procedure from E. co/i, however, yielded a monomer Tat protein (26). We found that Tat expressed in the wheat germ system exists as a monomer with the 72 residues of the first coding exon folded into a protease-resistant structure (26). It has been unclear how the quaternary structures of E. coh-expressed Tat and wheat germ-expressed Tat relate to the structure of Tat in HIV-l-infected cells. In this study, we have analyzed the quaternary structure of Tat expressed in viva in mammalian cells from a transfected eukaryotic expression vector. We provide evidence that Tat exists predominantly, if not exclusively, as a monomer when expressed in mammalian cells. We expressed the Tat protein in transfected COS cells from a plasmid called pBCl2/RSV/t23. This plasmid uses the powerful Rous Sarcoma virus LTR to direct synthesis of a Tat gene containing both coding exons from HIV-1 isolate HXB2 (27). The resulting fulllength Tat protein is 86 residues in size and has a calculated monomer molecular mass from sequence of 9784 Da. This Tat protein, however, migrates anomalously at 15 kDa in SDS-polyacrylamide gels (16, 26). We used a monoclonal antibody directed against the amino terminus of Tat (28) to detect the protein in COS cell extracts. COS cell extracts were prepared by a procedure developed to examine cellular proteins that associate with the adenovirus El A proteins (29). In this method, cells are lysed and nuclear and cytoplasmic proteins extracted under conditions that preserve stable protein-protein complexes. Briefly, COS monolayer cultures were transfected by a DEAE-dextran procedure (30) at 48 hr post-transfection culture dishes were washed twice with phosphate buffer saline (PBS), cells were then harvested in 1 .O ml of PBS by scraping with a rubber policeman and concentrated by centrifugation in a microfuge at 12,000 rpm for 15 sec. The cell pellet was gently resuspended in lysis buffer (100 ~1 lysis buffer/l O7 cells) containing 250 mM NaCI, 50 mM Hepes, pH 7.5, 1 mM DlT, and 0.1% NP-40. Cell extracts were incubated on ice for 30 min and then centrifuged at 12,000 rpm for 30 sec. Supernatants containing the cell extracts were removed and assayed for Tat expression by immunoblots. The specificity and sensitivity of the anti-Tat monoclonal antibody in immunoblots used in this study are demonstrated in Fig. 1. The antibody does not react

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FIG. 1. lmmunoblot assay to detect HIV-1 Tat protein in COS cell extracts. Extracts were prepared as described in the text from COS cells either mock-transfected or transfected with Tat expression plasmid pBClZ/RSV/t23 (27). Extracts were fractionated by electrophoresis in a 20% SDS-polyacrylamide gel, transferred to nitrocellulose, and reacted with a monoclonal antibody directed against the Tat amino terminus (28). Antigen-antibody complexes were detected with 1z51-labeled protein A. Lane M contains ?-labeled protein markers which were transferred to the nitrocellulose filter; lane 1 contains the extract from 1 X 1 O6 mock-transfected cells; lanes 2-6 contain; respectively, extracts from 1 X 10s, 5 X 105, 1 X 105, 5 X 104, and 1 X 1 O4 cells transfected with the Tat expression plasmid.

with any cellular proteins in extracts from mock-transfected cells (lane 1). In extracts from cells transfected with pBCl2/RSV/t23, the antibody reacts only with Tat expressed from the plasmid (lanes 2-6). In longer exposures of the Western blot shown in Fig. 1, Tat can be detected in lane 4 (extract from 1 X 1 O5cells) but not in lane 5 (extract from 5 X lo4 cells). In this experiment, therefore, Tat can be detected in extracts from 1 X 1 O5 transfected cells. In some transfections, a small amount of a slightly more rapidly migrating form of Tat was detected. Presumably, this represents the 72 residues of Tat from the first coding exon which is synthesized from a small amount of unspliced mRNA expressed from the vector (there is a translation termination codon after the first coding exon). Previously, we used gel filtration columns and glycerol gradients to determine that Tat exists as a monomer when expressed in vitro in the wheat germ cellfree translation system (26). We have used these biochemical techniques to analyze the oligomerization state of Tat expressed in COS cells. For gel filtration analysis, extracts were prepared from cells transfected

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b,a IOBKI

ovalbumln 146Kl

4

fraction

,,,,,l,,lll~ll,,,,.,,,,,I,i, no. 1 5

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1

10

15

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FIG. 2. Gel filtration analysis of Tat in COS cell extracts. The extract from approximately 3 X 10’ cells transfected with Tat expression plasmid pBCl2/RSV/t23 was mixed with marker proteins and filtered through a Superose 12 FPLC column as described previously (26). Column fractions were concentrated by trichloroacetic acid precipitation and Tat was detected by an immunoblot as described in Fig. 1. Marker proteins were detected in column fractions by Coomassie blue staining of residual proteins in the SDS-polyacrylamide gel after the transfer to nitroceiiulose. The Stokes radii of marker proteins are: bovine serum albumin (BSA) 3.62 nm; ovalbumin 2.83 nm; and cytochrome c 1.63 nm.

with pBC12/RSV/t23, mixed with marker proteins which serve as internal standards, and applied to a Superose 12 FPtC gel filtration column. The column was equilibrated and run in a buffer containing 500 mM KCI, 1.5 mll/l MgCI,, 10 mn/l Tris-HCI (pH 7.2) and 1 mM DlT as described previously (26). Fractions were collected and concentrated by trichloroacetic acid precipitation, and Tat was detected by immunoblots (Fig. 2). Although some Tat did trail throughout the column, a single major peak eluted after the cytochrome c marker. (The background signal in Fig. 2 which appears to be Tat in fractions coeluting with the ovalbumin marker was not observed in other experiments.) From the data shown in Fig. 2, we estimate that the Stokes radius of Tat is 1.25 nm, identical to the value measured for Tat expressed in the wheat germ system (26). Tat expressed in COS cells, therefore, appears to exist as a monomer. For glycerol gradient analysis, an extract from transfected COS cells was mixed with marker proteins and sedimented through a 7.5 to 17.5% glycerol gradient containing 500 mM KCI, 1.5 m/M MgCI,, 1 m&I DlT, and 10 m1\/1Tris-HCI (pH 7.2). Fractions were collected and concentrated by trichloroacetic acid precipitation, and Tat was detected by immunoblots (Fig. 3). Although Tat has a smaller molecular mass than cytochrome c, it sedimented in the glycerol gradient slightly ahead of the cytochrome c marker. Presumably, this is due to the different shapes and densities of the two proteins, as sedimentation rate is a function of molecular mass, density, and shape. From the data in Fig. 3, we estimate that the S,,,,value of Tat is approximately 2.0. We previously reported that the S20,wvalue of Tat expressed in the wheat germ system is 1.73 (26) indistinguishable from cytochrome c. In this study,

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however, we used lower concentrations of glycerol in gradients than previously (7.5-l 7.5% versus 15 35%). The lower concentration gradients provide better resolution of proteins with refativeiy em& S&W values. In gradients with this lower concentration of gfycerol, we have obsen/ed that Tat expressed in the wheat germ system also has an S20,w value of 2.0 (data not shown). Using standard hydrodynamic equations (37) we calculate from the data in Figs. 2 and 3 that the 86-residue Tat protein expressed in COS cells has a native molecular mass of approximately 10,500 Da. This is similar to the 9784 Da value calcuf&ed from sequence. Our results demonstrete that Tat expressed in mammalian cells is predominantly, if not exclusively, a monomer. In this study, we found that HIV-1 Tat extracted from mammalian cells exists as a monomer. Our results demonstrate that Tat expressed in viva in COS cells and in vitro in the wheat germ system (26) behaves indistinguishably in a Superose 12 gel filtration column and in glycerol gradients. These findings contrast with a report that Tat expressed and purifid from E. co/i formed a homodimer (79). In this purificatjon from E. co/i, Tat was expressed at very high levels, subjected to denaturing conditions, and then renatured at high Tat concentrations. This purification procedure, therefore, may promote Tat aggregation into timers or higher oligomeric complexes that are not biofogicaliy relevant. An alternative purification procedure from E. co/i has been reported which yields Tat in monomer form (26). This monomer Tat from E. co/i was also shown in vitro to form a one-to-one complex with TAR RNA, suggesting that Tat functions as a monomer during the transactivation process. Our results with Tat

cytochrome c 112.5KI

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FIG. 3. Glycerol gradient analysis of Tat in COS cell extracts. The extract from approximately 1 X 1O7 celk transfected with Tat expression plasmid pBC 12/RSV/t23 was mixed with marker proteins and sedimented through a 7.5 to 17.5% glycerol gradient. Fractions were collected from the bottom of the gradient and concentrated by trichloroacetic acid precipitation, and Tat was detected by an immunoblot as described in Fig. 1. Marker proteins in fraotions were detected by Coomassie blue staining of residual proteins in the SDS-polyacrylamide gel after the transfer to nitrocetlulose. The S20,W values of marker proteins are: bovine serum albumin (BSA) 4.3 S; ovalbumin 3.7 S; and cytochrome c 1.73 S.

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expressed in COS cells provide additional support for the idea that Tat functions as a monomer in HIV-l-infected cells. It is possible that some Tat proteins in COS cells exist as dimers or higher oligomers but are present at a concentration below the sensitivity of our experiments. Additionally, it is possible that Tat dimers or oligomers are present in COS cells in unstable or transient complexes that dissociate during preparation or analysis of cell extracts. In this study, however, cell extracts were prepared by a procedure developed to analyze protein-protein complexes (29), and gel filtration columns and glycerol gradients were carried out under standard conditions that preserve protein-protein interactions. Whether unstable or transient Tat dimers or oligomers are biologically relevant is questionable, especially in light of the finding that Tat appears to bind as a monomer to TAR RNA (25). The likelihood that Tat functions as a monomer underscores the remarkable potency of this small protein on the transcription rate of the HIV-1 LTR. In fact, a Tat protein consisting of only the first 57 residues possesses about 30% of the transactivation activity of the full-length protein (21-23). During transactivation, the Tat protein binds to the TAR RNA structure present in the 5’ ends of nascent HIV-l transcripts (3). While bound to TAR, Tat may function by simultaneously contacting a cellular protein associated with the transcription complex. Residues 48 through 57 of Tat are involved in formation of the protein-RNA complex (7, 7 7, 78) and at least one-third of the remaining residues are likely buried within the protein interior (32), leaving a limited number of residues available to make a protein-protein contact. In the cases where the three-dimensional structure of protein-protein recognition sites are known, from 10 to 30 residues of each protein are involved in binding (33). While bound to TAR RNA, therefore, Tat is likely to simultaneously contact only one or two cellular proteins. A 68-kDa HeLa cell protein which specifically binds to the TAR loop may be one such protein (8). Another candidate protein, call TBP-1 (Tat binding protein-l), has been identified by its ability to interact with Tat purified from f. co/i (34). The characterization of these or other cellular proteins that interact directly with Tat is crucial for further insights into the mechanism of Tat transactivation. ACKNOWLEDGMENTS We thank B. R. Cullen for providing the Tat expression plasmid and C. Debouck for the Tat antibody. We thank C. 0. Echetebu, C. H. Herrmann, and 1. W. Harper for critical comments on the manuscript. We also thank 0. Cole for help in manuscript preparation and C. H. Herrmann for art work. This work was supported by N.I.H. Grant Al-25308.

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