The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR.

We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.

The human immunodeficiency virus type 1 (HIV-1) 1 tat gene product trans-activates viral gene expression and is essential for HIV-1 replication (1)(2)(3). Tat strongly activates transcription from the HIV-1 long terminal repeat by binding to the Tatresponsive region (TAR), an RNA stem-loop structure located at the 5Ј end of HIV transcripts (4). Although the precise mechanism by which Tat exerts its effect is not yet known, it has been established that Tat regulates transcription at the level of initiation and elongation (5,6). The Tat protein exists in two forms, which in the HXB2 viral isolate consists of 72 and 86 amino acids. The 86-amino acid protein (Tat-86) is encoded by two exons, whereas the 72-residue protein (Tat-72), which is identical except for lacking 14 residues from the C terminus, is the product of the first tat exon. The shorter form is sufficient for trans-activation (7). The second exon of Tat has been proposed to play a role in activation of integrated long terminal repeats, regulation of MHC class 1 gene promoter activity, and TAR-independent trans-activation (8 -10). Mutational analysis of Tat has revealed three major regions that are important for function (Fig. 1); these include the N terminus, the cysteine rich-region which is important for metal binding, and a charged, arginine-rich region important for nucleolar localization and for binding to the cis-acting TAR element (11)(12)(13).
One of the primary cellular responses to viral infection is the production of interferon (14). The RNA-dependent protein kinase PKR, also referred to as DAI, P1 kinase, and p68 kinase, is a serine/threonine kinase that is induced by interferon and activated in the presence of dsRNA. PKR exerts a well-established regulatory effect on initiation of protein synthesis. Activation of PKR by dsRNA, or polyanions such as heparin and some structured single-stranded RNAs (15), is accompanied by autophosphorylation (16). Following its activation, PKR in turn catalyzes the phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2) on a serine residue at amino acid position 51 (17,18). Phosphorylation of eIF2 results in the sequestration of a second initiation factor, the guanosine nucleotide exchange factor eIF2B, leading to the inhibition of protein synthesis (18). This mode of translational shut down provides a mechanism of host defense and, as such, is detrimental to the viral life cycle. Many viruses have developed strategies to circumvent the action of PKR activation. The mechanisms by which viruses prevent the action of PKR vary as follows: adenovirus (19), vaccinia virus (20), and influenza virus (21) directly reduce PKR activity via different means, whereas poliovirus infection leads to PKR degradation (22). PKR appears to be down-regulated by HIV-1 (23), but the mechanism remains to be elucidated. PKR has also been implicated in oncogenic transformation and tumorigenesis (24,25) as well as differentiation (26) and apoptosis (27). The substrate specificity of PKR has been shown to extend beyond eIF2 to include IB (28,29) an inhibitor of the transcriptional activator NFB, as well as histone H2A (30), and a 90-kDa protein found in rabbit reticulocytes (31) which can be phosphorylated by PKR in vitro.
Tat binds a variety of cellular factors including a putative ATPase and DNA helicase (32), and a 36-kDa nuclear factor (33), as well as the transcription factors TFIID (34) and Sp1 (35). Several observations prompted us to evaluate the possibility of an interaction between Tat and PKR. First, both have been demonstrated to interact stably with TAR (14,36). Moreover, the TAR RNA binding protein can interact with PKR, preventing the activation of PKR (37,38). Second, recent reports indicate that Tat binds a novel cellular kinase (39,40), and Tat-mediated transcription is sensitive to the kinase inhibitor 5,6-dichloro-1-␤-D-ribofuranosyl benzimidazole (41). Third, PKR has been shown to activate NF-B by phosphoryl-ation of its inhibitor, IB (28,29). Previous data indicated that NF-B plays a role in Tat-regulated transcription from the HIV-1 long terminal repeat (42). Finally, the stable expression of Tat in HeLa cells treated with interferon is associated with reduced levels of PKR (23).
An interaction between Tat and the cellular kinase PKR would present an attractive regulatory mechanism. In this report, we describe the ability of PKR (activated in the presence of dsRNA) and of protein kinase C (PKC) to phosphorylate purified Tat-72 in vitro. We show that activated PKR is able to phosphorylate a series of Tat proteins expressed as glutathione S-transferase (GST) fusions. Binding studies indicate a correlation between phosphorylation and the ability of Tat to bind PKR. Furthermore, Tat competes with eIF2 as a PKR substrate, and preincubation with Tat prevents the activation of PKR by dsRNA. The significance of these interactions as they pertain to viral regulation and mechanisms by which HIV-1 is able to avoid the antiviral effect of interferon are discussed.

EXPERIMENTAL PROCEDURES
Purification of HIV-1 Tat-72 Protein-The region of Tat encoded by the first exon (amino acids 1-72) of the HIV-1 HXB2 isolate was overexpressed in Escherichia coli (43) and purified to greater than 90% homogeneity as assessed by silver staining, by C4 reverse phase high pressure liquid chromatography (HPLC) as described previously (44).
Cleavage of GST-Tat Fusion Protein-Bacterial extracts (50 l) were mixed with 250 l of EBC buffer and 25 l of glutathione S-Sepharose beads (equilibrated in EBC buffer). Samples were incubated on a rocking platform for 30 min at 4°C, centrifuged for 10 s at 12000 rpm in a microcentrifuge, and the supernatant discarded. Pelleted complexes were washed twice with 500 l of EBC buffer containing DTT (5 mM) and SDS (0.075%). Precipitates were resuspended in 20 ml of thrombin cleavage buffer (50 mM Tris-HCl, pH 7.6, 20 mM KCl, 1 mM DTT). Suspensions were centrifuged at 3000 rpm for 3 min at room temperature. Supernatants were discarded, and pellets were resuspended in 100-l volumes of cleavage buffer. Two units of human thrombin (Sigma) were added, and the reactions were allowed to proceed for 3 h at room temperature. Following the incubations, reactions were centrifuged in a microcentrifuge at 3000 rpm for 3 min and the supernatants collected. The concentration of Tat protein obtained following thrombin digestion was estimated by running samples of the supernatant on a 15% polyacrylamide gel containing SDS, staining the gel with Coomassie Blue, and comparing the band intensities with those of standard proteins of known concentrations.
Kinase Assays-Reactions (20 l) containing 2.5 Ci of [␥-32 P]ATP (ICN Biomedical Inc., Costa Mesa, CA) and 0.5 l of PKR (approximately 5 ng) purified to the mono-S stage (46) were conducted as described previously (47) in the presence of dsRNA derived from reovirus (a gift from A. Shatkin). Kinase reactions (20 l total volume) containing PKC (10 ng) were carried out as described by the manufacturer (Upstate Biotechnology Inc., Lake Placid, NY). Kinase reactions containing the ␤-insulin receptor kinase (a gift from A. Flint) were performed as described by Villalba et al. (48). Mammalian and yeast kinases involved in the Ras signal transduction pathway (STE 20, STE 11, MEK, BYR and ERK) were a gift from A. Polverino; activation assays were performed as described by Polverino et al. (49). In each case the enzyme was added last to the reaction components assembled on ice. Phosphorylation was visualized using SDS-polyacrylamide gel electrophoresis and autoradiography at Ϫ70°C with an intensifier screen.
Phosphorylation of Tat and eIF2 by PKR-Kinase assays in the presence of Tat were performed by the addition of 10 l from a 20-l PKR activation assay (described above) to 50 ng of purified Tat-72 or Tat from thrombin-cleaved GST-Tat protein. Substrate competition assays containing eIF2 and Tat-72 were carried out by mixing 50 ng of purified eIF2 (a gift from J. Hershey) with increasing concentrations of purified Tat-72 (up to 500 ng). Concurrently, 50 ng of purified Tat was mixed with increasing concentrations of purified eIF2 (up to 500 ng). To each reaction 10 l volumes from an kinase assay containing activated PKR was added. Reactions were incubated for 20 min at 30°C and stopped by the addition of Laemmli sample buffer. Phosphorylated proteins were resolved in a 20% polyacrylamide gel containing SDS. Dried gels were exposed at Ϫ70°C to x-ray film (Eastman Kodak Co.) in the presence of an intensifier screen.
Edman Degradation and Phosphoamino Acid Analysis-Phosphatelabeled Tat-72 was excised from the gel and processed as described by Beemon and Hunter (50). Labeled protein was digested "in-gel" for 20 h at 30°C (51) with either trypsin or Achromobacter protease I (final concentration of 1 g/reaction) and fractionated by HPLC. Fractions containing phosphate-labeled derivatives were subjected to Edman degradation using the modification of Russo et al. (52). Following acid hydrolysis, one-dimensional phosphoamino acid analysis was performed as described by Cooper et al. (53), using electrophoresis on glass thin layer chromatography plates (J. T. Baker, Inc.) in Buffer 3.5 (10:100:1890, pyridine:glacial acetic acid:H 2 O), for 30 min at 1000 V.
Binding of PKR to Tat-Aliquots (100 l) containing recombinant GST-Tat protein were mixed with 100-l volumes of glutathione S-Sepharose beads (equilibrated in EBC buffer) and incubated for 30 min at 4°C. Complexes were sedimented by centrifugation in a microcentrifuge for 20 s at 12,000 rpm. The pellets were washed five times in EBC buffer containing 5 mM DTT and 0.075% SDS. Activated PKR (10 l from a 20-l kinase assay) was added to the GST-Tat, glutathione S-Sepharose bead complexes, and incubated for 20 min at 30°C. 32 P-Labeled PKR that remained bound to GST-Tat was eluted by boiling in sample buffer and resolved in a 15% polyacrylamide gel containing SDS.

Phosphorylation of Tat by PKR-
The requirement for dsRNA in the activation and autophosphorylation of PKR is well established (14). In addition to its role in translational control via the phosphorylation of the ␣ subunit of eIF2 (17,18), activated PKR has been shown to phosphorylate histone 2A, IB, and a 90-kDa protein found in rabbit reticulocytes (28,30,31). To determine whether PKR is able to phosphorylate the HIV-1 trans-activating protein Tat, in vitro kinase assays were performed using combinations of dsRNA, PKR, and Tat-72 (Fig. 2). The intense phosphate-labeled band in lane 1 corresponds to the autophosphorylated (activated) form of PKR; faintly labeled bands correspond to degradation products of activated PKR. The reaction displayed in lane 4 was similar to that of lane 1, but was incubated for a further 20 min in the presence of purified Tat-72. It contains an additional labeled band with a mobility similar to that of Tat, suggesting that PKR previously activated in the presence of dsRNA is subsequently able to phosphorylate highly purified Tat-72. Phosphorylation of Tat by PKR increased linearly between 0.25 and 25 g/ml, but the intensity of phosphorylation was reduced at higher Tat concentrations (data not shown). No phosphorylation of either PKR or Tat occurred in the absence of either dsRNA (lanes 2 and 5) or PKR (lanes 3 and 6).
Additional tests were conducted to rule out the possibility that these observations resulted from contamination of either the Tat or PKR preparations. Initial attempts to confirm that the 32 P-labeled protein is indeed Tat, by using antibodies directed against Tat in immunoprecipitation or immunoblotting experiments, were unsuccessful. Three different anti-Tat antibody preparations all failed to react with the phosphoprotein. However, Tat-72 purified by reverse phase chromatography using a C18 column (6; data not shown), and both Tat-72 and Tat-86 isolated from GST-Tat fusion proteins (see Fig. 4), also served as substrates for activated PKR. The latter differ from one another in electrophoretic mobility, thereby eliminating potential contaminants from consideration. Two other HIV-1 encoded regulatory proteins, Rev and Nef, were not phosphorylated detectably by PKR (data not shown). A variety of mono-S fractions containing PKR (46), as well as PKR purified by an immunoaffinity column containing monoclonal antibody against PKR (54), all phosphorylated Tat-72 in a dsRNA-dependent manner (data not shown). Therefore, Tat-72 is a substrate for activated PKR.
Specificity of Tat Phosphorylation-To further assess the specificity of the interaction between Tat and PKR, several kinases were tested for their ability to phosphorylate Tat. Activated kinases were incubated in the presence of 50 ng of purified Tat-72. The results shown in Fig. 3 (lanes 8, 10, 12, 14,  16, and 18) suggest that neither the ␤-insulin receptor kinase nor a series of kinases (ST20, ST11, MEK, Byr, and Erk) required for Ras-regulated signal transduction was able to phosphorylate the purified form of Tat (lanes 7-18). Confirmation that these kinases were activated prior to incubation with Tat was achieved by observing the autophosphorylation of PKR, PKC, BIRK, ST20, and Erk (not shown). Of the kinases tested, only PKR and PKC phosphorylated Tat (lanes 1 and 5). As expected, activated PKR and PKC also phosphorylated histones, histone 2A only in the case of PKR (lanes 3 and 6).   1-18, except lanes 3 and 6). PKR and PKC kinase reactions were also performed in the presence of 50 ng of histone ( lanes  3 and 6). Phosphorylated proteins were resolved on 20% polyacrylamide gels containing SDS by autoradiography. Only the region corresponding to the position of phosphate-labeled Tat and histone are represented in the figure.
Several radiolabeled nucleotides were tested to further address the specificity of phosphorylation of Tat by activated PKR. Both [␥-32 P]ATP and [␥-32 P]GTP were utilized in Tat phosphorylation, but [␣-32 P]ATP, [␣-32 P]UTP, and [␣-32 P]CTP failed to phosphorylate Tat in a PKR-dependent manner. However, when Tat was present at high concentrations (greater than approximately 10 g/ml), it was nonspecifically labeled by all of these nucleotides even in the absence of PKR or dsRNA. This nonspecifically labeled Tat comigrated with the unphosphorylated marker Tat, unlike specifically labeled Tat which migrated slightly slower than its precursor (Fig. 7, lane 8).
Since the nonspecific labeling was reduced by addition of excess unlabeled ATP, we tentatively attributed it to an affinity of the highly basic Tat protein for nucleotides, rather than to a covalent modification.
Phosphorylation of Tat-86 and Mutant Tat Derivatives by Activated PKR-To address the sequence requirements for Tat phosphorylation, several mutant Tat proteins were expressed as GST-Tat fusions and selectively purified by absorption to glutathione S-Sepharose beads. Tat proteins were isolated from their GST parent vector by cleavage with thrombin, and equivalent concentrations of the released recombinant Tat proteins were incubated in the presence of activated PKR (Fig. 4). Cleavage of the GST-Tat fusion with thrombin was necessary as the intact Sepharose-bound fusion proteins were not labeled by PKR. Control reactions containing activated PKR, without and with reverse-phase HPLC purified Tat, are shown in lanes 1 and 2. PKR phosphorylated the HPLC purified Tat (lane 2) and the recombinant single exon form of Tat (Tat-72) released from GST (lane 7). The full-length, two-exon form of Tat (Tat-86) was also phosphorylated (lane 4). Furthermore, activated PKR phosphorylated mutant forms of Tat-86, namely Tat-86p18IS and Tat-86C22G (lanes 5 and 6), and the corresponding mutant forms of Tat-72, Tat-72p18IS and Tat-72C22G (lanes 7-9). However, the 48⌬ Tat truncation and its mutant derivatives 48⌬p18IS and 48⌬C22G (Fig. 1) were not labeled in the presence of activated PKR (lanes 10 -12). As expected, no proteins in the Tat size range were labeled when activated PKR was incubated with the product of the GST vector alone (lane 3). From these results it appears that residues 49 -72, containing the basic region, are important for phosphorylation by PKR. Mutations in the activation domain did not affect Tat phosphorylation, and in several repeats of this experiment Tat-86 and Tat-72 were labeled to an equivalent extent.
Identification of Tat-72 Phosphorylation Sites-There are 5 serine residues and 4 threonine residues in Tat-72. To determine which amino acids on Tat-72 are phosphorylated in vitro by PKR, labeled Tat was digested with trypsin, and the products were resolved by HPLC. A single predominant peak resulted (Fig. 5A). One-dimensional phosphoamino acid analysis on material from the HPLC peak showed that the label is associated with both serine and threonine residues (Fig. 5B). The same material was subjected to sequential Edman degradation, and the release of radioactive derivatives was monitored. Peaks of radioactivity were observed at cycles 6, 8, and 12, indicative of phosphorylation sites at these distances following an arginine or lysine residue. The only place that such a pattern occurs in the Tat-72 molecule is at serine 62, threonine 64, and serine 68 (Fig. 5C). These residues follow a run of basic residues at positions 49 -58; it seems that digestion took place preferentially after arginine 56, in accordance with previous observations (55). Digestion with an alternative protease in place of trypsin corroborated these assignments. Achromobacter protease I cleaves specifically after lysine residues, yielding peaks at cycles 11, 13, and 17 following a lysine residue at position 51 (Fig. 5B). These results indicate that the phosphorylation sites are clustered in the C terminus immediately after the basic region of Tat.
Correlation Between the Binding and Phosphorylation of Tat by Activated PKR-To investigate the sequence requirements for the binding of Tat to PKR, similar amounts of wild-type and mutant GST-Tat proteins were bound to glutathione S-Sepharose beads and then incubated with 32 P-labeled PKR. Fig. 6 shows that GST alone is unable to bind PKR. GST-Tat 86, GST-Tat 72, and their mutant variants, p18IS and C22G, all bound PKR, whereas the 48⌬ Tat truncation and its associated mutants failed to bind PKR. In addition the Tat construct Tat 86⌬2/36, an N-terminal deletion of residues 2-36 from GST-Tat 86, successfully bound activated PKR. These data suggest that the Tat sequence contained between amino acids 49 and 72 is important for binding PKR, but the N-terminal and C-terminal residues 2-36 and 73-86 are dispensable. In similar experiments with HIV-2 Tat (see Fig. 1), the intact molecule bound labeled PKR with an efficiency comparable with that of HIV-1 Tat (GST-Tat 130; Fig. 6). Removal of residues from the N terminus of HIV-2 Tat had little effect on PKR binding (GST- Tat 99 ⌬8/47), whereas a truncation in the basic region (GST 84⌬) or the mutation of four consecutive arginine residues in this region to alanines (GST-Tat 99 8184A) abolished binding. These data emphasize the importance of the Tat basic region for PKR binding and raised the possibility that the interaction of the two proteins might be mediated by an RNA bridge. No support for this idea was obtained from experiments with RNases, however. RNase treatment of activated PKR, or of the bacterial extract containing GST-Tat, or of the GST-Tat⅐PKR complex, had no discernible effect on the interaction. Both single-stranded RNA (RNase A) and double-stranded RNA (RNase III) -specific RNases were tested, singly and in combination (data not shown).
Substrate Competition Between Tat and eIF-2-PKR regulates protein synthesis by phosphorylating eIF2 on serine 51 of its ␣ subunit (17,18). The ability of Tat to serve as a substrate for PKR raised the possibility that Tat might compete with eIF2 for phosphorylation by activated PKR. To address this hypothesis, activated PKR was incubated with Tat and increasing concentrations of purified eIF2 (Fig. 7, lanes 1-4). The results suggest that at high concentrations, eIF2 reduces Tat phosphorylation by PKR (lane 4). Conversely, increasing the concentration of Tat protein, while maintaining a fixed concentration of eIF2, resulted in a marked reduction in eIF2 phosphorylation (lanes 5-8). These data suggest that Tat and eIF2 can compete as substrates for phosphorylation by autophosphorylated PKR. Considering the relative molecular masses of Tat and eIF2 (about 10 and 125 kDa, respectively), they appear to serve as substrates for PKR and as competitors on a comparable molar basis.
Autophosphorylation of PKR Is Inhibited by Purified Tat 72-Experiments to this juncture have shown that Tat is not only a substrate for PKR but that it is also able to inhibit eIF2 phosphorylation by this kinase. The activation of PKR is closely associated with its autophosphorylation (14). To determine whether Tat can inhibit this reaction as well, Tat was added to kinase reactions either before or after the introduction of dsRNA (Fig. 8). In the absence of Tat, PKR was autophospho- A, purified Tat-72 was labeled in a 20-fold scale-up in vitro kinase assay, isolated, and digested with trypsin. Peptides were resolved by HPLC. Fractions were collected at 0.5-min intervals. The graph represents the 32 P content (cpm) of the fractions eluted. B, fractions 46, 47, and 48 were pooled, subjected to acid hydrolysis, and examined by one-dimensional thin layer electrophoresis and autoradiography. The positions of nonradioactive phosphoamino acid standards, located by staining with 0.5% ninhydrin in acetone, are also indicated. C, Edman degradation was performed on a pool of three fractions in the major peak of 32 P label. Peaks of radioactivity were released at cycles 6, 8, 12 (denoted by asterisks). Similarly, peaks were observed at cycles 11, 13, and 17 upon analysis of a peptide released by Achromobacter protease I digestion. These sites are marked on the amino acid sequence of the region of Tat which contains phosphorylatable residues at these positions.

FIG. 6. Binding of activated PKR to Tat-GST fusion proteins.
Equivalent amounts of mutant and wild-type GST-Tat fusion proteins were bound to glutathione S-Sepharose beads and subsequently incubated with a kinase assay mixture containing activated PKR. Following incubation for 20 min at 30°C, complexes were washed extensively in EBC buffer containing SDS, and bound proteins were eluted by boiling in 2 ϫ concentrated Laemmli sample buffer. Bound PKR was detected by SDS-polyacrylamide gel electrophoresis and autoradiography and corresponds to the band migrating near the 14 C-labeled 69-kDa molecular mass marker. rylated in a dsRNA-dependent fashion (lanes 1 and 2). Preincubation of PKR with increasing concentrations of purified Tat-72 prior to the addition of dsRNA eliminated PKR autophosphorylation (lanes 3 and 4). As in previous experiments (Figs. 2-5), when the addition of Tat was delayed until after PKR activation by dsRNA had occurred, both Tat and PKR were phosphorylated (lane 5). These observations indicate that Tat-72 can inhibit the activation of PKR by dsRNA in vitro, as well as its activity in phosphorylating eIF2. DISCUSSION The molecular mechanisms governing the control of HIV-1 gene expression are complex and not fully understood. In the present study our aim has been to further characterize the properties of the HIV-1 regulatory protein, Tat, specifically with regard to its relationship with the interferon-induced dsRNA-activated protein kinase PKR. Several reports link these two regulatory proteins. First, both of them can bind to TAR RNA, the structured RNA segment that is the target for Tat trans-activation (56,57). Second, TAR RNA modulates the activity of PKR, although there is disagreement as to whether this RNA activates the kinase (57-59) or blocks its activation (36,60). Third, Tat stimulates the translation of TAR-contain-ing RNAs in vitro (61). Fourth, a synthetic Tat peptide that binds TAR RNA was reported to inhibit PKR activation (62).
Finally, Tat appears to down-regulate PKR in cells infected with HIV-1 or stably expressing Tat (23). We therefore set out to test the hypothesis that there is a direct interaction between Tat and PKR. Three lines of evidence support this hypothesis.
The results of in vitro kinase assays demonstrated that dsRNA-activated PKR phosphorylates Tat purified according to a number of different protocols. Both the one-exon and twoexon forms of Tat (Tat-72 and Tat-86, respectively) are substrates for the kinase. Mutations that prevent Tat from transactivating HIV-1 transcription (P18IS and C22G) do not affect Tat phosphorylation, but a mutation removing residues 49 -72 (⌬48) eliminates phosphate labeling by activated PKR. Without exception, the phosphorylation of Tat proteins by PKR was shown to be dependent on the prior activation of PKR by dsRNA. Tat-72 could also serve as an inhibitor of PKR, both by blocking its ability to autophosphorylate in response to dsRNA (and hence to become activated for phosphorylation of substrates) and by competing with its natural substrate eIF2. In the competition assay, the affinity for Tat and eIF2 seemed to be comparable in molar terms. Binding studies showed that PKR can form a complex with either Tat-72 or Tat 1-4). Alternatively, increasing concentration of purified Tat-72 (0, 5, 50, and 500 ng) were mixed with 50 ng of purified eIF2 (lanes 5-8). Activated PKR was added, and the reaction was incubated for 20 min at 30°C. Phosphorylated protein products were analyzed by electrophoresis in 20% SDS-polyacrylamide gels and autoradiography. The positions of autophosphorylated PKR, the phosphorylated ␣ subunit of eIF2, and phosphorylated Tat-72 are marked. The asterisk indicates nonspecific Tat phosphorylation, observed at high concentrations (Ͼ500 ng, lane 8). Note that the reduced intensity of the Tat band in lane 2 is an anomaly, attributable to the slightly decreased loading of sample in this lane. terminus of both Tat proteins led to increased PKR binding, suggesting that a conformational change may render the interaction site more available to PKR. While this report was in preparation, McMillan and co-workers (63) reported the interaction of Tat with PKR. In their study, Tat-86 was phosphorylated but labeling of Tat-72 was not, although its ability to inhibit PKR was observed. Although we cannot provide an explanation for this discrepancy, we speculate that it might be attributable to the different sources of Tat employed (McMillan et al. (63) used synthetic Tat-72).
PKR phosphorylates two serine and one threonine residues immediately adjacent to the basic region of Tat, consistent with the known preference of PKR for serines in the context of a basic amino acid environment (17,18,64). This suggested that Tat phosphorylation might influence its ability to trans-activate via its interaction with TAR, or via effects on its cellular localization, or both. When tested in a Tat-dependent transcription assay (65), phosphorylation by PKR elicited no discernible effect on trans-activation 2 ; however, this negative result is difficult to interpret as only a fraction of the Tat was modified. On the other hand, in this study we demonstrate the phosphorylation of Tat by the mitogen-activated kinase PKC. PKC also phosphorylates Nef (66), although PKR did not. Several researchers have proposed that PKC plays a role in Tatmediated trans-activation (67)(68)(69). Depletion of PKC in Jurkat and 293 cells resulted in a reduction in Tat trans-activation, and a PKC mutant lacking a functional ATP-binding site failed to support trans-activation (69). It is also possible that PKC influences HIV-1 transcription via the phosphorylation of IB (70) or another unidentified PKC substrate (71). Since both PKR and PKC phosphorylate Tat and IB in vitro, it will be important to learn whether they modify the same sites on these proteins and whether they mediate similar responses in vivo. In this connection, we note that a number of laboratories have reported their inability to detect Tat phosphorylation in vivo (67,68). Our failure to observe reaction of antibodies with PKR-phosphorylated Tat suggests that these negative results may be due to the inability of anti-Tat antibodies to recognize the modified form of Tat. Indeed, phosphorylation of the Tat protein of HIV-2 has been observed both in vivo and in vitro (39).
The ability of Tat to inhibit PKR also has potential biological implications. Dominant negative forms of PKR can cause malignant transformation of 3T3 cells (24,25). Therefore it is conceivable that down-regulation of the kinase by Tat could contribute to the deregulation of growth control seen in Tattreated cells derived from Kaposi's sarcoma lesions of HIV-1infected individuals (72,73). Furthermore, PKR plays a recognized role in the interferon-induced antiviral response (14). It has been reported that HIV-1 replication is sensitive to interferon and that Tat confers partial resistance to the effect of interferon (3). Our data suggest that Tat may exert its effect via its ability to inhibit the activation of the interferon-induced protein kinase PKR, thereby reducing the PKR-mediated phosphorylation of eIF2, or of IB, or other substrates of this kinase. Moreover, the ability of Tat to form a complex with PKR provides a possible mechanism for the repression of PKR levels that have been observed in HeLa cells stably expressing Tat or in T-cells infected with HIV-1 (23).
Although both PKR and Tat are both RNA-binding proteins and can interact with TAR RNA, the PKR/Tat interaction documented here is evidently independent of TAR RNA and of RNA in general. In that both Tat and eIF2 are substrates for PKR and interact with similar affinities, it would appear that Tat resembles the vaccinia virus PKR inhibitor K3L (74). This early protein displays sequence homology to the ␣ subunit of eIF2 in the region of its phosphorylation site; lacking an appropriately placed serine residue, it acts as a pseudo-substrate for PKR. Like Tat, K3L down-regulates PKR by directly binding to the kinase and preventing its activation (i.e. its autophosphorylation) and inhibiting its activity (74). This mechanism is distinct from the competition that can occur between Tat and PKR for binding to TAR RNA but not dsRNA (59,61,62).
In conclusion, we have demonstrated that both the virally activated protein kinase PKR and the mitogen-activated kinase, PKC, are able to phosphorylate the HIV-1 trans-activation protein Tat. Protein phosphorylation constitutes an important mechanism for the regulation of intracellular events (75). Although the consequences of Tat phosphorylation with respect to the regulation of HIV-1 gene expression remain to be established, the ability of Tat to control the activity of PKR may have far-reaching implications. These potentially include effects on both transcription and translation, mediated by the phosphorylation of IB and eIF2, respectively, and suggest a mechanism whereby Tat may contribute to the ability of HIV to evade the action of PKR. The regulation of Tat by a kinase may provide the trigger whereby latent integrated virus is activated and elicits exciting possibilities for targeted intervention.