Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5 of the RNA Polymerase II...

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/00/$04.0010

July 2000, p. 5077–5086 Vol. 20, No. 14

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Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5of the RNA Polymerase II Carboxyl-Terminal Domain during

Human Immunodeficiency Virus Type 1 TranscriptionMEISHENG ZHOU, MATTHEW A. HALANSKI, MICHAEL F. RADONOVICH, FATAH KASHANCHI,†

JUNMIN PENG,‡ DAVID H. PRICE,‡ AND JOHN N. BRADY*

Virus Tumor Biology Section, LRBGE, Division of Basic Sciences, National Cancer Institute,Bethesda, Maryland 20892

Received 24 March 2000/Returned for modification 8 April 2000/Accepted 18 April 2000

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment ofcarboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay,we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb)both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAPII) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5.Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylatesserine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity ofCDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphor-ylation by CDK9 is strongly inhibited by low concentrations of 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole,an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexesdemonstrates that CDK7 is released from the transcription complex between positions 114 and 136, prior tothe synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complexthrough 179. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preini-tiation complex and the other between 136 and 179. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptionalelongation may be due to its ability to modify the substrate specificity of the CDK9 complex.

Human immunodeficiency virus type 1 (HIV-1) encodes atransactivator protein, Tat, which stimulates transcriptionelongation through interaction with the transactivation re-sponse (TAR) RNA element located at the 59 end of nascenttranscripts (12, 28, 68, 75). In view of the observations thathyperphosphorylation of the carboxyl-terminal domain (CTD)of the large subunit of RNA polymerase II (RNAP II) corre-lates with the formation of processive elongation complexes(11) and that Tat transactivation requires the CTD (6, 44, 46,79), it has been proposed that a critical step in Tat transacti-vation is mediated through a cellular kinase(s) (68, 80). Twocyclin-dependent kinase (CDK)–cyclin pairs, present in twodistinct transcription factor complexes, have been implicatedas Tat cofactors which could phosphorylate the CTD (54, 68).TFIIH, a general transcription factor which contains ninepolypeptides (ERCC3/XPB, ERCC2/XPD, p62, p54, p44,CDK7 [MO15], cyclin H, MAT1, and p34) (13, 24), possessesCTD kinase activity (14, 37). The kinase activity of TFIIHresides in the CDK7 subunit (15, 58, 61, 62). In associationwith cyclin H and Mat1, CDK7 forms the CDK-activatingkinase (CAK) complex that phosphorylates CDKs involved inthe regulation of the cell cycle (19, 42, 43, 53, 67). The asso-

ciation of CAK with core TFIIH switches its substrate speci-ficity from CDKs to the CTD of RNAP II (57, 81). Interest-ingly, the yeast homologue of CDK7, Kin28, is found only in acomplex with TFIIH and is devoid of CAK activity (8).

The second CTD kinase, TAK (Tat-associated kinase), wasfirst reported by Herrmann and Rice (22). It was later foundthat the human positive transcription elongation factor com-plex called P-TEFb, first identified in and purified from Dro-sophila extracts, is actually equivalent to TAK (41, 85). P-TEFbmost likely functions by phosphorylating RNAP II CTD andpreventing polymerase arrest (40). Cloning and sequence anal-ysis of the small subunit of the Drosophila P-TEFb complexrevealed its extensive sequence identity (72%) to a previouslyidentified cdc2-related human kinase termed PITALRE (nowreferred to as CDK9) (18, 85). Importantly, immunodepletionof CDK9 from HeLa nuclear extract eliminated basal tran-scription elongation and Tat transactivation (39, 84, 85). Fur-ther, the addition of the affinity-purified human P-TEFb com-plex completely restored these two processes (84).

The interaction between Tat and P-TEFb is mediatedthrough human cyclin T1 (51, 76). The function of the Tat–P-TEFb complex is mediated through the high-affinity, loop-specific binding of the Tat–P-TEFb complex to the TAR RNAstructure, and the formation of the tripartite complex betweenTat, cyclin T1, and TAR depends on the 59 bulge and centralloop in TAR (2, 16, 17, 26, 32, 54, 68, 76). The P-TEFb-associated CDK9 kinase then induces phosphorylation ofRNAP II CTD and perhaps of other proteins present in thetranscription complex, leading to a transition from nonproces-sive to processive transcription. More recent observations in-dicate that a critical cysteine residue (C261) which is not con-

* Corresponding author. Mailing address: Virus Tumor Biology Sec-tion, LRBGE, Division of Basic Sciences, National Cancer Institute,Bethesda, MD 20892. Phone: (301) 496-0986. Fax: (301) 496-4951.E-mail: [email protected].

† Present address: UMDNJ-New Jersey Medical School, Newark,NJ 07103.

‡ Present address: Department of Biochemistry, University of Iowa,Iowa City, Iowa 52242.

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served in the murine cyclin T1 protein (Y261) is important forthe formation of the tripartite complex between Tat, cyclin T1,and TAR (2, 16, 17, 26, 32). Interestingly, a reciprocal exchangeof a cysteine to a tyrosine at position 261 (C2617Y261) be-tween human cyclin T1 (hT1) and the murine cyclin T1 (mT1)renders hT1 inactive and mT1 active for human Tat transac-tivation (2, 16, 17, 26, 32). Thus, the ability of Tat to recruitcyclin T1-CDK9 to TAR not only stimulates HIV-1 transcrip-tional elongation but also governs the species specificity ofHIV-1 Tat transactivation. It was originally proposed that bothTFIIH and P-TEFb may act sequentially and in a concertedmanner, promoting hyperphosphorylation of RNAP II CTDand increasing polymerase processivity (27, 84). At present,however, the role of TFIIH in HIV-1 transcription is contro-versial (5).

The mammalian CTD consists of 52 repeats of heptapep-tide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 and is phosphorylatedmostly at serine residues during transcription. However, theexact mechanism of CTD phosphorylation remains elusive (11,71). Our results indicate that CDK7 (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex(PIC) and function to hyperphosphorylate the CTD of RNAPII. In basal transcription, CDK7 and CDK9 facilitate transcrip-tion activity: CDK7 phosphorylates CTD serine 5, and CDK9phosphorylates CTD serine 2. In the presence of Tat, CDK7 isnot required for HIV-1 transcription. Remarkably, Tat modi-fies the substrate specificity of PIC CDK9, allowing CDK9 tophosphorylate serine 2 and serine 5. The substrate specificity ofCDK9 was confirmed using recombinant P-TEFb. In the ab-sence of Tat, P-TEFb phosphorylates the CTD at serine 2. Inthe presence of Tat, P-TEFb phosphorylates the CTD at po-sitions 2 and 5. Phosphorylation at positions 2 and 5 is sensitiveto low concentrations of 5,6-dichloro-1-b-D-ribofuranosylben-zimidazole (DRB), an inhibitor of transcription elongation byRNAP II. These observations provide significant insight intothe complex process of Tat transactivation and provide the firstexperimental evidence that Tat modifies the substrate speci-ficity of P-TEFb.

MATERIALS AND METHODS

Antibodies. Anti-CTD RNAP II monoclonal antibodies 8WG16, H5 (phos-phoserine 2), H14 (phosphoserine 5) (49, 69), and anti-Tat monoclonal antibodyare products of BAbCO. Anti-CDK9 (PITALRE) antibody was purchased fromBiodesign Company. Anti-CDK7, anti-cyclin H, anti-Mat1, anti-p62 (subunits ofTFIIH), and anti-CDK8 antibodies are products of Santa Cruz Biotechnology.

Biotinylation of template DNAs. The wild type, TATA box mutant, and TARmutant TM26 (4) HIV-1 long terminal repeat (LTR) templates (nucleotides [nt]2110 to 1168) were amplified by PCR with the forward primer 59 biotinylated-TAT GGA TTT ACA AGG GAC TTT C-39 and the reverse primer 59-GATCCG ATT ACT AAA AGG G-39. The primers were synthesized and biotinyl-ated by Lofstrand.

Expression and purification of recombinant P-TEFb proteins. The productionof recombinant P-TEFb proteins was carried out as described by Peng et al. (51).

Immunodepletion of CDK8, CDK7, and/or CDK9 from HeLa nuclear extract.HeLa nuclear extract (100 ml) in 0.8 M KCl buffer D (20 mM HEPES [pH 7.9],15% glycerol, 800 mM KCl, 10 mM MgCl2, 0.2 mM EDTA [pH 8.0], 0.1% NP-40,and 1 mM dithiothreitol [DTT]) was incubated with 20 ml of protein A-Sepha-rose beads to which anti-CDK8, anti-CDK7 and/or anti-CDK9 had been pre-bound (10 mg of immunoglobulin G). Antigen-antibody complexes were removedby centrifugation. After the procedure was repeated twice, depleted nuclearextract was dialyzed against 0.1 M KCl buffer D and assayed by Western blotanalysis.

Purification of PICs. Reaction mixtures (30 ml) contained 15 ml of HeLanuclear extract, 1.0 mg of biotinylated templates and 1.0 mg of poly(dI-dC) in theabsence or presence of Tat protein (100 ng). The in vitro transcription (IVT)buffer contained 50 mM KCl, 6.25 mM MgCl2, 20 mM HEPES (pH 7.9), 2 mMDTT, 0.5 mM EDTA (pH 8.0), 10 mM ZnSO4, 10 mM creatine phosphate, 100mg of creatine kinase/ml, and 8.5% glycerol (13 IVT buffer). After a 30-minincubation at 30°C, streptavidin-coated magnetic beads (Dynabeads; Dynal) pre-equilibrated in binding buffer (20 mM HEPES [pH 7.9], 80 mM KCl, 10 mMMgCl2, 2 mM DTT, 10 mM ZnSO4, 100 mg of bovine serum albumin/ml, 0.05%NP-40, and 10% glycerol) were then added to the reactions, and the mixtures

were further incubated for 30 min at 30°C. The immobilized templates were thenharvested using a magnetic stand, and the PICs were washed extensively with 13IVT buffer. In vitro transcription and Western blot analysis could then be per-formed using the purified PICs assembled on the immobilized templates.

Western blot analysis of the purified PICs. The purified PICs assembled onthe immobilized templates were heated for 10 min at 100°C in sodium dodecylsulfate (SDS)-loading buffer. The released proteins were fractionated by elec-trophoresis on SDS–4 to 20% polyacrylamide gels and then transblotted ontopolyvinylidene fluoride (PVDF) membranes (Millipore). CDK9 (a subunit ofP-TEFb), CDK7, cyclin H, Mat1, and p62 (subunits of TFIIH), CDK8, and Tatwere detected with specific antibodies as indicated above.

In vitro transcription with the purified PICs. In vitro transcription reactions(100 ml) were set up by resuspending the purified PICs in 100 ml of 13 IVTbuffer, 50 mM ATP, 50 mM CTP, 50 mM GTP, 10 mCi of [a-32P]UTP, and 10units of RNasin (Promega). The transcription reactions were allowed to takeplace for 60 min at 30°C. The radiolabeled transcripts were fractionated byelectrophoresis on 6% denaturing polyacrylamide gels and detected by Phos-phorImager.

Kinase reactions in the purified PICs and immunoprecipitation of the phos-phorylated RNAP II. Kinase reactions were performed by mixing the purifiedPICs with 10 mCi of [g-32P]ATP in 100 ml of 13 IVT buffer. After an incubationof 10 min at 30°C, the PICs were separated from the supernatants and washedextensively. Five hundred microliters of RIPA buffer (50 mM Tris-HCl [pH 7.5],150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) was thenadded into the tubes containing PICs immobilized by streptavidin-coated beads,and the mixtures were incubated for 120 min at 4°C with rocking. The superna-tants were saved, and the phosphorylated RNAP II was immunoprecipitated bymonoclonal antibody 8WG16.

Kinase reactions in the purified PICs and Western blot analysis of the phos-phorylated RNAP II. Kinase reactions were done by mixing the purified PICswith 50 mM ATP in 100 ml of 13 IVT buffer. After an incubation of 10 min at30°C, the PICs were separated from the supernatants and heated for 10 min at100°C in SDS-loading buffer. The released proteins were fractionated onSDS–4% polyacrylamide gels and then transblotted onto PVDF membranes.RNAP II was detected with anti-CTD monoclonal antibody 8WG16, H5, or H14.

DRB sensitivity assay of distinct RNAP II CTD phosphorylation sites. Bio-tinylated HIV-1 LTR templates were incubated with CDK7-depleted extract,and the PICs were then purified with streptavidin-coated magnetic beads. Forthe inhibition assays, the purified PICs were incubated with ATP in the presenceof different concentrations of DRB and washed extensively. The PICs were thenheated 10 min at 100°C in SDS-loading buffer. The released proteins werefractionated on SDS–4% polyacrylamide gels and then transblotted onto PVDFmembranes. Phosphorylated RNAP II was detected with anti-CTD monoclonalantibody H5 or H14. Alternatively, the kinase reaction buffer contained[g-32P]ATP, and phosphorylated RNAP II was immunoprecipitated by anti-CTDmonoclonal antibody H5 or H14. The activity was determined by direct quanti-tation using the Molecular Dynamics ImageQuant system.

Stepwise transcription. The purified PICs were incubated with 50 mM ATP for10 min and then washed extensively with 13 IVT buffer. The PICs were walkedto position U14 by incubation with 50 mM CTP, GTP, and UTP for 5 min at 30°Cand then washed extensively with 13 IVT buffer. The transcriptional elongationcomplexes (TECs) stalled at U14 were walked stepwise along the DNA byrepeated incubation with different sets of three nucleoside triphosphates (NTPs)and then washed extensively with 13 IVT buffer to remove the unincorporatedNTPs. When indicated, RNA transcripts were labeled with [a-32P]UTP andanalyzed on 15% denaturing polyacrylamide gels. To analyze the components ofTECs, the TECs were elongated with cold NTPs. The TECs that were stalled atdifferent stages were analyzed by Western blot. To detect phosphorylation ofRNAP II CTD during transcription, the phosphorylated CTD was labeled with[g-32P]ATP during stepwise transcription.

CTD kinase assay. The CTD kinase assays were performed by mixing 100 ngof glutathione S-transferase (GST)–CTD, 100 ng of P-TEFb, 10 mM ATP, and 10mCi of [g-32P]ATP in the absence or presence of Tat and incubating for 60 minat 23°C. The total reaction volume was 30 ml, and the final conditions were 50mM Tris-HCl (pH 7.5), 5 mM DTT, 5 mM MnCl2, 4 mM MgCl2, and 10 mMZnSO4. The phosphorylated GST-CTD was then immunoprecipitated with anti-CTD monoclonal antibody H5 or H14 and fractionated by electrophoresis onSDS–8% polyacrylamide gels. The labeled products were detected by Phosphor-Imager.

RESULTS

Tat stimulates the formation of transcriptionally activePICs on the biotinylated HIV-1 LTR templates. We have usedan immobilized template assay to isolate basal and Tat PICsand study their transcription and CTD kinase activities. Wefirst demonstrated that Tat specifically activates transcription.HIV-1 LTR promoter templates were 59 end labeled withbiotin at position 2110 as described in Materials and Methods.The biotinylated templates were incubated with HeLa nuclear

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extract in the absence or presence of Tat. PICs were subse-quently isolated using streptavidin-coated magnetic beads, andin vitro transcription assays were performed. In the absence ofTat, a low level of basal HIV-1 transcription was observed (Fig.1A, lane 1). The addition of increasing amounts of Tat proteinto the preincubation mixture significantly increased transcrip-tion from the HIV-1 promoter (Fig. 1A, lanes 2 to 5). Opti-mum Tat transactivation was observed when approximately100 ng of Tat was added to the reaction mixture (Fig. 1A, lane4). The addition of the adenosine analogue DRB to the tran-scription reaction mixture inhibited Tat transactivation (datanot shown).

To demonstrate the specificity of the in vitro transcriptionsystem, we utilized a template which contains a base substitu-tion in the TAR RNA bulge. This mutation knocks out theability of Tat to bind TAR RNA, inhibiting Tat transactivationin vitro and in vivo (4). Consistent with previous reports, theTAR RNA mutation (TM26) inhibited the ability of Tat totransactivate the template (Fig. 1B, lanes 4 to 6). The TM26mutation did not significantly affect the level of basal transcrip-tion. To further demonstrate the specificity of the Tat trans-activation, we utilized Tat mutants with single amino acid sub-stitutions at lysine 41 or cysteine 22. Consistent with previousresults, the mutants failed to activate transcription (data notshown).

CDK7 and CDK9 associate with HIV-1 PICs. We next an-alyzed the relative protein composition of PICs formed in theabsence or present of Tat, especially with respect to CTDkinases. Biotinylated templates were incubated with HeLa nu-clear extract in the absence or presence of Tat as described inMaterials and Methods. Parallel binding reactions were carriedout with a TATA box mutant HIV-1 LTR template as a neg-ative control for nonspecific binding of protein to the tem-plates. Figure 2 shows the Western blot analysis for P-TEFb(CDK9), TFIIH (CDK7, cyclin H, Mat1, and p62 subunits),CDK8, and Tat. The results of these assays demonstrate thatboth CDK7 and CDK9 are present in the HIV-1 PICs. Inter-estingly, the amount of either kinase is equal in the absence orpresence of Tat with the wild-type HIV-1 LTR template (Fig.2A and B, lanes 1 and 3). The appearance of proteins bound tothe templates is specific. Parallel assays performed with aTATA box mutant HIV-1 LTR, which is transcriptionally in-active, failed to precipitate proteins associated with either theP-TEFb complex or the TFIIH complex (Fig. 2, lanes 2 and 4).In contrast to the results obtained with CDK7 and CDK9, we

find that CDK8 is not present in the HIV-1 PIC in the absenceor presence of Tat (Fig. 2C, lanes 1 and 3).

Basal and Tat-activated transcription differ in their require-ments for CDK7 and CDK9. To determine the relative impor-tance of CDK7 and CDK9 in HIV-1 transcription, HeLa nu-clear extracts were treated with anti-CDK7 and/or anti-CDK9antibodies and subsequently used for in vitro transcriptionassays. Western blot analysis of the antibody-treated extractsdemonstrated that CDK7 and CDK9 had been specificallydepleted (Fig. 3A, panels 1 and 2). CDK7 was detected in themock-depleted control (panel 1, lane 1) and CDK9-depleted(lane 3) extracts, but not in the CDK7-depleted extracts (lanes2 and 4). Similarly, CDK9 was detected in the mock-depleted(panel 2, lane 1) and CDK7-depleted (lane 2) extracts, but notin the CDK9-depleted extracts (lanes 3 and 4). Western blotanalysis of the same extracts with anti-CDK8, anti-TBP, oranti-RNAP II antibody demonstrated the specificity of theclearing, since no change in the level of CDK8, TBP, or RNAPII was observed in the immunodepleted extracts (Fig. 3A,panels 3, 4, and 5).

Biotinylated HIV-1 LTR templates were incubated with themock-depleted, CDK8-depleted, CDK7-depleted, CDK9-de-pleted, and CDK7- and CDK9-depleted extracts in the absenceor presence of Tat, and PICs were purified with streptavidin-coated magnetic beads. First, in vitro transcription reactionswere performed with the purified PICs, and the result is shownin Fig. 3B (top). In the absence of Tat, the depletion of CDK7or CDK9 decreased HIV-1 transcription approximately 3- and10-fold, respectively (lanes 1 to 4). In the presence of Tat, noquantitative decrease in transcription was observed in the ab-sence of CDK7 (lanes 6 and 8). In contrast, CDK9 depletiondecreased Tat transactivation by more than 16-fold to essen-tially background levels (lanes 6 and 9). When both CDK7 andCDK9 were depleted from the extract, the basal transcriptionand Tat transactivation were eliminated (lanes 5 and 10).

It has been reported that CDK8 has in vitro CTD phosphor-ylation activity. To investigate whether CDK8 functions in

FIG. 1. Tat-stimulated transcription from HIV-1 LTR. In vitro transcriptionreactions were performed with the purified PICs, and the transcripts were la-beled with [a-32P]UTP. The runoff transcripts are 168 nt, as indicated. (A) Tatstimulated transcription from the wild-type HIV-1 LTR. (B) Tat was not able toactivate transcription from the TAR mutant (TM26) HIV-1 LTR. A comparisonof the transcription activities of wild-type HIV-1 LTR (lanes 1 to 3) and TM26(lanes 4 to 6) is shown.

FIG. 2. CDK7 and CDK9, but not CDK8, are components of the HIV-1 PIC.Association reactions (30 ml) were performed with 15 ml of HeLa nuclear extract,1.0 mg of biotinylated HIV-1 LTR templates, and 1.0 mg of poly(dI-dC) in theabsence (lanes 1 and 2) or presence (lanes 3 and 4) of Tat. PICs were purifiedwith streptavidin-coated magnetic beads. Western blot analysis of the purifiedHIV-1 PICs was then done with anti-CDK9, anti-CDK7, anti-cyclin H, anti-Mat1, anti-p62, anti-CDK8, and anti-Tat antibodies. An HIV-1 LTR TATA boxmutant (Mut) was used as a parallel control (lanes 2 and 4). WT, wild type.

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HIV-1 transcription, in vitro transcription was also performedwith the CDK8-depleted extract (Fig. 3B, bottom). The resultsof this experiment demonstrate that CDK8 depletion does notaffect either basal transcription or Tat transactivation (Fig. 3B,top, lanes 1, 2, 6, and 7). Consistent with these results, theexperiments presented above and in Fig. 2C demonstrate thatCDK8 is barely detected in HIV-1 PICs (Fig. 2, compare withCDK7 and CDK9). It is also important to point out that CDK7and/or CDK9 depletions did not affect the level of CDK8 in

extracts (Fig. 3B, bottom). These results suggest that CDK8does not play a critical role in HIV-1 transcription.

CDK7 and CDK9 phosphorylate the CTD in the HIV-1PICs. We next analyzed the effect of CDK7 and CDK9 onphosphorylation of RNAP II in the HIV-1 PICs. Consistentwith the recent publication by Isel and Karn (25), the presenceor absence of Tat does not affect the extent of CTD phosphor-ylation in the PICs. Equal CTD phosphorylation and conver-sion of RNAP II from form IIa to form IIo were observed inboth the basal and Tat PICs (Fig. 4, lanes 1 and 5). Themigration position of RNAP II form IIa was determined byWestern blot analysis of HeLa nuclear extracts.

Hyperphosphorylation of the CTD in the basal transcriptioncomplex was decreased approximately twofold by depletion ofeither CDK7 or CDK9 (Fig. 4, lanes 1 to 3). In the presence ofTat, no detectable quantitative difference in hyperphosphory-lation of the RNAP II CTD was observed in PICs from extractfrom which CDK7 was immunodepleted (compare lane 6 withlane 5). However, immunodepletion of CDK9 decreased thehyperphosphorylation of the RNAP II CTD by approximatelytwofold (lane 7). Depletion of both CDK7 and CDK9 from theHeLa extract decreased hyperphosphorylation of the CTD inthe PICs 10-fold (lanes 4 and 8). This result demonstrates thatboth the CDK7 and CDK9 kinases phosphorylate the RNAP IICTD in the HIV-1 transcription complex. It is important topoint out that the extent of CTD phosphorylation may notstrictly correlate with transcription, in part because it is difficultto determine the number of active PICs under different exper-imental conditions.

CDK9 and CDK7, respectively, phosphorylate serine 2 andserine 5 of the RNAP II CTD in HIV-1 PICs. The RNAP IICTD is composed of multiple repeats of the heptapeptidesequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (11, 45, 71). Pat-turajan et al. (49) have described monoclonal antibodies whichrecognize different phosphoamino acid epitopes on the CTD.To investigate the specificity of CTD phosphorylation, we havecompared the phosphorylation pattern of the RNAP II CTD inHIV-1 PICs with antibodies specific for phosphorylated serine2 (H5 antibody) and serine 5 (H14 antibody). Monoclonalantibody 8WG16 was used to detect both the unphosphory-lated IIa and phosphorylated IIo forms of RNAP II. Incuba-tion of the PICs with ATP resulted in the conversion of RNAPII from the IIa to the IIo form (Fig. 5, 8WG16 antibody, inputversus lanes 1 and 5). With parallel assays run with extractswith either CDK7 or CDK9 depleted, demonstrated in the

FIG. 3. The effects of the CTD kinase activities of CDK7 and CDK9 onHIV-1 transcription. (A) Western blot analysis of mock-depleted, CDK7-de-pleted, CDK9-depleted, or CDK7- and CDK9-depleted extracts with anti-CDK7,anti-CDK9, anti-CDK8, anti-TBP, or anti-CTD of RNAP II antibody. Panels 3,4, and 5 demonstrate that depletions did not change the level of CDK8, othergeneral transcription factors, or RNAP II. (B) The effects of the CTD kinaseactivities of CDK7 and CDK9 on HIV-1 transcription. Biotinylated HIV-1 LTRtemplates were incubated with mock-depleted, CDK8-depleted, CDK7-depleted,CDK9-depleted, and CDK7- and CDK9-depleted extracts in the absence (lanes1 to 5) or presence (lanes 6 to 10) of Tat, and PICs were then purified withstreptavidin-coated magnetic beads. In vitro transcription was done with thepurified PICs, and transcripts were labeled with [a-32P]UTP and fractionated on6% denaturing polyacrylamide gel containing 7 M urea in 13 TBE buffer (top).The Western blot analysis of kinase-depleted extracts was done with anti-CDK8antibody (bottom).

FIG. 4. Phosphorylation of RNAP II CTD in HIV-1 PICs. BiotinylatedHIV-1 LTR templates were incubated with mock-depleted, CDK7-depleted,CDK9-depleted, or CDK7- and CDK9-depleted extracts in the absence (lanes 1to 4) or presence (lanes 5 to 8) of Tat, and PICs were then purified withstreptavidin-coated magnetic beads. Kinase reactions were performed with thepurified PICs, and phosphorylated RNAP II was labeled with [g-32P]ATP andimmunoprecipitated (IP) with anti-CTD monoclonal antibody 8WG16.



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basal HIV-1 transcription initiation complex, serine 2 wasphosphorylated by CDK9 (H5 antibody, lanes 2 and 3), whileserine 5 was phosphorylated by CDK7 (H14 antibody, lanes 2and 3). Depletion of both CDK7 and CDK9 from the extractsabolished the phosphorylation of the CTD in the HIV-1 PIC(Fig. 5, lane 4).

The analysis of kinase activity in HIV-1 PICs formed in thepresence of Tat provided evidence that the substrate specificityof CDK9 is altered in the Tat complexes. As with the resultsobtained with the basal HIV-1 PICs, CDK9 was required tophosphorylate serine 2 (Fig. 5, middle, lanes 6 and 7). Remark-ably, the phosphorylation of serine 5 was maintained in theHIV-1 Tat PICs assembled from CDK7-depleted extracts (Fig.5, bottom, compare lane 2 with lane 6). Since the serine 5kinase activity was lost when CDK9 was also depleted from theextract (Fig. 5, bottom, lane 8), this result suggests that CDK9phosphorylates serine 5 in the HIV-1 Tat transcription com-plex. In the absence of Tat, CDK9 phosphorylates only serine2. It should be stressed that these studies, which clearly showthe specificity of CDK9 kinase activity in the absence or pres-ence of Tat, are done in the context of the transcription com-plex and the authentic substrate, the CTD of RNAP II.

CTD phosphorylation by Tat-modified CDK9 is sensitive toDRB. Tat transactivation has been shown to be preferentiallysensitive to the adenosine analogue DRB, which inhibitsRNAP II elongation. We next, therefore, tested the sensitivityof serine 2 and serine 5 phosphorylation to DRB. CDK7-depleted extract specific for CDK9 serine 2 and serine 5 kinaseactivities was used in the inhibition assay. The results of thisexperiment demonstrate that serine 2 and serine 5 phosphor-ylation by Tat-induced CDK9 is sensitive to low concentrationsof DRB. At a concentration of 5 mM DRB, serine 2 phosphor-ylation and serine 5 phosphorylation were inhibited by approx-imately 70 or 90%, respectively (Fig. 6).

CDK7 is released between positions 114 and 136, whileCDK9 remains stably associated with the HIV-1 TECs. SinceCDK7 and Tat-induced CDK9 phosphorylate serine 5, theability of Tat to modify the substrate specificity of the CDK9complex would most likely become important when CDK7 was

released from the transcription complex. Interestingly, onother polymerase II promoters, TFIIH is released once thepolymerase has traveled approximately 30 nt (83). To seewhether the same phenomenon was observed with the HIV-1transcription complex, we used the immobilized template assayand isolated TECs stalled at 114, 136, or 179 (see Materialsand Methods). The protein composition of transcription com-plexes was subsequently analyzed by Western blotting. Theresults of this experiment demonstrate that CDK7 is releasedfrom the template between 114 and 136 (Fig. 7A). The ab-sence or presence of Tat does not affect the stability of TFIIH(CDK7) with the template. In contrast, CDK9 is stably asso-ciated with the TECs through position 179 (Fig. 7A). Theamount of CDK9 associated with the complex is not modifiedby the presence or absence of Tat.

A second phase of RNAP II CTD phosphorylation occursbetween 136 and 179. The above-described results suggestthat Tat-induced phosphorylation of serine 5 by CDK9 mightbe important after transcription has reached the 136 position,at which time CDK7 has been released from the complex. Toanalyze phosphorylation of the RNAP II CTD during tran-scription elongation, PICs were first incubated with cold ATP.The transcription complexes were then stalled at various posi-tions downstream of the RNA initiation site by sequentialincubation with different combinations of three NTPs. Kinasereactions were subsequently performed in the presence of[g-32P]ATP. RNAP II was immunoprecipitated from the reac-tions and analyzed by SDS gel electrophoresis. The results ofthis study provided two very important observations. First, inthe transcription complex assembled in the absence of Tat,there was no subsequent phosphorylation of RNAP II once theelongation complex had reached 136 to 179 (Fig. 7B, lanes 1and 2). Second, in the presence of Tat there was an additionalphase of CTD phosphorylation that occurred between nt 136and 179 (Fig. 7B, lanes 3 and 4). This phosphorylation must bedue to CDK9 activity, since CDK7 had been released from thetemplate.

Of importance, the secondary phase of CTD phosphoryla-

FIG. 5. CDK9 and CDK7 phosphorylated serine 2 and serine 5, respectively,of the RNAP II CTD in HIV-1 PICs. Biotinylated HIV-1 LTR templates wereincubated with mock-depleted, CDK7-depleted, CDK9-depleted, or CDK7- andCDK9-depleted extracts in the absence (lanes 1 to 4) or presence (lanes 5 to 8)of Tat. PICs were then purified with streptavidin-coated magnetic beads. ThePICs were then incubated with 50 mM ATP for 10 min in order to have RNAPII CTD phosphorylated and washed extensively. Western blot analysis of thePICs was done with anti-CTD monoclonal antibodies 8WG16, H5, or H14.

FIG. 6. CTD phosphorylation by Tat-modified CDK9 is sensitive to DRB.Biotinylated HIV-1 LTR templates were incubated with CDK7-depleted extractin the presence of Tat, and PICs were then purified with streptavidin-coatedmagnetic beads. The inhibition assays were performed by incubating the purifiedPICs with ATP in the presence of different concentrations of DRB. Western blotanalyses of the complexes were done with anti-CTD monoclonal antibody H5 orH14, and the activities were determined by direct quantitation using the Molec-ular Dynamics ImageQuant. The top curve (») indicates the inhibition of serine2 phosphorylation, while the bottom curve (n) indicates the inhibition of serine5 phosphorylation.



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tion also correlates with the synthesis of TAR RNA structurethat has the binding site for Tat. The TM26 HIV-1 LTR carriesbase substitutions in the bulge of TAR structure which havebeen shown to inhibit Tat interaction with the RNA (4). WhenRNA was synthesized from this template, no postinitiationphosphorylation of RNAP II was detected (Fig. 7C).

Finally, the results presented in Fig. 7D suggest that thesecond phase of RNAP II CTD phosphorylation correlateswith Tat transactivation. HIV-1 transcription PICs were iso-lated and incubated in the presence of sequential nucleotidemixtures to stall complexes at different stages of transcription.[a-32P]UTP was added to the transcription reactions to labelthe nascent RNA. Tat did not have any detectable effect ontranscription through the first 36 bases (lanes 1 and 2). In

contrast, when the complexes were allowed to synthesize theTAR RNA enhancer and proceed to nt 179, an increase in thelevel of transcription was observed in the presence of Tat (Fig.7D, lanes 3 and 4) which represents the initial stages of Tattransactivation. The rather modest increase in [a-32P]UTP in-corporation is due to the fact that we are only analyzing tran-scription of 43 bases of RNA. Importantly, the sizes of the 36-and 79-base RNA transcripts confirm that the transcriptioncomplexes are indeed stalled at the indicated sites on thetemplate DNA.

The recruitment of the Tat–P-TEFb complex by TAR stim-ulates HIV-1 transcription elongation. To investigate whetherthe Tat–P-TEFb complex can be recruited after initiation torescue transcription from the stalled transcription complexes,the experiment was designed as indicated in the legend to Fig.8. Biotinylated wild-type or TAR mutant (TM26) HIV-1 LTRtemplates were incubated with CDK9-depleted extract, andthe PICs were purified with streptavidin-coated magneticbeads. The transcription complexes were then walked stepwisealong the templates to 179 (as described in Materials andMethods). The runoff transcription was performed by incubat-ing the TECs stalled at 179 with ATP, CTP, GTP, and[a-32P]UTP. P-TEFb and Tat were added at different sites asindicated in Fig. 8. Interestingly, the results presented hereimply that P-TEFb can indeed be recruited during elongationas a complex with Tat (lanes 10 to 12) and that the recruitmentof the Tat–P-TEFb complex was TAR dependent (lanes 4 to12). It should be noted that the level of transcription facilitatedby the recruitment of Tat–P-TEFb through TAR binding dur-ing elongation was significantly lower than that observed whenTat and P-TEFb were present during the assembly of the PICs(lanes 12 and 15). These results suggest that while Tat–P-TEFbmay enter at a later point, perhaps the most efficient entry isduring the PIC formation, so that efficient conversion to theelongation complex is achieved.

Tat directly modifies the substrate specificity of CDK9 inthe recombinant P-TEFb complex. The above-described exper-iments suggest that Tat modifies the substrate specificity of theCDK9-containing P-TEFb complex. To demonstrate this pointdirectly, CTD kinase assays were performed with recombinantP-TEFb (Fig. 9A) (51). Following the kinase reaction, equalaliquots of the same kinase assay were immunoprecipitatedwith phosphoserine 2 (H5)- or phosphoserine 5 (H14)-specificantibody. The results presented in Fig. 9B demonstrate severalimportant points about the functional interaction between Tatand CDK9. First, in the absence of Tat, CDK9 phosphorylates

FIG. 7. Stepwise walking of RNAP II elongation complexes and TAR-de-pendent rephosphorylation of RNAP II CTD during elongation. The purifiedPICs were incubated with 50 mM ATP for 10 min and then washed extensively.The PICs were walked to position U14 by incubation with 50 mM CTP, GTP, andUTP for 5 min at 30°C and then washed extensively. The TECs stalled at U14were walked stepwise along the DNA by repeated incubation with different setsof three NTPs and then washed extensively to remove the unincorporated NTPs.(A) Western blot analysis of PICs and stalled TECs. (B and C) TAR-dependentrephosphorylation of RNAP II CTD during elongation. (D) Transcription facil-itated by TAR-dependent rephosphorylation of RNAP II CTD during elongation.

FIG. 8. The recruitment of the Tat–P-TEFb complex by TAR binding during elongation. Biotinylated wild-type (WT) or TAR mutant (TM26) HIV-1 LTRtemplates were incubated with CDK9-depleted extract, and PICs were purified with streptavidin-coated magnetic beads. The transcription complexes were walkedstepwise along the templates to 179 (as described in Materials and Methods). The runoff transcription was then performed by incubating the TECs stalled at 179 withATP, CTP, GTP, and [a-32P]UTP. P-TEFb and Tat were added at different sites, as indicated.



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the CTD at serine 2 but not at serine 5 (Fig. 9B, lane 1). In thepresence of Tat, a slight increase in the level of serine 2 phos-phorylation was observed. The addition of 200 ng of Tat in-creased serine 2 phosphorylation approximately twofold (top

panel, lane 8). The addition of a Tat mutant containing anamino acid substitution at cysteine 22 failed to increase CDK9activity (Fig. 9B, top, compare lane 1 with lanes 2 to 5).

Remarkably, in the presence of Tat, CDK9 was also able to

FIG. 9. Tat directly modified the substrate specificity of P-TEFb in a CTD kinase assay. (A) A silver-stained SDS-polyacrylamide gel electrophoresis of recombinantP-TEFb fractions. (B) CTD kinase assay. The assays were performed by mixing 100 ng of GST-CTD, 100 ng of P-TEFb, 10 mM ATP, and 10 mCi of [g-32P]ATP inthe absence or presence of Tat and incubating for 60 min at 23°C. The total reaction mixture volume was 30 ml, and the final conditions were 50 mM Tris-HCl (pH7.5), 5 mM DTT, 5 mM MnCl2, 4 mM MgCl2, and 10 mM ZnSO4. The phosphorylated GST-CTD was then immunoprecipitated (IP) with anti-CTD monoclonalantibody H5 (top) or H14 (bottom) and fractionated by electrophoresis on SDS–8% polyacrylamide gels. Numbers at left represent molecular masses in kilodaltons.The labeled products were detected by PhosphorImager. Numbers at the top show the amounts of Tat (GST-Tat 72) or a Tat mutant (GST-Tat72Cys22) that wereadded, expressed in nanograms. Lane M, molecular mass marker. (C) DRB sensitivity assay of serine 2 phosphorylation (lanes 1 to 4) and serine 5 phosphorylation(lanes 5 to 8). Micromolar concentrations of DRB are expressed. Numbers at left, molecular masses in kilodaltons.



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phosphorylate serine 5 (bottom, compare lane 1 with lanes 6 to9). Importantly, the Tat mutant Cys22 failed to activate serine5 phosphorylation (bottom, lanes 2 to 4). The fold increase inTat-induced serine 5 phosphorylation is difficult to calculate,since there is no serine 5 phosphorylation in the absence ofTat. However, the level of serine 5 phosphorylation was equiv-alent to the level of serine 2 phosphorylation. In results similarto those presented in Fig. 6, the addition of low concentrationsof DRB to the kinase reactions inhibited serine 2 and serine 5phosphorylation by Tat-induced CDK9 (Fig. 9C). These resultsprovide direct evidence that Tat modifies the substrate speci-ficity of the CDK9 enzyme and that the induction of kinaseactivity observed in the presence of Tat is primarily due tophosphorylation at serine 5. It is important to point out that wecannot eliminate the possibility that Tat simply markedly en-hances the activity of P-TEFb kinase. The activation must,however, be selective toward serine 5 phosphorylation.

DISCUSSION

The RNAP II CTD heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 contains three serine residues at positions 2, 5, and 7.The CTD is heavily phosphorylated in vivo, and substitution ofnonphosphorylatable amino acids at position 2 or 5 of theSaccharomyces cerevisiae CTD is lethal (77, 82). Phosphoryla-tion of the CTD is temporally linked to the transition betweentranscription initiation and elongation. Human CTD kinasesspecific for serine 5, serines 2 and 5, or serines 2 and 7 havebeen characterized (11, 71). It has been reported that theTFIIH kinase phosphorylates serine 5 of the CTD (58, 71).The analyses of TFIIH kinase activity in HIV-1 transcriptioncomplexes presented in this manuscript are consistent withthese findings. The substrate specificity of the P-TEFb complexhas been more difficult to distinguish. Ramanathan et al. haverecently reported that serine 5 is essential for phosphorylationby the Tat-TAK complex (Tat–P-TEFb complex) (55). Usingan in vitro kinase assay, the investigators demonstrated thatphosphorylation of the CTD peptide was abolished when amutation was introduced at serine 5. Mutation of serines 2 and7 did not affect the activity of the Tat-TAK complex. Theauthors did not include reactions which contained P-TEFbalone in the absence of Tat. Thus, it was not obvious that theserine 5 phosphorylation observed in their assay is the Tat-modified function of P-TEFb. Moreover, the specificity of theP-TEFb kinase alone cannot be distinguished. Patturajan et al.have recently reported that the deletion of yeast CTDK-I, aCDK kinase closely related to P-TEFb, eliminates the increasein CTD serine 2 phosphorylation during a response to nutrientdepletion (48). In our studies, we provide clear evidence thatrecombinant P-TEFb phosphorylates the CTD at serine 2. Inthe presence of Tat, the substrate specificity of P-TEFb isaltered such that it phosphorylates serine 2 and serine 5. It isimportant to point out that the change in substrate specificityis reproduced in the natural setting of the kinase, the transcrip-tion complex, and the natural substrate, the RNAP II CTD.

A unique feature of Tat transactivation of HIV-1 transcrip-tion has been observed, which is that it is preferentially inhib-ited by DRB, an adenosine analogue that targets RNAP II-mediated elongation. P-TEFb has been distinguished fromother transcription factors by its sensitivity to very low doses ofDRB. In this regard, the results presented in this report dem-onstrate that serine 2 phosphorylation and serine 5 phosphor-ylation by Tat-activated P-TEFb are sensitive to DRB and thatat a concentration of 5 mM DRB, serine 2 and serine 5 phos-phorylation was inhibited by approximately 70 or 90%, respec-tively.

It will be of interest to determine whether the initial phos-phorylation at serine 2 and serine 5 within the PICs is impor-tant for Tat transactivation. Okamoto et al. (44) have shownthat the CTD is not required for basal transcription and for theformation of short, attenuated transcripts. In contrast, tran-scriptional activation by Tat in vivo and in vitro requires theCTD. It is possible that the critical step of CTD phosphoryla-tion takes place in the elongation complex, after CDK7 hasbeen released, TAR RNA has been synthesized, and Tat–P-TEFb has been recruited to the complex. TAR likely acts torecruit the Tat–P-TEFb complex to the elongation complex inthis activation process. It will also be of interest to determinewhether phosphorylation of serine 2, serine 5, or serines 2 and5 is required for Tat transactivation at this stage.

In a very elegant analysis of the fate of transcription factorsduring the transition from initiation to elongation, Zawel et al.have demonstrated that TFIID remains promoter bound,wherease TFIIB, TFIIE, TFIIF, and TFIIH are released rap-idly (83). TFIIH release occurs after the complex reaches 130to 150. Consistent with these studies, our analysis indicatesthat TFIIH is released between 114 and 136 (Fig. 7A). Pre-vious observations suggest that Tat interacts with a target cel-lular protein (as a cofactor of Tat) and that the interaction ofTat with its cellular cofactor is a prerequisite for TAR binding(1, 38). Recent studies strongly imply that the Tat cofactor is acellular protein kinase termed TAK which interacts with theactivation domain of Tat and phosphorylates CTD (21, 22, 79).It has been found that a human positive-acting transcriptionelongation factor complex called P-TEFb is actually equivalentto TAK (85). Several observations further suggest that therecruitment of the Tat–P-TEFb complex by TAR binding toelongating complexes is the critical step in Tat transactivation(22, 29, 40, 41). Similar to the results obtained in this study,recent observations indicate that the entry of the Tat–P-TEFbcomplex into the transcription complex comes at the time ofPIC assembly (47, 52). Our results presented here are consis-tent with the observations that Tat–P-TEFb associates withHIV-1 PICs. Importantly, in contrast to the TFIIH-CDK7complex, which is released from the complex between 114 and136, P-TEFb remains stably associated with the TECs. Theresults presented in this study further suggest that the ability ofTat to alter the substrate specificity of CDK9 would allow thecontinued hyperphosphorylation of the RNAP II CTD atserine 2 and serine 5 in the Tat transcription elongation com-plex. As a general transcription elongation factor, it will also beimportant to determine how P-TEFb acts on other promotersand whether other activators affect the activity of P-TEFb.

Genetic data from several groups show that TAR can befunctionally replaced by heterologous RNA structures. Thesubsequent recruitment of Tat to these RNA targets by fusionof Tat to an RNA binding domain can clearly fully activateHIV-1 LTR-dependent transcription (59, 70). The results sug-gest that TAR acts only as an interface. Further, the recruit-ment of P-TEFb to an HIV-1 LTR containing a heterologouspromoter-proximal target by fusion of cyclin T1 to an RNAbinding domain is both necessary and sufficient for full activa-tion of transcription from HIV-1 LTR in vivo. The authors ofthe latter study suggest that Tat does not activate the P-TEFbin any specific way but rather serves as an interface betweenthe RNA and the enzyme complex (3). The results presented inthe latter study demonstrate that Tat, in fact, does modify theactivity of the P-TEFb-associated CDK9 kinase, altering thespecificity of CTD phosphorylation to allow CDK9 to phos-phorylate serine 5.

Multiple kinases appear to be involved in phosphorylatingthe CTD in vivo (7, 35, 50). In yeast, at least three distinct



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complexes have been described, Kin28-CCL1 (8, 15, 72, 73),SRB10-SRB11 (36), and CTDK1 (a possible yeast homologueof P-TEFb) (48, 63). In higher eukaryotes, there are threehomologues, CDK7-cyclin H (14, 37, 58, 60–62), CDK8-cyclinC (34, 56, 65), and P-TEFb (40, 41). The observations demon-strate that CDK8-cyclin C (SRB10-SRB11, the yeast homo-logue) associates with the RNAP II holoenzyme (34, 36, 56).However, only a small portion (less than 10% in mammals and6% in yeast) of total RNAP II was found to be associated in aholoenzyme form with CDK8 (SRB10) in vivo (30, 34). It hasbeen reported that SRB10-SRB11 (CDK8-cyclin C) is a neg-ative regulator of transcription (20, 65) and that SRB10 is nota general repressor of protein-coding genes (36, 66). CTDphosphorylation by SRB10-SRB11 kinase prevents the assem-bly of the PIC and thereby represses the transcription of spe-cific genes (20), including those involved in cell type specificity(74), meiosis (64, 66), sugar utilization (31, 36), and stressresponse (9). Our results presented in this report suggest thatCDK8 may not function in HIV-1 transcription and Tat trans-activation.

Finally, it is of interest to consider the possibility that phos-phorylation of the RNAP II CTD may have a direct effect oncapping of the pre-mRNAs. Capping is targeted to nascentRNAs through binding of the guanyltransferase to the phos-phorylated CTD. Guanyltransferase binds CTD peptides con-taining phosphate groups at either serine 2 or serine 5. Inter-estingly, it has recently been reported that binding ofguanyltransferase to CTDs containing a phosphorylated serine5 specifically stimulates enzymatic activity by enhancing theaffinity for GTP and increasing the yield of enzyme-GMP in-termediate (23). A CTD containing phosphorylated serine 2has no effect on enzymatic activity. It will be of importance todetermine if the Tat-directed P-TEFb phosphorylation atserine 5 contributes to the capping of the viral pre-mRNA.Several studies have previously shown that Tat enhances thetranslation of mRNAs synthesized from the HIV-1 LTR (10,33, 78). As capping is known to markedly increase the effi-ciency of translation of mRNAs, it will be interesting to deter-mine whether Tat enhances recruitment of capping enzymes tothe hyperphosphorylated CTD.

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