Proc. Natl. Acad. Sci. USAVol. 93, pp. 11575-11579, October 1996Biochemistry

Trans-activation by human immunodeficiency virus Tat proteinrequires the C-terminal domain of RNA polymerase IIHIROSHI OKAMOTO*, CHRISTIAN T. SHELINEt, JEFFRY L. CORDENt, KATHERINE A. JONESt,AND B. MATIJA PETERLIN*§*Howard Hughes Medical Institute, Departments of Medicine, Microbiology, and Immunology, University of California, San Francisco, CA 94143-0724;tThe Salk Institute for Biological Studies, La Jolla, CA 92037; and *Department of Molecular Biology and Genetics, Johns Hopkins University Schoolof Medicine, Baltimore, MD 21210

Communicated by Michael J. Chamberlin, University of California, Berkeley, CA, August 9, 1996 (received for review May 2, 1996)

ABSTRACT Human immunodeficiency virus (HIV)-encoded trans-activator (Tat) acts through the trans-activation response element RNA stem-loop to increase greatlythe processivity of RNA polymerase II. Without Tat, tran-scription originating from the HIV promoter is attenuated. Inthis study, we demonstrate that transcriptional activation byTat in vivo and in vitro requires the C-terminal domain (CTD)ofRNA polymerase II. In contrast, the CTD is not required forbasal transcription and for the formation of short, attenuatedtranscripts. Thus, trans-activation by Tat resembles enhanc-er-dependent activation of transcription. These results sug-gest that effects of Tat on the processivity ofRNA polymeraseII require proteins that are associated with the CTD and mayresult in the phosphorylation of the CTD.

Trans-activator (Tat) of primate and equine lentiviruses is aunique RNA-binding transcriptional activator (1). Through itsinteractions with trans-activation response element (TAR), ahairpin structure that forms in the 5'-untranslated region of allviral RNAs, Tat increases rates of elongation by RNA poly-merase 11 (2, 3). Although its mechanism of action is stillunknown, several studies have suggested direct roles for RNApolymerase II, general transcription factors, and various tran-scriptional co-activators in Tat trans-activation (4-7). Atpresent, it is not known whether effects of Tat resembleenhancer-dependent regulation of transcription in their re-quirement for a multicomponent RNA polymerase II complex.The large subunit of RNA polymerase II contains a regu-

latory C-terminal domain (CTD), which consists of multipleheptapeptide repeats of the sequence YSPTSPS, which is thetarget for phosphorylation by cellular kinases (8, 9). Theunphosphorylated form of RNA polymerase II, designatedIIA, is exclusively found in preinitiation complexes, whereasthe highly phosphorylated form of RNA polymerase II, des-ignated IIO, is found in elongating transcription complexes (8,9). It has been suggested that phosphorylation of the CTDcontributes directly to promoter clearance and the processivityof RNA polymerase 11 (10-12). In addition, the CTD isrequired for the binding of suppressors of RNA polymerase IIto form the RNA polymerase II holoenzyme complex that iscritical for enhancer activity in vivo (13, 14). In this study, usingdifferent forms of RNA polymerase II that contained variousnumbers of the heptapeptide repeats, we studied the require-ment of the CTD for trans-activation by Tat of human immu-nodeficiency virus (HIV) type 1 (HIV-1) and for the produc-tion of short, attenuated transcripts that are transcribed in theabsence of Tat.

(GIBCO/BRL) using 5 ,ug of reporter DNA, 5 ,ug ofpSVTATor pSVTATZX, and 10 gg of the indicated RP011215-CTDplasmids for 5 hr. After an additional 10 hr required for theexpression of encoded proteins, a-amanitin was added to themedium (2.5 ,ug/ml) and the cells were incubated for anadditional 48 hr (14). Trypan blue staining revealed no in-creased mortality of cells transfected with the c-amanitin-resistant RNA polymerases II containing only 31 or 5 CTDrepeats (A31 and A5) compared with the wild-type RNApolymerase II after 60 hr of a-amanitin treatment. RNA forRNase protection experiments was prepared from the cyto-plasmic fractions of transfected cells as described previously(15).RNase Protection Assay. Twenty micrograms of RNA were

used for the RNase protection assay. To make rabbit 3-globinor HIV long terminal repeat (LTR) probe, Sp6,BTS orpGEMI/WT vector was linearized with EcoRI and transcribedwith SP6 RNA polymerase or T7 RNA polymerase to produce[a-32P]UTP-labeled RNA probe, respectively (15, 16). Theseassays were performed with the Guardian RNase protectionkit (Clontech). Protected fragments were separated on 6% or11% polyacrylamide/urea sequencing gels and exposed tox-ray film. Bands were quantified by the Image Analysis system(Alpha Innotech, San Leandro, CA). Background intensity wassubtracted from the intensity of each band and relative inten-sity was calculated as the percentage of the intensity of eachband relative to the most intense band in the lane on the gel.Western Blots. Samples (20 ,ug) of whole-cell lysates were

analyzed by Western blot analysis after SDS/15% polyacryl-amide gel electrophoresis, electroblotted onto Immobilon-Pmembranes (Millipore) and probed with the a-hemagglutininantibody 12CA5 (Boehringer Mannheim). Immune complexeswere visualized with enhanced chemiluminescence (Amer-sham).

In Vitro Transcription. In vitro transcription reactions werecarried out using the HIV-1 promoter in the presence orabsence of Tat, as described previously by Sheline et al. (17).Endogenous RNA polymerase II activity was inhibited bypreincubating 50 jig of HeLa nuclear extract with 1.7 ,ug ofmAb 8WAG16 in TM/0.1 M KCl for 10 min at 4°C (TM = 50mM Tris-HCl, pH 7.9/12.5 mM MgCl2/1 mM EDTA/20%glycerol/0.1 M phenylmethylsulfonyl fluoride/i mM DTT).Reactions were supplemented with 1.4 ,ug (10 milliunits) ofRNA polymeriase IIA or RNA polymerase IIB, and incubated10 min at 30°C prior to the addition of the DNA template,purified Tat protein (100 ng per reaction), and nucleotides.

MATERIALS AND METHODSCell Culture and Transfection. COS cells were maintained

in 10-cm dishes. They were transfected with Lipofectin

Abbreviations: HIV, human immunodeficiency virus; LTR, long ter-minal repeat; TAR, trans-activation response element; CTD, C-terminal domain of RNA polymerase II; Tat, trans-activator; SV40,simian virus 40; HIV3glo, the HIV-1 LTR linked to the ,B-globin gene.§To whom reprint requests should be addressed at: Howard HughesMedical Institute, University of California, U-426, 3rd and ParnassusAvenues, San Francisco, CA 94143-0724. e-mail: matija@itsa.ucsf.edu.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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The RNA polymerase IIB fraction had been preincubated witheither 1.4 or 2.3 ,ug of mAb 8WG16 for 10 min at 4°C, asindicated in the legend of Fig. 5. Purified RNA polymerase IIAand RNA polymerase IIB were generously provided by NancyThompson and William Burgess (University of Wisconsin,Madison). The RNA polymerase IIA fraction was purifiedfrom calf thymus using immunoaffinity chromatography withmAb 8WG16, as described by Thompson et al. (18) andmodified by Buermeyer et al. (19). RNA polymerase IIB waspurified from calf thymus extracts following the protocoldescribed by Hodo and Blatti (20).

RESULTSThe CTD of RNA Polymerase II Is Required for Tat

Trans-Activation in Cells. The effect of the CTD on Tattrans-activation in vivo was tested by expressing differenta-amanitin-resistant forms of RNA polymerase II, whichcontained various numbers of heptapeptide repeats (HA-WT,HA-A31, and HA-A5, encoding 52, 31, or 5 heptapeptiderepeats, respectively, Fig. 1A), in COS cells. HIV promoteractivity was monitored with a reporter plasmid in which theHIV-1 LTR was linked to the 13-globin gene (HIVf3glo, Fig.1B), and HIVf3glo transcription was measured by an RNaseprotection assay with an RNA probe complementary to therabbit f3-globin gene. As presented in Fig. 2, the wild-typeRNA polymerase II (with 52 heptapeptide repeats) and the

A. Plasmid effectors

pSVTAT

pSVTATZX TATZX

CTD

HA-WT (52) CR _

HA-A31 (31) KA-R iI E3...

HA-A5 (5) aA-R

B. Plasmid reporters

4XSpl

SV40

4XSpl

SV40

33TElHIVI LTR A TAR

pHIV,glo _

FIG. 1. Plasmid effectors and reporters used in this study. (A)Expression of wild-type and mutant Tat was directed by pSVTAT andpSVTATZX, respectively. pSVTATZX contained a frame-shift mu-

tation at the 5' end of Tat (2). HA-WT(52), HA-A(31), and HA-A(5)directed the expression of a-amanitin-resistant RNA polymerase II,whose CTD contained 52, 31, and 5 heptapeptide repeats, respectively(14). aA-R, a-amanitin-resistant; pA, poly(A) site. (B) Plasmid re-

porters contained only promoter elements (4XSpl), the simian virus40 (SV40) 72-bp repeat, which is a strong transcriptional enhancer, or

the HIV-1 LTR, which responds to Tat (pHIV,3glo) linked to thefull-length ,B-globin gene (14). T, TATA box; EN, enhancer; Pro,promoter; pA, poly(A) site.

52 31 5 N length ofCTD

4-(-

... .......

1 2 3 4

Relative Intensity

100 70 2 0o100 70 70

l l .03 1nd1 2 3 4

HIV-globin(A)

52 31 5 N |noTat

--X Zs. TAT

1 2 3 4 15-( globin

(B)

HIV-globinA -globin ntB

A/BRNA probe -Q

protectedRNAs

216193179

FIG. 2. Tat trans-activation in cells requires the CTD of RNApolymerase II. Increased levels of ,B-globin transcripts from the HIV-1LTR trans-activated by Tat require the CTD. RNase protection assayswere performed with the rabbit f3-globin probe depicted in LowerRightbelow the diagram of the generic reporter plasmid. COS cells ex-pressed the indicated RNA polymerases II, plasmid effectors pS-VTAT, pHIV,Bglo, and 4XSpl. Cells were treated as described inMaterials and Methods. Lane 1, the wild-type RNA polymerase II, 52;lane 2, a CTD with 31 heptapeptide repeats, 31; lane 3, a CTD with5 heptapeptide repeats; and lane 4, no a-amanitin-resistant RNApolymerase II. Relative Intensity of bands [HIV-,Bglo reporter (A) and4XSpl reporter (B)] and their ratios (A/B) are given below theautoradiogram. Twenty micrograms of RNA were analyzed by theRNase protection assay. The expression of Tat proteins in the presenceof the different RNA polymerases II was analyzed by using theanti-HA antibody 12CA5 and Western blot analysis. The doublet ofTat resulted from posttranslational modification of Tat in its Cterminus (21). Arrows indicate sizes of protected RNA species. Theprobe was 216 nucleotides long and hybridized to transcripts of 193nucleotides (HIV,Bglo, designated as HIV-globin) and transcripts of179 nucleotides (4XSpl, designated as ,3-globin). pA, poly(A) site.

truncated CTD possessing 31 heptapeptide repeats both sup-ported Tat-activated transcription (Fig. 2 Left, lanes 1 and 2).In sharp contrast, RNA polymerase II carrying only 5 hep-tapeptide repeats dramatically abolished effects of Tat (Fig. 2Left, lane 3). As reported previously, transcription from 4XSpl(Fig. 1B) was unaffected by the truncation of the CTD (Fig. 2Left) (14). Additionally, Tat had no effect on transcriptionfrom 4XSpl. Whereas the ratio of relative intensities ofHIV-globin to the internal control of the sample with thewild-type RNA polymerase II or RNA polymerase II with 31heptapeptide repeats was 1, that with the RNA polymerase IIwith 5 heptapeptide repeats was only 0.03. Equivalent amountsof Tat protein were expressed in these cells (Fig. 2 UpperRight). We conclude that transcriptional activation by Tatrequires the CTD of RNA polymerase II in vivo.

Basal Transcription from the HIV-1 LTR Does Not Requirethe CTD of RNA Polymerase II in Cells. We next tested therequirement of the CTD for the basal transcription in theabsence of Tat from the HIV-1 LTR in vivo. The wild-typeRNA polymerase II and the truncated CTD possessing 31heptapeptide repeats supported basal and Tat-activated tran-scription (Fig. 3). As presented in Fig. 2, RNA polymerase IIcarrying only 5 heptapeptide repeats dramatically abolishedeffects of Tat (Fig. 3 Upper Left, lane 3) but did not affect basallevels of transcription observed in the absence of Tat (Fig. 3Upper Right, lane 7). This result mirrored the activity of theSV40 enhancer, which also required the CTD (Fig. 1B, SV40;Fig. 3 Lower Left, lane 3). However, the activity of 4XSpl wasnot affected by the truncation of the CTD (Fig. 3 Lower Right)(14). Faint bands shown in lane 4 or lane 8 of Fig. 3 Upper andLower Left panels represent transcripts that have been madewithin 10 hr of incubation before the ca-amanitin-containingmedium was added. We conclude that the transcriptional

Proc. Natl. Acad. Sci. USA 93 (1996)

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52 31 5 N length of CTD + + + + ~- - Tat

52 31 5 N 52 31 5 N lengthofCTD

pHIVoglo+

pSVTATZX

LT

> ST

89 100 72 15

1 2 3 4 5 6 7

48 50 3 3 2 3 2

8nd (LT + ST) (

--bi

RelativeIntensity

1 2 3 4

100 100 3 10

5 6 78

93 95 100 0

FIG. 3. Tat trans-activation but not basal transcription from HIV-1LTR requires the CTD of RNA polymerase II. RNase protectionassays were performed with the rabbit ,3-globin probe as in Fig. 2. COScells expressed the indicated RNA polymerases II, plasmid effectorspSVTAT or pSVTATZX and pHIV,Bglo. Cells were treated as de-scribed in Materials and Methods. Lanes 1 and 5, the wild-type CTD,52; lanes 2 and 6, a CTD with 31 heptapeptide repeats, 31; lanes 3 and7, a CTD with 5 heptapeptide repeats; and lanes 4 and 8, noa-amanitin-resistant RNA polymerase II. As controls, COS cellsexpressed the same RNA polymerases and SV40 or 4XSpl. Twentymicrograms of RNA were analyzed by RNase protection assay.

effects of Tat but not basal transcription from the HIV-1 LTRrequires the CTD of RNA polymerase II in vivo. Thus, Tattrans-activation resembles the activity of transcriptional en-

hancers.Promoter-Proximal (Attenuated) Transcription from the

HIV-1 LTR Is Also Independent of the CTD of RNA Polymer-ase II. Whereas most viral transcripts terminate in the 5' LTRand result in the accumulation of short, nonpolyadenylylatedtranscripts of 55-59 nucleotides in the absence of Tat, more

than 99% of transcripts are full-length in the presence of Tat,indicating that Tat principally affects rates of elongation ratherthan initiation of transcription (2). To examine the possibilitythat the CTD is required only for the formation of long(promoter-distal) transcripts and not for the attenuated (pro-moter-proximal) transcripts, we analyzed short and long tran-scripts from the HIV-1 LTR, using an RNA probe correspond-ing to TAR and U5 sequences. As presented in Fig. 4,full-length transcripts which formed in the presence of Tatwere dramatically reduced with the shortest CTD (Fig. 4, lanes1-4). Ratios of long transcripts to total transcripts [LT/(LT +ST)] were calculated by dividing the intensity of long tran-scripts by the sum of intensities of long and short transcripts.Whereas the percentages of long transcripts with the wild-typeRNA polymerase II and RNA polymerase II with 31 hep-tapeptides repeats were 48 and 50, respectively, that with RNApolymerase II with 5 heptapeptides repeats was only 3. On theother hand, the formation of prematurely terminated tran-scripts was unaffected by the length of the CTD in the presenceor absence of Tat (Fig. 4, lanes 1-8). LT/(LT + ST) of thesebands were almost the same (2 or 3). Similar results were

obtained using a stably transformed HeLa-Tat cell line, whichexpresses Tat constitutively (data not presented). These resultsindicate that whereas promoter-proximal transcription doesnot require the CTD, the activation of transcriptional elonga-tion by Tat is absolutely dependent on the CTD.

+1 +80 nt

0-A 220 RNA probe

protectedRNAs -_-__

80 long transcripts (LT)55-59 short transcripts (ST)

FIG. 4. Promoter-distal transcripts that depend on Tat but notpromotor-proximal transcripts from the HIV-1 LTR require the CTDof RNA polymerase II. COS cells co-expressed different RNA poly-merases II, plasmid effectors pSVTAT or pSVTATZX, andpHIV,Bglo. Lanes are as in Fig. 3. Ratio of long transcripts to totaltranscripts [LT/(LT + ST)] are given below the autoradiogram. RNAprobe and hybridizing species from the HIV-1 LTR are depicted belowthe diagram of pHIVI3glo. The probe was 220 nucleotides long andhybridized to full-length transcripts of 80 nucleotides or prematurelyterminated transcripts of 55-59 nucleotides. pA, poly(A) site.

Tat Trans-Activation in Vitro Also Requires the CTD ofRNAPolymerase II. To assess further the role of the CTD in Tattrans-activation, in vitro transcription reactions were carriedout using HeLa nuclear extracts in which the endogenous RNApolymerase II was inactivated by incubation with anti-CTDantibodies (mAb 8WG16) according to the procedure de-scribed by Buermeyer et al. (19). Reaction mixtures were thensupplemented with purified fractions of wild-type RNA poly-merase IIA or a form ofRNA polymerase IIB lacking the CTD(16, 17). As presented in Fig. 5, the addition of the anti-CTDantibodies reduced both basal and Tat-activated transcription(compare lanes 3 and 4 with lanes 1 and 2). However, theaddition of RNA polymerase IIA fully restored basal andTat-activated transcription (Fig. 5, lanes 5 and 6), whereas theaddition of RNA polymerase IIB (lacking the CTD) partiallyrestored only basal transcription (Fig. 5, compare lane 7 withlanes 1 and 3) and did not promote Tat trans-activation (Fig.5, compare lanes 8 and 4). Control reactions indicated thatRNA polymerase IIA and RNA polymerase IIB were equallycapable of activating basal transcription from the adenovirusmajor late promoter (Fig. 5, compare lanes 11 with 12 and 13),and similar results were obtained using the HIV-1 LTR athigher levels of DNA, where basal levels of transcription can

be readily detected in the absence of Tat (data not presented).These results indicate that Tat trans-activation, but not basalHIV-1 transcription, depends on the CTD. As the CTDrequirement could reflect the need for transcription factorsthat are bound to the CTD (13) or CTD bound kinases, wenoted that neither basal nor Tat-activated transcription was

inhibited by recombinant glutathione S-transferase-CTD pro-tein, indicating that the CTD alone is not sufficient to bindfactors necessary for effects of Tat (P. Wei and K.A.J.,unpublished data). Taking these observations together withour in vivo data, we conclude that the increased processivity of

52 31 5 N

pHlV,glo+

pSVTAT

RelativeIntensity

100 100 5 10

SV40 f* 4XSpl

Biochemistry: Okamoto et al.

..........

R

R: -A ........

................

1:'. 1:;z.f:I:IM-,

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11578 Biochemistry: Okamoto et al.

Pol IIA Pol IIBNone a-CTD a-CTD a-CTD

i i

V

:F.'

Tat: - + - + - + - +1 2 3 4 5 6 7 8

,;lec c IC

AdMLP-_ 41 41

9 10 11 12 13

FIG. 5. The CTD of RNA polymerase II is required for Tat trans-activation in vitro. In vitro transcription reactions were carried out with theHIV-1 LTR in the absence or presence of Tat, as indicated below the autoradiogram. Reactions in lanes 1 and 2 displayed basal and Tat-activatedRNA levels, respectively, in the absence of any anti-CTD antibody. Reactions presented in lanes 3-8 were preincubated with 1.7 ,jg of the anti-CTDantibodies to inactivate the endogenous RNA polymerase II. These reaction mistures were then supplemented with either 1.4 ,ug (10 milliunits)of purified RNA polymerase IIA (lanes 5 and 6) or with an equivalent amount of purified RNA polymerase IIB (lanes 7 and 8). The arrow denotesspecific HIV run-off transcripts (480 nucleotides). Whereas the reaction in lane 9 did not contain any anti-CTD antibody, reactions in lanes 10-13were each preincubated with 1.7 jig of the anti-CTD antibody. The reaction in lane 11 was supplemented with 1.4 ,tg of purified RNA polymeraseIIA and lanes 12 and 13 contained 1.4 ,g of RNA polymerase IIB along with additional aliquots of 1.4 and 2.3 ,ug of the anti-CTD antibodies,respectively. The arrow denotes specific adenovirus major late promoter run-off transcripts (540 nucleotides).

RNA polymerase II by Tat requires the CTD of RNA poly-merase II.

DISCUSSIONOur results indicate that the CTD of RNA polymerase II isrequired for effects of Tat but not for promoter-proximal tran-scription from the HIV-1 LTR both in vivo and in vitro. Thus,effects of Tat resemble those of previously described enhancerelements (14). They also reveal that initiation complexes canassemble on the viral promoter and transcribe TAR in theabsence of the CTD. Thus, the RNA polymerase II with aseverely truncated CTD is still capable of significant transcriptionon the HIV-1 LTR and the adenovirus major late promoter.The CTD is a unique feature of RNA polymerase II, and it

is not present in RNA polymerases I or III or the bacterial orviral RNA polymerases, although its function has not beenelucidated completely (8, 9). The CTD has been proposed tostabilize preinitiation complexes by interacting with generaltranscription factors, including the TATA-binding protein, andit is also required for the association of suppressors of RNApolymerase II, general transcription factors, and other proteinsin the RNA polymerase II holoenzyme complex (22, 23).Recently, we observed that Tat is part the RNA polymerase IIholoenzyme and that immobilized Tat can purify transcrip-tional complexes, which with addition of transcription factorIIB and TATA-binding protein support Tat trans-activation onthe wild-type but not mutant TAR templates (T. P. Cujec andB.M.P., unpublished data).

Because the formation of preinitiation complexes requiresthe unphosphorylated CTD, whereas the CTD in elongatingcomplexes is extensively phosphorylated, the CTD has alsobeen reported to play a role in regulating the transition frominitiation to elongation of transcription (8-12). Thus, our datasuggest that Tat might affect transcriptional elongation byinfluencing the state of phosphorylation of the CTD. Indeed,Rice and colleagues have detected a Tat-associated kinase thatis capable of phosphorylating the CTD in vitro, which mightplay a role in Tat trans-activation (24). An attractive modelcompatible with these data is that a single-step assembly of Tatand RNA polymerase II holoenzyme is followed by theactivation of Tat at TAR leading to the phosphorylation of theCTD and subsequent transcriptional elongation. Additionally,Tat might remain associated with the elongating RNA poly-

merase II, and this association is also CTD dependent (25).However, the determination of the precise role of the CTD inTat trans-activation must await the identification of relevanttranscriptional co-activators of Tat.

We thank Nancy Thompson and Lee Strasheim of the Burgesslaboratory for the RNA polymerase IIA and IIB fractions that wereused in this study. We are also grateful to Michael Armanini forsecretarial assistance and to members of the Peterlin laboratory fortheir comments on the manuscript. This work was supported in part bya grant from the National Institutes of Health (AI33824). H.O. is arecipient of a fellowship from the Japanese Foundation for AIDSPrevention.

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