MOLECULAR AND CELLULAR BIOLOGY, Aug. 1988, p. 3175-3182 Vol. 8, No. 80270-7306/88/083175-08$02.00/0Copyright © 1988, American Society for Microbiology

An RNA Polymerase II Transcription Factor Inactivated inPoliovirus-Infected Cells Copurifies with Transcription Factor TFIID

STEVEN KLIEWER AND ASIM DASGUPTA*Department of Microbiology and Immunology and Jonsson Comprehensive Cancer Center, School of Medicine,and The Molecular Biology Institlute, University of California, Los Angeles, Los Angeles, California 90024-1747

Received 3 March 1988/Accepted 11 May 1988

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro.Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficientin a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidencesuggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required tospecifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID throughthree chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derivedchromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fractionprepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infectedcells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extractswere shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation ofTFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

Infection of mammalian cells with picornaviruses resultsin the inhibition of host cell RNA synthesis (2, 3, 17).Infection of HeLa cells with poliovirus, for example, causesa dramatic decrease in total cellular transcription within 3 hof infection (7, 16, 29). Transcription mediated by each of thethree polymerase systems (RNA polymerases [pol] I, II, andIII) is affected.

Early attempts to elucidate the mechanism of picorna-virus-induced inhibition of host cell transcription focused onthe polymerases. RNA pol I, II, and III solubilized frominfected-cell extracts, however, were shown to be fullyactive when assayed under conditions requiring only non-specific initiation (27). Furthermore, no differences wereobserved in the chromatographic properties of partiallypurified RNA polymerases prepared from infected and unin-fected cells (1, 27). Finally, two-dimensional gel electropho-resis revealed no differences in the subunit structure ofRNApol II isolated from infected and uninfected cells (1). Theseresults suggested that a transcriptional component otherthan the elongating polymerase was inactivated by picorna-virus infection.Crawford et al. first showed that the poliovirus-induced

inhibition of transcription observed in vivo could be studiedin vitro (7). In contrast to mock-infected cell extracts,poliovirus-infected cell extracts prepared 3 h postinfectionwere unable to support RNA pol II-mediated transcriptionfrom the adenovirus type 2 (Ad2) major late promoter(MLP). Addition of purified RNA pol II to poliovirus-infected cell extracts failed to restore transcription. How-ever, pol II transcription in infected-cell extracts was re-stored by the addition of an S100 extract containingtranscription factors. When the S100 extract was fraction-ated by chromatography on phosphocellulose, the restoringactivity eluted between 0.35 and 1.0 M KCI. These resultssuggested that at least one transcription factor required forspecific transcription by RNA pol II was deficient in polio-virus-infected cell extracts.The promoters recognized by RNA pol II, termed class II

* Corresponding author.

promoters, contain several DNA sequence elements up-stream of the transcription start site (12). An A+T-richsequence element, designated the TATA box, is usuallycentered between positions -25 and -30 relative to the startsite of transcription. The TATA box, in conjunction withsequences near the CAP site, appears to position the tran-scriptional start site and is required for minimal promoteractivity. Sequence elements located farther upstream arerequired for optimal expression of particular genes.

Purified RNA pol II must be supplemented with factorspresent in crude cellular extracts in order to recognize andaccurately initiate transcription from class II promoters invitro. Chromatographic fractionation of HeLa cell extractshas shown that at least four transcription factors, designatedTFIIA, -B, -E, and -D (nomenclature of Roeder and co-workers [11, 20]), are minimally required in addition to RNApol II for specific transcription from the Ad2 MLP in areconstituted system (11, 20, 22-25). Incubation of templatecontaining the Ad2 MLP with the four fractions and RNApol II results in the formation of a highly stable preinitiationcomplex capable of rapidly initiating transcription in thepresence of ribonucleoside triphosphates (13, 22, 23). Thereis evidence that TFIID interacts specifically with the TATAbox and surrounding sequences (26). An additional MLP-specific transcription factor, designated USF/MLTF, hasbeen shown to interact with sequences upstream of theTATA box and to stimulate transcription from the Ad2 MLP10- to 20-fold (5, 6, 26).

In this report we show that extracts prepared from polio-virus-infected HeLa cells are deficient in an RNA pol IItranscription factor required for specific transcription fromthe Ad2 MLP. With an in vitro transcription assay reconsti-tuted with phosphocellulose-derived fractions, poliovirus-infected cell extracts are shown to be deficient in an activityeluting in the TFIID-containing fraction. Furthermore,TFIID and the activity required to specifically restore tran-scription in poliovirus-infected cell extracts are shown tocopurify through three columns and to have the samekinetics of heat inactivation.

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3176 KLIEWER AND DASGUPTA

MATERIALS AND METHODS

Cells and viruses. HeLa cells were grown in Spinnerculture in SMEM (Gibco Laboratories) supplemented with6% newborn calf serum and 1 g of glucose per liter. Cellswere infected as described previously (9) with poliovirustype 1 (Mahoney strain) at a multiplicity of infection of 40.After suspension in SMEM without serum, mock-infectedcells were treated identically to poliovirus-infected cells.

Preparation of extracts and fractionation on phosphocellu-lose. All procedures were performed at 4°C. Whole-cellextracts were prepared from mock- and poliovirus-infectedHeLa cells essentially as described by Manley et al. (19),except that all buffers contained 0.5 mM phenylmethylsulfo-nyl fluoride. Approximately 20 ml each of mock- and polio-virus-infected whole-cell extracts (20 mg of protein per ml)were fractionated by chromatography on phosphocellulose(Whatman P11, loaded at 10 mg of protein per ml of packedresin) as described in Dignam et al. (11). Flowthrough (at 0.1M) and 0.3, 0.5, and 1.0 M KCl step fractions were collectedand designated fractions A, B, C, and D, respectively. Theprotein concentration of each fraction was typically 4.0 mg/ml for fraction A, 4.2 mg/ml for fraction B, 2.5 mg/ml forfraction C, and 1.8 mg/ml for fraction D. The B, C, and Dfractions were dialyzed against buffer D (20 mM HEPES[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH7.9], 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol,0.5 mM phenylmethylsulfonyl fluoride) to a salt concentra-tion of 0.1 M KCl.

Purification of TFIID from uninfected cells. The scheme forpurification of TFIID was based on the work of Reinberg etal. (22). Whole-cell extracts and the derived 1.0 M KCIphosphocellulose fraction were prepared from uninfectedHeLa cells as described above. The 1.0 M KCl fractionprepared from approximately 30 ml of whole-cell extract (35mg of protein per ml) was dialyzed against buffer D to a saltconcentration of 100 mM KCl and loaded onto a DEAE-Sephacel column (5 mg protein per ml of resin) equilibratedwith buffer D. The column was washed with 2 columnvolumes of buffer D, and the proteins were eluted with alinear gradient (4 column volumes) of KCl (0.1 to 0.5 M) inbuffer D. Aliquots were collected and assayed for TFIIDactivity in combination with phosphocellulose-derived A andC fractions. Active fractions (eluting between 0.19 and 0.24M KCl) were pooled, dialyzed against buffer D containing 50mM KCl, and loaded onto a heparin-Sepharose column (2mg of protein per ml of resin) equilibrated with buffer Dcontaining 50 mM KCl. The column was washed with 2column volumes of 50 mM KCl in buffer D, and proteinswere eluted with a linear gradient (4 column volumes) of KCI(0.05 to 0.6 M) in buffer D. Aliquots were collected andassayed for TFIID activity. Active fractions (eluting be-tween 0.27 and 0.33 M KCl) were pooled, concentrated witha microconcentrator (Amicon), dialyzed to a salt concentra-tion of 0.1 M KCl, and stored at -70°C.

Pol II purification and nonspecific pol II assay. RNA pol IIwas purified from calf thymus as described previously (14).Phosphocellulose column-purified enzyme was used in allexperiments. Nonspecific pol II activity was assayed asdescribed previously (14). This assay depends on the abilityof pol II to elongate a small RNA primer, oligo(U), hybrid-ized to a single-stranded homopolymeric DNA template,poly(dA).Template DNA. The circular template DNAs pML

(C2AT)19 and p(C2AT)19 were a generous gift from M.Sawadogo and R. G. Roeder, Rockefeller University. pML

(C2AT)19 was constructed by Sawadogo and Roeder (25) bycloning the Ad2 MLP (nucleotides -404 to + 10 of the MLP)directly upstream of a synthetic 380-base-pair (bp) DNAfragment lacking C residues in the transcribed strand. Thistemplate generates a transcript with no G residues. In vitrotranscription reactions with either circular or linearizedtemplate are performed in the absence of GTP or, if GTP ispresent, in the presence of RNase T1 and the chain termina-tor 3'-O-methyl-GTP. Under these conditions, the onlyproduct that can accumulate is the approximately 400-bp,RNase T1-resistant transcript resulting from specific initia-tion at the MLP. The control plasmid p(C2AT)19 lacks onlythe MLP sequences of pML(C2AT)19.

In vitro transcription assays. Transcription reaction mix-tures (25 p1I) contained 20 mM HEPES (pH 8.4), 60 mM KCI,5 mM dithiothreitol, 12% glycerol, 5% polyethylene glycol8000, 7.5 mM MgCl2, 20 mM (NH4)2SO4, 0.6 mM each ATPand CTP, 0.025 mM UTP, 6 p.Ci of [at-32P]UTP (Amersham;3,000 Ci/mmol), 0.1 mM 3'-O-methyl-GTP (Pharmacia), 20 Uof RNase T1 (Boehringer Mannheim), 0.4 pLg of circulartemplate DNA, and protein (either whole-cell extract orfractions A, B, C, and D) as described in the figure legends.Reactions were terminated after 45 min of incubation at 30°Cby addition of 175 p.l of stop buffer (7 M urea, 0.5% sodiumdodecyl sulfate, 10 mM EDTA, 100 mM LiCl, 10 mM Tris,pH 7.9) and extracted with phenol-chloroform. The RNAwas ethanol precipitated, suspended in 15 pL1 of loadingbuffer (80% formamide, 0.01% xylene cyanol, 0.01% bromo-phenol blue in 1x TBE [89 mM Tris borate, 89 mM boricacid, 2 mM EDTA]), and loaded onto an 8% acrylamide-8 Murea gel. Full-length transcripts were quantitated by liquidscintillation counting of excised gel slices in Filtron-X (Na-tional Diagnostics).

RESULTS

Inhibition of transcription by poliovirus. In order to inves-tigate the inhibition ofRNA pol TI-mediated transcription bypoliovirus, whole-cell extracts were prepared from mock-and virus-infected HeLa cells at 3 h post-infection. SpecificRNA pol II activity was assayed by addition of a DNAtemplate containing the Ad2 MLP. As discussed in Materialsand Methods, specific transcription from this template gen-erates an approximately 400-nucleotide-long transcript. La-beled RNA product was resolved by 8% polyacrylamide-8M urea gel electrophoresis and visualized by autoradiogra-phy.

Transcription reactions performed with poliovirus-in-fected cell extracts prepared 3 h postinfection were inhibitedapproximately fourfold relative to reactions performed inmock-infected cell extracts (Fig. 1, compare lanes 2 and 3).Synthesis of the 400-nucleotide-long specific initiation prod-uct was sensitive to concentrations of a-amanitin (1.0 ,ug/ml)required to specifically inhibit pol II transcription (Fig. 1,lane 5). No specific transcription was detectable when thetemplate p(C2AT)19, lacking the Ad2 MLP, was substitutedfor pML(C,AT)19 (Fig. 1, lane 4). In vitro transcriptionreactions performed with virus-infected cell extracts consis-tently produced a distinct, high-molecular-weight RNA (ar-rowhead in Fig. 1) that was not detected in reactionsperformed with mock-infected cell extracts. Synthesis of thisproduct did not require the presence of template containingthe Ad2 MLP in the reaction mix (Fig. 1, lane 6) and was notaffected by actinomycin D (5 p.g/ml) (Fig. 1, lane 7). Theproduct was also observed in runoff transcription assaysperformed with linearized pML(C2AT)19 (data not shown).

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INHIBITION OF TRANSCRIPTION BY POLIOVIRUS 3177

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infected-cell extracts. In vitro transcription reactions from the Ad2MLP were performed with HeLa whole-cell extracts (50 ,ug) pre-pared 3 h postinfection (p) or post-mock infection (m). Lanes: 2,mock-infected-cell extract; 3, poliovirus-infected-cell extract; 4,mock-infected-cell extract in the presence of template p(C2AT)19,lacking the MLP; 5, mock-infected-cell extract in the presence ofa-amanitin (a-A; 1 ,ug/ml); 6, poliovirus-infected-cell extract in theabsence of template DNA; 7, poliovirus-infected-cell extract in thepresence of actinomycin D (Act. D; 5 ±Lg/ml). Marker DNAsprepared by digesting pBR322 with MspI are shown in lanes 1 and 8.The arrow indicates the position of the correctly initiated in vitrotranscript (approximately 400 nucleotides). The arrowhead indicatesthe position of poliovirus-specific RNA synthesized by infected-cellextracts.

These results suggest that the infected-cell extract wasmaking poliovirus-specific RNA. It is unclear at present whythe virus-specific RNA product was not digested by theRNase T1 present in the transcription reaction mixtures (seeMaterials and Methods). It is possible that the product isformed by the addition of UMP residues to preexisting,largely double-stranded RNA replicative-form molecules. Aprevious report suggested that whole-cell extracts preparedfrom poliovirus-infected cells contain viral replication com-

plexes which are capable of incorporating ribonucleosidetriphosphates to complete synthesis of poliovirus RNAmolecules (7).The results of the following experiments (data not shown)

were consistent with previously published results (7). (i) Theinhibition of transcription was not due to decreased stabilityof the synthesized transcript in virus-infected-cell extractsrelative to that in mock-infected-cell extracts. (ii) Varyingthe concentration of template DNA or ribonucleoside tri-phosphates did not reverse the inhibition of transcription invirus-infected cell extracts. (iii) Mixing mock- and virus-infected whole-cell extracts failed to reveal the presence of atrans-inhibitory activity in the extracts prepared from in-fected cells.

Experiments were performed to determine whether non-

specific pol II elongation activity was impaired in poliovirus-infected cell extracts. No significant differences in pol II

elongation activity could be detected between extracts pre-pared from mock- and poliovirus-infected cells (data notshown).Assay of individual components. The factors required for in

vitro transcription from the Ad2 MLP by pol II can bepartially resolved by chromatography on phosphocellulose(11, 24, 25). In an effort to determine which component(s) of

the pol II transcriptional machinery is inactivated by polio-virus infection, extracts prepared from mock- and poliovi-rus-infected cells at 3 h postinfection or post-mock infectionwere fractionated in parallel on phosphocellulose columns asdescribed in Materials and Methods. Four fractions, termedA (flowthrough at 0.1 M KCI), B (eluate at 0.3 M KCI), C(eluate at 0.5 M KCI), and D (eluate at 1.0 M KCI) wererecovered. Fractions A, C, and D have been shown to berequired to direct accurate transcription from the Ad2 MLPin an in vitro reconstituted system (11, 23). Fraction Acontains the pol II transcription factor TFIIA (11, 20, 22).Fraction C contains at least two pol II transcription factors,termed TFIIB and TFIIE, in addition to RNA poly II (11,23). Fraction D has been shown to contain TFIID, the TATAbox-binding factor (22, 26). The B fraction contains the bulkof the pol II (14, 24). Low levels of B fraction have nosignificant effect on transcription in vitro (due to the pres-ence of pol II in the C fraction), while higher levels of Bfraction inhibit transcription in vitro (24). Finally, the MLP-specific transcription factor USF/MLTF elutes in the A andB fractions (5, 6, 26).The specific activities of fractions A, B, C, and D prepared

from either mock- or virus-infected cells were determined inan in vitro reconstituted transcription system. Each fraction(prepared from either mock- or virus-infected cells) requiredfor specific transcription from the Ad2 MLP was titrated intoan excess of the remaining fractions prepared from mock-infected cells.

Figure 2A shows titrations of fraction A prepared fromeither mock-infected (lanes 2 to 6) or poliovirus-infected(lanes 7 to 11) cells into an excess of fractions B, C, and Dprepared from mock-infected cells. Transcription reactionmixtures containing only fractions B, C, and D resulted in alow level of specific transcription (Fig. 2A, lane 1), indicatingslight contamination with fraction A in one or more of thesefractions. However, the addition of fraction A prepared fromeither mock- or poliovirus-infected cells greatly stimulatedspecific transcription (Fig. 2A, lanes 2 to 11). Quantitation ofthe transcripts revealed no significant reduction in the spe-cific activity of fraction A prepared from virus-infected cellscompared with that of the same fraction prepared frommock-infected cells.As discussed above, specific transcription from the Ad2

MLP was not dependent on added B fraction (Fig. 2B). Thelevel of transcription seen in reaction mixtures containingonly fractions A, C, and D (prepared from mock-infectedcells) did not change significantly in the presence of Bfraction prepared from either mock- or poliovirus-infectedcells (Fig. 2B). Thus, we were unable to determine whetherfraction B activity prepared from infected cells was alteredwith respect to that of the same fraction prepared frommock-infected cells. However, as discussed below, theaddition of fraction B prepared from either mock- or virus-infected cells failed to restore transcription in whole-cellextracts prepared from infected cells (see Fig. 4B, lanes 9 to16).

Figure 3A shows titrations of C fractions prepared frommock- or virus-infected cell extracts into an excess offractions A, B, and D derived from mock-infected cellextracts. C fraction prepared from virus-infected cells wasfound to have the same specific activity as C fractionprepared from mock-infected cells.

Titrations of fraction D prepared from mock- or virus-infected cells into an excess of fractions A, B, and C derivedfrom mock-infected cells are shown in Fig. 3B. Transcriptionreaction mixtures lacking D fraction resulted in a low level of

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FIG. 2. Titrations of fractions A and B prepared from mock-andpoliovirus-infected cells in an in vitro reconstituted system. Frac-tions A, B, C, and D were prepared from mock- and poliovirus-infected cells at 3 h postinfection as described in Materials andMethods. Fractions A and B, prepared from mock- or virus-infectedcells, were titrated into an excess of the other three fractionsprepared from mock-infected cells. Open and solid circles representquantitation of specific transcripts formed with fractions A and Bprepared from mock- and virus-infected cells, respectively. Quanti-tation was performed by scintillation counting of excised gel slices,and the counts were normalized to a value of 100 for the darkestband in each titration. (A) Transcription reaction mixtures contained4.2 ,ug of fraction B, 12.5 iLg of fraction C, and 7.0 iLg of fraction D,all prepared from mock-infected cells. Lanes: 1, no added Afraction; 2 to 6, 0.7, 1.4, 3.2, 5.0, and 6.5 ,ug, respectively, offraction A prepared from mock-infected cells (Amock); 7 to 11, 0.7,1.4, 3.2, 5.0, and 6.5 ,ug, respectively, of fraction A prepared frompoliovirus-infected cells (Apv). (B) Transcription reaction mixturescontained 7.0 ,ug of fraction A, 12.5 ,xg of fraction C. and 7.0 ,ug offraction D, all prepared from mock-infected cells. Lanes: 1, noadded B fraction; 2 to 6, 0.42, 0.84, 1.7, 2.5, and 4.2 ig, respec-tively, of fraction B prepared from mock-infected cells; 7 to 11, 0.42,0.84, 1.7, 2.5, and 4.2 ,ug, respectively, of fraction B prepared frompoliovirus-infected cells.

transcription, indicating slight contamination with fraction Din one or more of the other fractions. However, addition ofD fraction prepared from mock-infected cells stimulatedspecific transcription approximately eightfold (Fig. 3B, lanes2 to 6). Unlike fractions A and C, fraction D prepared frompoliovirus-infected cells had an approximately fourfold-lower specific activity than the same fraction prepared frommock-infected cells (Fig. 3B, lanes 7 to 11). High concentra-tions of D fraction consistently resulted in inhibition oftranscription (Fig. 3B, lanes 5 and 6), as reported previously(24).

Restoration of transcription in infected-cell extracts. In anapproach complementary to the experiments with an in vitroreconstituted system, attempts were made to restore tran-scription in poliovirus-infected-cell extracts prepared 3 hpostinfection with partially purified pol II and transcriptionfactors. Figure 4 shows the effect of adding increasingconcentrations of phosphocellulose-derived fractions A (Fig.4A) and B (Fig. 4B) prepared from either mock- or virus-infected cells to whole-cell extracts derived from mock- orpoliovirus-infected cells. Addition of fraction A preparedfrom mock- or virus-infected cells failed to restore transcrip-tion in infected whole-cell extracts (Fig. 4A, lanes 9 to 16).Likewise, addition of B fraction prepared from mock- or

FIG. 3. Titrations of fractions C and D prepared from mock- andpoliovirus-infected cells in an in vitro reconstituted system. Frac-tions A, B, C, and D were prepared from mock- and poliovirus-infected cells at 3 h postinfection as described in Materials andMethods. Fractions C and D, prepared from mock- or virus-infectedcells, were titrated into an excess of the other three fractionsprepared from mock-infected cells. Open and solid circles representquantitation of specific transcripts formed with fractions C and Dprepared from mock- and virus-infected cells, respectively. Quanti-tation was performed by scintillation counting of excised gel slices,and the counts were normalized to a value of 100 for the darkestband in each titration. (A) Transcription reaction mixtures contained7.0 ig of fraction A, 4.2 p.g of fraction B, and 7.0 Rg of fraction D,all prepared from mock-infected cells. Lanes: 1, no added Cfraction; 2 to 6, 2.5, 5.0, 7.5, 10, and 12.5 jig, respectively, offraction C prepared from mock-infected cells (Cmock); 7 to 11, 2.5,5.0, 7.5, 10, and 12.5 ,ug, respectively, of fraction C prepared frompoliovirus-infected cells (CPv). (B) Transcription reaction mixturescontained 7.0 ,ug of fraction A, 4.2 ,ug of fraction B, and 12.5 p.g offraction C, all prepared from mock-infected cells. Lanes: 1. noadded D fraction: 2 to 6, 2.0, 4.0. 6.0, 8.0, and 10 jig, respectively,of fraction D prepared from mock-infected cells; 7 to 11, 2.0, 4.0,6.0, 8.0, and 10 ,ug, respectively, of fraction D prepared frompoliovirus-infected cells.

virus-infected cells failed to restore transcription in infected-cell extracts (Fig. 4B, lanes 9 to 16). Addition of fractions Aor B prepared from either mock- or virus-infected cells didnot stimulate transcription in mock-infected whole-cell ex-tracts (Fig. 4A and B, lanes 1 to 8). Addition of purified polII also failed to restore transcription in virus-infected cellextracts (data not shown) (7). These results suggest that polII and transcription factors present in the A and B fractionsare not inactivated in poliovirus-infected cells within 3 h ofinfection.

Figure 5 shows the effect of adding increasing concentra-tions of fractions C (Fig. 5A) and D (Fig. SB) prepared fromeither mock- or virus-infected cells to whole-cell extractsderived from mock- or virus-infected cells. Transcription ininfected-cell extracts was stimulated by the addition of Cfraction prepared from either mock- or virus-infected cells(Fig. SA, lanes 9 to 16). C fractions prepared from eithermock- or virus-infected cells were found to be equallycapable of stimulating transcription in a concentration-de-pendent manner when added to mock-infected cell extracts(Fig. SA, compare lane M with lanes 1 to 4 and 5 to 8). Theseresults suggest that (i) transcription factors present in the Cfraction are present in limiting quantities in whole-cell ex-tracts prepared from mock- and poliovirus-infected cells and

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FIG. 4. Fractions A and B do not restore transcription in polio-virus-infected-cell extracts. (A) Increasing concentrations of Afraction prepared from mock-infected (Amock; lanes 1 to 4 and 9 to12) or poliovirus-infected (Apv; lanes 5 to 8 and 13 to 16) cells wereadded to whole-cell extracts (WCE) derived from mock-infected(MOCK) (lanes 1 to 8) or virus-infected (INF.) (lanes 9 to 16) cells.Reactions performed in mock-and virus-infected-cell extracts with-out added fraction A are shown in lanes M and P, respectively.Amounts of fraction A added were: 2.6 ,ug (lanes 1, 5, 9, and 13), 5.2jig (lanes 2, 6, 10, and 14), 7.8 jig (lanes 3, 7, 11, and 15), or 10.4 ,ug(lanes 4, 8, 12, and 16). (B) Increasing concentrations of B fractionprepared from mock-infected (Bmock; lanes 1 to 4 and 9 to 12) orpoliovirus-infected (Bpv; lanes 5 to 8 and 13 to 16) cells were addedto whole-cell extracts prepared from mock-infected (lanes 1 to 8) orvirus-infected (lanes 9 to 16) cells. Amounts of fraction B addedwere: 2.1 ,ug (lanes 1, 5, 9, and 13), 4.2 jig (lanes 2, 6, 10, and 14),6.3 ,ug (lanes 3, 7, 11, and 15), or 8.4 jig (lanes 4, 8, 12, and 16).

(ii) poliovirus-infected cells are not deficient in transcriptionfactors present in the C fraction at 3 h postinfection.

Addition of increasing amounts of fraction D preparedfrom mock-infected cells stimulated transcription signifi-cantly in whole-cell extracts derived from virus-infectedcells (Fig. 5B, compare lane P with lanes 9 to 12). Fraction Dprepared from poliovirus-infected cells, however, failed tostimulate transcription when added to virus-infected whole-cell extracts (Fig. 5B, lanes 13 to 16). D fraction preparedfrom mock- or virus-infected cells failed to stimulate tran-scription when added to mock-infected whole-cell extracts(Fig. 5B, lanes 1 to 8). Together, these results suggest thatthe activity of a transcription factor(s) present in the Dfraction is significantly reduced in poliovirus-infected cellswithin 3 h of infection. We note that addition of increasingamounts of fraction D prepared from virus-infected cells toeither mock- or virus-infected cell extracts (Fig. 5B, lanes 5to 8 and 13 to 16) resulted in a decrease in transcript levels,suggesting that an activity present in fraction D preparedfrom poliovirus-infected cells was interfering with transcrip-tion. We are at present unable to explain these results.

Copurification of TFIID and restoring activity. As men-tioned, the D fraction derived from chromatography onphosphocellulose has been shown to contain the pol IItranscription factor TFIID. However, as the D fractioncontains roughly 2 to 5% of the total protein present in thewhole-cell extract, we wished to determine whether theactivity required to specifically restore transcription in po-liovirus-infected whole-cell extracts (transcription-restoring

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+ +4 44+ + +

FIG. 5. Fraction D but not fraction C specifically restores trail-scription in poliovirus-infected-cell extracts. (A) Increasing concen-trations of C fraction prepared from mock-infected (Cmack; lanes 1 to4 and 9 to 12) or poliovirus-infected (CP,; lanes S to 8 and 13 to 16)cells were added to whole-cell extracts (WCE) derived from mock-infected (MOCK) (lanes 1 to 8) or virus-infected (INF.) (lanes 9 to16) cells. Reactions performed in mock- and virus-infected cellsextracts without added C fraction are shown in lanes M and P,respectively. Amounts of fraction C added were: 2.3 ,ug (lanes 1, 5,9, and 13), 4.6 ,ug (lanes 2, 6, 10, and 14), 6.9 jig (lanes 3, 7, 11, and15), or 9.2 ,ig (lanes 4, 8, 12, and 16). (B) Increasing concentrationsof D fraction prepared from mock-infected (Dmock; lanes 1 to 4 and9 to 12) or poliovirus-infected (Dpv; lanes S to 8 and 13 to 16) cellswere added to whole-cell extracts derived from mock-infected (lanes1 to 8) or virus-infected (lanes 9 to 16) cells. Amounts of fraction Dadded were: 2.1 jig (lanes 1, 5, 9, and 13), 4.2 jg (lanes 2, 6, 10, and14), 6.3 jig (lanes 3, 7, 11, and 15), or 8.4 jig (lanes 4, 8, 12, and 16).

activity) would further copurify with TFIID activity. Bystarting with phosphocellulose-derived D fraction preparedfrom uninfected cells, TFIID activity and transcription-restoring activity were further purified by gradient elutionfrom DEAE-Sephacel. As shown in Fig. 6A, the transcrip-tion-restoring and TFIID activities coeluted precisely fromthe column. Addition of DEAE-purified TFIID to poliovirus-infected whole-cell extracts stimulated transcription signifi-cantly (>4-fold), restoring transcription to levels seen inmock-infected extracts (Fig. 6B, compare lane 1 with lanes 6to 10). In contrast, addition of DEAE-purified TFIID tomock-infected extracts stimulated transcription only slightly(<1.5-fold) (Fig. 6B, lanes 1 to 5).DEAE-purified TFIID activity was further purified by

gradient elution from a heparin-Sepharose column. Frac-tions were assayed for TFIID activity as described above.Although TFIID activity was detectable, it was dilute. Dueto the background level of transcription in reactions per-formed with poliovirus-infected-cell extract, it was difficultto detect transcription-restoring activity in the individualfractions. Thus, the fractions containing TFIID activity werepooled and concentrated. This concentrated TFIID activity(Fig. 7A) was shown to specifically restore transcription inpoliovirus-infected whole-cell extracts (Fig. 7B, lanes 3 to7). Addition of heparin-purified TFIID to mock-infected cellextracts failed to stimulate transcription (Fig. 7B, lanes 1 and2).

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A.RESTORING ACT

2 3 4 5 6 7 8 9 10

FRACTION- 6 7 8 9101i2 13 1415

A. TF lID ACT B RESTOR ING AC T

1 2 3 4 5 1 2 3 4 5 6 7

-0- .- me 4M-O M 4W**t

-_.- 40 _ * _.- ---

TFIID ACT

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EXTRACTr - - -D P 0

TF.ID, O 2 4 A h 2 4 6 8FIG. 6. TFIID and transcription-restoring activity copurify from

DEAE-Sephacel. Phosphocellulose-derived TFIID was further pu-rified by gradient elution from DEAE-Sephacel as described inMaterials and Methods. (A) Aliquots (4 jil) of the column fractionswere assayed for the activity (ACT.) required to restore transcrip-tion in poliovirus-infected cell extracts (50 ,ug) (top); aliquots of thefractions were also assayed for TFIID activity in transcriptionreactions reconstituted with phosphocellulose-derived A (7.0 ,ug)and C (12.5 [ig) fractions (bottom). Fraction numbers are indicated.Arrows indicate the position of the correctly initiated transcript. (B)Increasing amounts of DEAE-purified TFIID were added to whole-cell extracts (50 ,ug) prepared from mock-infected (m) (lanes 1 to 5)or poliovirus-infected (p) (lanes 6 to 10) cells. Amounts of TFIIDadded were 0 ,ug (lanes 1 and 6), 1.2 ,ug (lanes 2 and 7), 2.4 ,ug (lanes3 and 8), 3.6 ,ug (lanes 4 and 9), or 4.8 ,ug (lanes 5 and 10).

Heat lability of TFIID and transcription-restoring activity.Workman and Roeder recently showed that moderate heattreatment of nuclear extracts results in the preferentialinactivation of TFIID (28). The transcriptional activity ofnuclear extracts was abolished by incubation at 47°C for 15min; addition of purified TFIID to the heat-treated extractswas shown to completely restore transcriptional activity.Using DEAE-purified TFIID, we wished to determinewhether the transcription-restoring and TFIID activities hadthe same kinetics of heat inactivation. As shown in Fig. 8,the transcription-restoring and TFIID activities were inacti-vated at the same rate by incubation at 47°C (half-life ofapproximately 1.5 min). The phosphocellulose-derived Aand C fraction activities used in the reconstituted assayswere not significantly affected by incubation for 15 min at47°C (data not shown). These results are consistent with theidea that TFIID is responsible for restoring transcription inpoliovirus-infected-cell extracts.

DISCUSSION

We have investigated the mechanism of inhibition of hostcell RNA pol II-mediated transcription by poliovirus infec-tion. Three lines of evidence presented here suggest thattranscription factor TFIID is deficient in poliovirus-infectedHeLa cells. First, we have shown that the activity requiredto specifically restore transcription from the Ad2 MLP in

EXTRACT

TF IID.Ak'J 0 2 4 6 8

. mf p p P P P

0 8 0 2 4 6 8

FIG. 7. TFIID and transcription-restoring activity copurify fromheparin-Sepharose. DEAE-purified TFIID was further purified bygradient elution from heparin-Sepharose as described in Materialsand Methods. (A) Increasing amounts of heparin-purified TFIIDwere assayed for the ability to complement phosphocellulose-derived A (7 ,ug) and C (12.5 jig) fractions in a reconstitutedtranscription assay. Amounts of TFIID added were 0 ,ug (lane 1), 0.9,ug (lane 2), 1.8 pLg (lane 3), 2.7 jig (lane 4), and 3.6 jig (lane 5). (B)Increasing amounts of heparin-purified TFIID were added to whole-cell extracts (50 ,ug) prepared from mock-infected (m) (lanes 1 and 2)or poliovirus-infected (p) (lanes 3 to 7) cells. Amounts of TFIIDadded were 0 ,ug (lanes 1 and 3), 0.9 pLg (lane 4), 1.8 ,ug (lane 5). 2.7jig (lane 6), and 3.6 jig (lanes 2 and 7). The arrow indicates theposition of the correctly initiated transcript.

virus-infected cell extracts copurifies with TFIID throughthree chromatographic steps. Second, transcription reactionmixtures reconstituted with phosphocellulose-derived chro-matographic fractions revealed a fourfold decrease in thespecific activity of the D fraction (1.0 M KCl eluate) pre-pared from virus-infected cells at 3 h postinfection comparedwith that of the same fraction prepared from mock-infectedcells. The D fraction has been shown to contain TFIID (11,22, 24, 25). We note that this fourfold reduction in thespecific activity of the D fraction parallels the reduction intranscriptional activity seen in whole-cell extracts prepared3 h postinfection. The specific activities of fractions A and C(flowthrough and 0.5 M KCl eluate, respectively) wereunchanged at 3 h postinfection. Finally, we have shown thatpartially purified TFIID and the activity required to specifi-cally restore transcription in poliovirus-infected cell extractshave similar, if not identical, kinetics of heat inactivation.Both activities were rapidly inactivated by incubation at47°C. Fractions containing other transcription factors wererelatively resistant to the same heat treatment. Workmanand Roeder recently showed that TFIID is rapidly andpreferentially inactivated by heat treatment at moderatetemperatures (28). Together, these results are consistentwith the idea that TFIID is inactivated in cells infected withpoliovirus.Crawford et al. first demonstrated that pol II-mediated

transcription from the Ad2 MLP in poliovirus-infected-cellextracts could be restored by the addition of a chromato-graphic fraction eluting from phosphocellulose between 0.35and 1.0 M KCI (7). This fraction, however, contains at leastfour transcription factors (IIB, IIE, IID, and USF/MLTF)required for efficient transcription from the Ad2 MLP invitro. Our results indicate that chromatographic fractionscontaining TFIID (and not IIB, IIE, or USF/MLTF) aresufficient to restore transcription in virus-infected-cell ex-tracts. One or more transcription factors present in thephosphocellulose-derived C fraction (0.5 M KCl eluate)appears to be present in limiting quantities in both the mock-

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INHIBITION OF TRANSCRIPTION BY POLIOVIRUS 3181

RESTORATION

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FIG. 8. TFIID and the transcription-restoring activity have thesame heat lability. (A) Aliquots (30 ,ul) of DEAE-purified TFIIDactivity were incubated at 47°C for the indicated times. Heat-treatedportions were then assayed for TFIID activity in a reconstitutedtranscription system (open circles) and transcription restoring activ-ity was assayed in poliovirus-infected cell extracts (solid circles).Labeled transcripts synthesized in the reactions were quantitated byliquid scintillation counting of excised gel slices. The percentrelative activity for the transcription-restoring activity was calcu-lated after correcting for the background level of transcriptionobserved in reactions performed with virus-infected-cell extractsonly. (B) (Top) Heat-treated, DEAE-purified TFIID (4.8 ,ug) wasadded to whole-cell extracts (50 ,ug) prepared from poliovirus-infected cells (lanes 3 to 8). Reactions performed with mock-(m) andpoliovirus-infected-cell (p) extracts only are shown in lanes 1 and 2,respectively. (Bottom) Aliquots of heat-treated TFIID were assayedfor TFIID activity in transcription reactions mixtures reconstitutedwith phosphocellulose-derived A (7 ,ug) and C (12.5 ,ug) fractions(lanes 2 to 7). No TFIID was added in lane 1.

and virus-infected whole-cell extracts, as addition of Cfraction prepared from mock-infected cells significantlystimulated transcription in both mock- and virus-infectedcell extracts (Fig. 5A). Clearly, the transcription restorationassay alone is not sufficient to distinguish between factorspresent in limiting amounts in cell extracts and those that areinactivated by infection with poliovirus. Only by assayingthe specific activities of fractions C and D prepared frommock- and poliovirus-infected cell extracts in an in vitroreconstituted transcription system were we able to deter-mine that poliovirus specifically inactivated a transcriptionfactor(s) present in the D fraction.

Reinberg et al. recently showed that the phosphocellulose-derived D fraction contains a second pol II transcriptionfactor, designated TFIIX (22). Transcription from the Ad2MLP was shown to be dependent on TFIIX, but only whenviral sequences downstream of the CAP site (+33 to +536)were present. As the template used in our studies[pML(C2AT)19] lacks these downstream viral sequences, itseems unlikely that the inhibition of pol II transcription bypoliovirus is due to a reduction in TFIIX activity.Template competition and order-of-addition experiments

indicate that TFIID is involved at an early step in preinitia-tion complex formation (10, 13, 22). Footprinting analyseswith highly purified TFIID have revealed that the factorinteracts specifically with the TATA box and surroundingsequences of the Ad2 MLP (26). It is interesting to speculatethat infection of cells with poliovirus disrupts the DNA-binding activity of TFIID. Alternatively, poliovirus infectioncould disrupt TFIID function without altering its DNA-binding function. It was recently shown in the RNA pol IIIsystem that poliovirus infection results in the inactivation oftranscription factor TFIIIC without a concomitant reductionin the ability of TFIIIC to bind specifically to internalpromoter sequences of the adenovirus VA gene (15). Deter-mining whether poliovirus infection affects the DNA-bindingactivity of TFIID may prove difficult, however, as we havebeen unable to footprint TFIID onto the Ad2 MLP by usingpartially purified factor. Under similar conditions we wereable to observe a USF/MLTF footprint on the Ad2 MLP;extracts derived from either mock- or virus-infected cellswere equally capable of protecting the upstream binding sitefrom digestion by DNase I (data not shown). A directcomparison of the DNA-binding activity of TFIID isolatedfrom mock- and virus-infected cell extracts may requireextensive purification of the factor from these extracts. Wenote that relatively high concentrations of extensively puri-fied TFIID were required to observe a footprint on the Ad2MLP (26).Three subspecies ofRNA pol II, termed II0, IIA, and IIB,

have been described and shown to differ in the molecularweight of their largest subunit (18). There is evidence thatspecific transcription from class II promoters is catalyzed bythe 110 subspecies (large subunit, Mr 240,000) of the enzyme(4, 8). Rangel et al. recently reported that levels of RNApolymerase II0 were significantly reduced following infec-tion of HeLa cells with poliovirus (21). RNA polymerase 110levels were reduced by approximately 20% at 3 h postinfec-tion and by 75% at 4 h postinfection. Our observations that(i) phosphocellulose-derived D fraction activity is reducedby approximately 75% as early as 3 h postinfection, (ii) theinhibition of pol II transcription in virus-infected whole-cellextracts is closely paralleled by the loss of phosphocelluloseD fraction activity, and (iii) partially purified TFIID specif-ically restores transcription in extracts prepared from cells at3 h postinfection suggest that inactivation of a pol II tran-scription factor precedes the observed reduction in the levelsof the 110 subspecies of pol II. The results of two additionalexperiments further suggest that the observed inhibition oftranscription in poliovirus-infected cell extracts at 3 h post-infection cannot be accounted for by inactivation of RNApol II. First, addition of purified pol II failed to restoretranscription in virus-infected cell extracts (data not shown)(7). Second, mock- and virus-infected whole-cell extractsdisplayed comparable pol II elongation activities in a non-specific pol II assay (data not shown).The minimal conclusion of this study is that at least one

pol II transcription factor, which copurifies with TFIID, isinactivated in cells infected with poliovirus. It is not knownat present whether the loss of TFIID activity reflects adeficiency of the protein per se (e.g., via proteolysis) or achange in the activity state of the protein. Although TFIID islikely to be required for the transcription of all pol IIpromoters containing the TATAA motif, the results pre-sented here are based on transcription reactions performedwith only the Ad2 MLP, and therefore the possibility thatadditional factors required for the efficient transcription ofother pol II promoters are also inactivated by poliovirus

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3182 KLIEWER AND DASGUPTA

infection cannot be ruled out. Future studies will be directedtowards characterizing the factor(s) deficient in poliovirus-infected cells by using the Ad2 MLP as well as other pol II

promoters and elucidating the mechanism of poliovirus-mediated inactivation of pol II transcription factors.

ACKNOWLEDGMENTS

We thank members of the Dasgupta Laboratory for helpfuldiscussions, Steve Yoshinaga for pBR322 Mspl markers, M. Sawa-dogo and R. G. Roeder for the generous gift of templatespML(C2AT)19 and p(C2AT)19, and Richard Gaynor for criticallyreading the manuscript. We are particularly indebted to Lee Fradkinfor advice offered throughout the course of this work and forcomments on the manuscript.

This work was supported by Public Health Service grant AI-18272from the National Institute of Allergy and Infectious Diseases toA.D. A.D. is supported by an American Cancer Society FacultyResearch Award. S.K. was supported by a Public Health ServiceNational Research Service Award (GM-07185).

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10. Davison, B. L., J. M. Egly, E. R. Mulvihill, and P. Chambon.1983. Formation of stable preinitiation complexes betweeneukaryotic class B transcription factors and promoter se-

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14. Fradkin, L. G., K. Leong, C. D. Morrow, A. J. Berk, and A.Dasgupta. 1985. Primer-dependent eukaryotic RNA polymerasecapable of accurate transcription from the adenovirus major latepromoter in a reconstituted system. Proc. Natl. Acad. Sci. USA82:7979-7983.

15. Fradkin, L. G., S. K. Yoshinaga, A. J. Berk, and A. Dasgupta.1987. Inhibition of host cell RNA polymerase IIl-mediatedtranscription by poliovirus: inactivation of specific transcriptionfactors. Mol. Cell. Biol. 7:3880-3887.

16. Holland, J. J., and J. A. Peterson. 1964. Nucleic acid and proteinsynthesis during poliovirus infection of human cells. J. Mol.Biol. 8:556-573.

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20. Matsui, T., J. Segall, P. A. Weil, and R. G. Roeder. 1980.Multiple factors required for accurate initiation of transcriptionby purified RNA polymerase II. J. Biol. Chem. 255:11992-11996.

21. Rangel, L. M., C. Fernandez-Tomas, M. E. Dahmus, and P.Gariglio. 1987. Modification ofRNA polymerase IIO subspeciesafter poliovirus infection. J. Virol. 61:1002-1006.

22. Reinberg, D., M. Horikoshi, and R. G. Roeder. 1987. Factorsinvolved in specific transcription in mammalian RNA polymer-ase lI. Functional analysis of initiation factors IIA and IID andidentification of a new factor operating at sequences down-stream of the initiation site. J. Biol. Chem. 262:3322-3330.

23. Reinberg, D., and R. G. Roeder. 1987. Factors involved inspecific transcription by mammalian RNA polymerase II. Puri-fication and functional analysis of initiation factors IIB and IIE.J. Biol. Chem. 262:3310-3321.

24. Samuels, M., A. Fire, and P. A. Sharp. 1982. Separation andcharacterization of factors mediating accurate transcription byRNA polymerase II. J. Biol. Chem. 257:14419-14427.

25. Sawadogo, M., and R. G. Roeder. 1985. Factors involved inspecific transcription by human RNA polymerase II: analysis bya rapid and quantitative in vitro assay. Proc. Natl. Acad. Sci.USA 82:4394-4398.

26. Sawadogo, M., and R. G. Roeder. 1985. Interaction of a gene-specific transcription factor with the adenovirus major latepromoter upstream of the TATA box region. Cell 43:165-175.

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28. Workman, J. L., and R. G. Roeder. 1987. Binding of transcrip-tion factor TFIID to the major late promoter during in vitronucleosome assembly potentiates subsequent initiation by RNApolymerase II. Cell 51:613-622.

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