Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription

Rajeev Banerjee1, Mary K. Weidman1, Sonia Navarro2, Lucio Comai2 and Asim Dasgupta1

1 Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
2 Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Correspondence
Asim Dasgupta
dasgupta{at}ucla.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3Cpro appeared to cleave the TATA-binding protein-associated factor 110 (TAF110), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF110 and purified 3Cpro indicated that the Q265G266 and Q805G806 sites were cleaved by 3Cpro. Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Poliovirus (PV) is the type species of a large group of medically important viruses (picornaviruses), including those inducing infectious hepatitis (hepatitis A virus), the common cold (rhinoviruses) and encephalitis and myocarditis (coxsackieviruses). The single-stranded, positive-polarity RNA genome (~7500 nt) of PV (Kitamura et al., 1981; Racaniello & Baltimore, 1981) is translated into one large polyprotein, which is processed co-translationally by the virally encoded proteases 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural proteins. The viral proteases have been studied extensively and found to be highly specific in their polyprotein cleavage: 3Cpro and 3CDpro cleave the polyprotein at glutamine–glycine (QG) sites, whilst 2Apro cleaves only at tyrosine–glycine (YG) bonds (Krausslich & Wimmer, 1988). The proteases do not cleave every potential cleavage site within the polyprotein; other determinants, such as accessibility and context of the cleavage site, are also important.

Accurate initiation from the rDNA promoter requires two factors in addition to pol I: the upstream binding factor (UBF) and the selectivity factor (SL-1). UBF (Jantzen et al., 1990), a sequence-specific DNA-binding protein, interacts with two regions of the template rDNA: the upstream control element, located between nt –200 and –170 on the rDNA, and the core promoter element, located between nt +20 and –45 relative to the site of transcription initiation. Human UBF consists of two different polypeptides of 97 and 94 kDa. The 94 kDa form results from a 37 aa deletion due to differential splicing. UBF is known to activate rDNA transcription by recruiting pol I to the rDNA promoter, stabilizing binding of SL-1 and competing with non-specific DNA-binding proteins, such as histone H1 (Kuhn & Grummt, 1992; Kuhn et al., 1993). SL-1, a protein complex containing the TATA-binding protein (TBP) and three pol I-specific TBP-associated factors (TAFs), TAF48, TAF63 and TAF110 (Comai et al., 1992), confers promoter specificity. The TBP subunit of SL-1 does not bind to DNA, and promoter recognition is carried out by the associated TAFs. Both UBF and SL-1 activities are regulated, at least in part, by phosphorylation during the M and G1 phases (Grummt, 2003).

Infection of HeLa cells with PV causes a severe decrease in cellular transcription catalysed by all three cellular RNA polymerases (Dasgupta et al., 2003). Both biochemical and genetic evidence has shown that the viral protease 3Cpro is responsible for pol I transcription shut-off by PV (Weidman et al., 2003). Our studies have also shown that the inhibition of rDNA transcription by PV can be demonstrated in vitro by using mock- and virus-infected cell extracts. By fractionation of mock-infected cell extracts, we were able to show that a partially purified fraction containing UBF and the species-specific SL-1 could restore pol I transcription in virus-infected cell extracts (Rubinstein et al., 1992).

Here, we have demonstrated that purified UBF and SL-1 are both required for complete restoration of pol I transcription in PV-infected extracts. In vitro studies demonstrated that TAF110, but not TAF63 or TAF48, is cleaved at at least at two sites, Q265G266 and Q805G806, by the PV-encoded protease 3Cpro leading to the generation of four products having approximate molecular masses of 95, 65, 26 and 8 kDa. Endogenous UBF and TAF110 in HeLa cells were depleted by 3–4 h post-infection (p.i.). These results suggested that both SL-1 and UBF are targeted by PV to bring about RNA pol I transcription shut-off.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 5 % newborn calf serum (NCS) and 5 % fetal calf serum. Cells were split in a 1 : 10 ratio in fresh medium every 3 days. HeLa suspension cells were grown in minimum essential medium (Sigma) with 5 % NCS at 37 °C with constant stirring. Cells were re-inoculated into fresh medium every 3 days at a ratio of 1 : 5 to 1 : 10. Cells were infected with PV type 1 (Mahoney strain) at an m.o.i. of 20, as described previously (Clark et al., 1991; Rubinstein et al., 1992).

In vitro transcription/translation and protease assays.
In vitro coupled transcription and translation in the presence of [35S]methionine were performed with the TNT Quick Coupled Transcription/Translation system (Promega) as described previously (Yalamanchili et al., 1996). In a 25 µl reaction volume, 20 µl TNT master mix, 10 µCi (370 kBq) [35S]methionine and 1 µg plasmid DNA were added. Reactions were carried out at 30 °C for 90 min. One microlitre of this reaction mixture was digested with 1 µg purified 3Cpro for 3–4 h at 30 °C in a 10 µl reaction volume, as described previously (Yalamanchili et al., 1996). The reaction mixture also contained 20 mM HEPES, 100 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT). Control reactions were incubated for the same time, but without 3Cpro. Products were separated by SDS-PAGE, fixed and detected by fluorography.

Pol I transcription assay.
Specific transcription assays were performed basically as described by Tower et al. (1986). The 25 µl reaction mixture contained a final concentration of 80 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7·9, 1 mM DTT, 0·1 mM EDTA, 500 µM each of ATP, CTP and GTP, 5–10 µCi (185–370 kBq) [{alpha}32P]UTP [specific activity, 3000 Ci (111 TBq) mmol–1], 100 µg {alpha}-amanitin ml–1, 2·5 µg rDNA template pUCHU3 ml–1 (Rubinstein & Dasgupta, 1989) and 50–60 µg mock- or PV-infected transcription extracts. The template was truncated with BamHI so that correct initiation of transcription at the promoter produced a 1500 nt run-off transcript. Reaction mixtures were incubated at 30 °C for 1 h. The reactions were then terminated and RNA was isolated, denatured with glyoxal and DMSO, electrophoresed on 1·4 % agarose gels and autoradiographed as described by Manley et al. (1983).

Plasmid constructs.
The plasmid prHU3 was kindly provided by R. Tjian (Learned & Tjian, 1982). The complete 2·0 kb EcoRI–BamHI insert, which includes the transcription-initiation region of a human rRNA gene, was subcloned into pUC18 between the EcoRI and BamHI sites for greater plasmid yields. The two templates, prHU3 and pUCHU3, were found to be equivalent in directing in vitro transcription. The cDNA clones that encode TAF110, TAF63 and TAF48 have been reported previously (Comai et al., 1992).

Pol I, UBF and SL-1 purification.
Pol I used in the reconstituted transcription reaction was prepared from HeLa cells. Nuclear extracts were loaded on a heparin–agarose column and the proteins were eluted with a salt gradient from 0·1 to 0·7 M KCl. Fractions eluted at 250 mM KCl were pooled, dialysed against TM buffer [50 mM Tris/HCl (pH 7·9), 12·5 mM MgCl2, 1 mM EDTA, 20 % glycerol] containing 0·1 M KCl and fractionated on a Sepharose 300 (Pharmacia Biotech) gel-filtration column. Active fractions were then loaded onto a Q-Sepharose column (Poros HQ) equilibrated with TM buffer containing 0·1 M KCl. Proteins were eluted with a salt gradient from 0·1 to 0·7 M KCl in TM buffer. The active fractions were pooled, dialysed to 0·125 M KCl, aliquotted and stored at –80 °C. This pol I preparation contained no detectable UBF or SL-1 activity. The human UBF and SL-1 proteins were purified as described previously (Bell et al., 1988; Comai et al., 1992; Zhai et al., 1997). The purity of UBF and SL-1 preparations was checked by 10 % SDS-PAGE followed by silver staining.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Both SL-1 and UBF are required to restore rDNA transcription in PV-infected cell extracts
Previous studies from our laboratory have shown that the inhibition of host-cell transcription by RNA polymerases observed in PV-infected HeLa cell extracts could be rescued by partially purified pol I transcription factors (Rubinstein & Dasgupta, 1989; Rubinstein et al., 1992). We used a similar approach to examine whether purified pol I factors could restore transcription from an rDNA template containing a human pol I promoter in PV-infected extracts. Both SL-1 and UBF were purified as described previously (Bell et al., 1988; Comai et al., 1992). Whilst purified UBF showed mainly one major polypeptide at approximately 97 kDa, purified SL-1 consisted of four major polypeptides with approximate molecular masses of 110, 63, 48 and 38 kDa (Fig. 1a, lanes 1 and 2). A polypeptide migrating slightly faster than TAF63 appeared to be a contaminant that co-purified with the SL-1 complex (Fig. 1a, lane 2). The 38 kDa polypeptide co-migrated with purified human TBP (Fig. 1a, lane 3). The purified factors were examined in an in vitro transcription assay (Rubinstein & Dasgupta, 1989). As shown in Fig. 1(b), accurate transcription from the rDNA template containing a human pol I promoter required pol I, SL-1 and UBF (lane 4). Omission of any of the three proteins in the reaction resulted in a dramatic decrease in transcription (Fig. 1b, lanes 1–3). Thus, there was very little, if any, cross-contamination between the three protein preparations. SL-1, but not UBF, was found to be present in limiting quantities in mock-infected extracts, as transcription in mock extracts was stimulated significantly by SL-1 compared with UBF (Fig. 1c, lanes 1–3). Transcription from the human rDNA promoter was stimulated 5·2-fold over the control in mock-infected cell extracts in the presence of both factors (Fig. 1b, lanes 1 and 4). We then examined whether purified SL-1 and UBF could restore pol I transcription from the rDNA template in PV-infected cell extracts. Both SL-1 and UBF-1 stimulated pol I transcription to some extent when added to PV-infected transcription extracts individually; however, neither purified SL-1 nor UBF was able to restore pol I transcription completely in PV-infected extracts (Fig. 1c, lanes 6 and 7). In the presence of both factors, transcription in infected extract was restored almost to the level seen in mock-infected extracts (Fig. 1c, compare lanes 4 and 8). The high-molecular-mass transcript seen in PV-infected extracts is PV RNA, which results from elongation of pre-initiated viral RNA present in the infected extract and is visualized due to incorporation of labelled UMP by the PV RNA-dependent RNA polymerase during the pol I transcription run-off assay (Rubinstein & Dasgupta, 1989). The purified pol I could not restore pol I transcription in PV-infected extracts, suggesting that the transcriptional activity of pol I in HeLa cells was not affected by PV infection (Fig. 1d). These results suggested that transcriptional activity of both SL-1 and UBF was compromised in PV-infected cells.



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Fig. 1. Restoration of pol I transcription in PV-infected cell extracts by SL-1 and UBF. (a) Purification of UBF and SL-1. UBF and SL-1 were purified from HeLa cells as described previously (Comai et al., 1992). Purified UBF (lane 1), SL-1 (lane 2) and TBP (lane 3) were analysed by SDS-PAGE (10 % gel) followed by silver staining. (b) Reconstitution of in vitro transcription by RNA polymerase I. Approximately 75 ng each of purified UBF and SL-1 and 500 ng purified RNA pol I were used in the in vitro transcription assay using the pUCHU3 rDNA template. Transcription was carried out in the presence of all three components (lane 4), pol I and SL-1 (lane 1), pol I and UBF (lane 2) or SL-1 and UBF (lane 3). (c) Transcription from a human rDNA template (pUCHU3) was performed as described in Methods. S100 extract (50 µg) prepared at 4 h p.i. from mock-infected (lanes 1–4) and PV-infected (lanes 5–8) HeLa cells was used in the transcription assay in the absence (lanes 1 and 5) or presence of approximately 75 ng UBF (lanes 2 and 6), 75 ng SL-1 (lanes 3 and 7) or 75 ng each of UBF and SL-1 (lanes 4 and 8). The arrowheads indicate the position of the correctly initiated (1500 nt) in vitro-synthesized transcript. The arrow indicates the position of PV-specific RNA synthesized by infected cell extracts. (d) Transcription from the rDNA template was performed at 4 h p.i. using 100 µg mock-infected (lane 1) or PV-infected (lanes 2 and 3) extracts in the absence (lane 2) or presence (lane 3) of 500 ng purified pol I.

 
SL-1 is cleaved by 3Cpro in vitro
SL-1 is composed of TBP and three TAFs with molecular masses of 105/110, 63 and 48 kDa (Fig. 2a). By using in vitro-translated TAF proteins and purified 3Cpro, we were able to show that TAF110 was cleaved by 3Cpro into four main fragments with approximate molecular masses of 95, 65, 26 and 8 kDa (Fig. 2c, lanes 1 and 2; Fig. 3b, lane 6). A catalytically inactive 3Cpro mutant (Hämmerle et al., 1991) and the PV protease 2Apro did not cleave TAF110, ruling out the possibility that a contaminating protease in the 3Cpro preparation was responsible for TAF110 cleavage (Fig. 2c, lanes 3 and 4). No significant proteolytic cleavage was observed when TAF48 and TAF63 were incubated with either 3Cpro or 2Apro(Fig. 2b, lanes 1–8). These results suggested that PV 3Cpro could cleave TAF110, but not TAF63 or TAF48, in vitro.



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Fig. 2. Cleavage of the TAF110 subunit of SL-1 by 3Cpro in vitro. (a) Schematic representation of the SL-1 complex showing the three pol I TAFs complexed to TBP. (b) In vitro-translated, [35S]methionine-labelled TAF48 (~100 000 c.p.m.) (lanes 1–4) and TAF63 (~100 000 c.p.m.) (lanes 5–8) were incubated in the presence of buffer alone (lanes 1 and 5) or 500 ng each of purified 3Cpro (lanes 2 and 6), 2Apro (lanes 3 and 7) or a catalytically inactive 3Cpro point mutant (lanes 4 and 8). (c) In vitro-translated TAF110 (~50 000 c.p.m.) was incubated with buffer (lane 1) or 500 ng each of 3Cpro (lane 2), 2Apro (lane 3) or 3Cpro mutant (lane 4). Labelled products were analysed by SDS-PAGE followed by autoradiography as described in Methods.

 


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Fig. 3. Mutagenesis of QG cleavage sites in TAF110. (a) The 869 aa TAF110 is shown. The downward arrowheads indicate the potential 3Cpro cleavage sites in TAF110. The numbers above indicate the positions of the QG sites. The 3Cpro-mediated cleavage sites that could generate proteolytic products with approximate molecular masses of 95, 65, 26 and 8 kDa are shown by upward arrows. (b) Kinetics of TAF110 cleavage by 3Cpro. Approximately 75 000 c.p.m. of in vitro-translated, [35S]methionine-labelled TAF110 was incubated with 1 µg purified 3Cpro for 0, 15, 30, 60, 90 or 120 min, as indicated. (c) Mutational analysis of QG cleavage sites in TAF110. Wild-type (wt; lanes 1, 2, 7 and 8) TAF110 and Q510A (lanes 3 and 4), Q737A (lanes 5 and 6), Q265A (lanes 9 and 10), Q277 (lanes 11 and 12), Q805 (lanes 13 and 14) and Q414A (lanes 15 and 16) point mutants and the Q265A plus Q805A double mutant (lanes 17 and 18) were incubated with buffer (odd-numbered lanes) or 3Cpro (even-numbered lanes).

 
To determine whether all of the proteolytic cleavage products of TAF110 were produced at the same time following incubation with 3Cpro or whether the cleavages were sequential, a time-course analysis of TAF110 cleavage by 3Cpro was performed. As shown in Fig. 3(b), there was no significant cleavage of TAF110 after a 15 min incubation with 3Cpro (Fig. 3b, lane 2). After 30 min, two TAF110 cleavage products with molecular masses of 95 and 8 kDa were apparent (Fig. 3b, lane 3). Two additional proteolytic products with molecular masses of 65 and 26 kDa were detected after a 60 min incubation (Fig. 3b, lane 4). The intensities of the 65, 26 and 8 kDa products increased after 90 and 120 min incubation with 3Cpro (Fig. 3b, lanes 5 and 6). The intensity of full-length TAF110 reduced significantly, starting at 60 min, and almost all full-length TAF110 disappeared by 120 min, concomitant with the increased generation of the 65, 26 and 8 kDa proteolytic products. The intensity of the 95 kDa polypeptide did not change significantly at later time points compared with full-length TAF110. Increasing the incubation time to 4 h did not change the intensity of the 3Cpro-digested products significantly compared with the 2 h time point (data not shown).

3Cpro-mediated cleavage at 265 and 805 QG sites of TAF110
Examination of the primary sequence of TAF110 (aa 1–869) revealed that it contained six glutamine–glycine (QG) sites at positions 265, 277, 414, 510, 737 and 805 (Fig. 3a; Comai et al., 1994). Because PV 3Cpro preferentially cleaves glutamine–glycine sites in the viral precursor polypeptides, we wished to determine which QG sites were cleaved by 3Cpro in the in vitro proteolysis assay. The six QG sites were mutated individually by substituting the Q residue with alanine. Previous studies have shown that substitution of the Q residue (of the QG bond) with alanine blocks 3Cpro-mediated cleavage of both viral precursor proteins and transcription factor TBP (Krausslich & Wimmer, 1988; Das & Dasgupta, 1993). Alteration of the QG sites at positions 277, 414, 510 and 737 did not change the TAF110 cleavage pattern by 3Cpro compared with the wt protein, suggesting that these sites were not cleaved by 3Cpro to generate the four polypeptides (Fig. 3c, lanes 1–6, 11, 12, 15 and 16). In contrast, 3Cpro-mediated generation of the 26 kDa product was inhibited completely by the Q265A mutation (Fig. 3c, lanes 9 and 10). Additionally, the intensity of the 65 kDa polypeptide was reduced significantly in the Q265A mutant compared with wt TAF110 (Fig. 3c, compare lanes 8 and 10). Similarly, the Q805A mutation inhibited formation of the 95, 65 and 8 kDa proteolysed products (Fig. 3c, lanes 13 and 14). The intensity of the 26 kDa product was also reduced to some extent in the Q805A mutant (lane 14). The Q805A mutation appeared to change the conformation of TAF110, which apparently induced cleavage at an alternative site, as evidenced by the appearance of two new proteolysis products of 100 and 85 kDa (Fig. 3c, lanes 13 and 14, indicated by dots). Mutating both the Q265 and Q805 sites resulted in total disappearance of the 95, 65, 26 and 8 kDa polypeptides (Fig. 3c, lanes 17 and 18). However, the generation of the two polypeptides of 100 and 85 kDa was still evident in the double mutant, as seen earlier with the Q805A single mutant. These results suggested that proteolytic cleavage by 3Cpro primarily at the QG sites at positions 265 and 805 leads to the generation of the four proteolytic products of TAF110 that are seen in vitro.

Both SL-1 and UBF are modified in PV-infected cells
Cell-free transcription extracts prepared from HeLa cells at 0, 1, 2, 3 and 4 h p.i. were used to transcribe a human rDNA template in a run-off transcription assay. Analysis of labelled transcripts showed that rDNA transcription was not inhibited significantly up to 2 h p.i. (Fig. 4a, lanes 1–3). At 3 h p.i., rDNA transcription was inhibited approximately fourfold compared with the control (lane 4). No pol I transcript was detectable at 4 h p.i. (Fig. 4a, lane 5). To assess whether TAF110 was cleaved in PV-infected cells, transcription extracts prepared from PV-infected cells at 0, 1, 2, 3 and 4 h p.i. were examined by Western blot analysis using a polyclonal antibody against TAF110 (Comai et al., 1992). The antibody recognized a polypeptide migrating at 105/110 kDa and an additional 80 kDa polypeptide at 0 h p.i. (Fig. 4b, lane 1). The 110 kDa polypeptide co-migrated with purified TAF110 in the Western blot (Fig. 4b, compare lanes 1 and 6). Both the 110 and 80 kDa polypeptides disappeared between 3 and 4 h p.i. (Fig. 4b, lanes 4 and 5). Despite repeated attempts, we were unable to detect the TAF110 proteolytic products in PV-infected cells. This suggested that 3Cpro cleavage products were either not recognized by the TAF110 antibody or that the proteolytic products were degraded quickly in infected cells.



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Fig. 4. Kinetics of pol I transcription shut-off correlate with disappearance and modification of TAF110 and UBF in PV-infected cells. (a) HeLa cells were infected with type 1 PV at an m.o.i. of 20 and the infections were stopped at 0, 1, 2, 3 and 4 h p.i. The transcription extracts (60 µg) from various time points were used for transcription of the pUCHU3 template as described in Methods. The arrowhead indicates the position of migration of the 78 kDa TAF110-associated polypeptide. (b) Cell-free extracts (50 µg) prepared from various time points were analysed by Western blotting using anti-TAF110 antibody. Lane 6 (C) is the positive control showing the Western blot of purified TAF110. (c) The same extracts as used in (a) were used to detect UBF in a Western blot using anti-UBF antibody. These samples were run for longer times than in (b) during SDS-PAGE to resolve the two forms of UBF clearly. The arrowheads indicate the position of migration of the cleaved UBF products.

 
Previous results from our laboratory have shown that purified recombinant UBF and in vitro-translated, [35S]methionine-labelled UBF are not cleaved by purified 3Cpro in vitro (Rubinstein et al., 1992), suggesting that UBF might not be a target for PV 3Cpro or that the in vitro-translated UBF was not modified properly. However, our transcription analysis in PV-infected extracts showed that UBF was required for full restoration of pol I transcription in the presence of SL-1 (Fig. 1). We therefore examined whether UBF was being modified by PV infection. The level of UBF was examined by Western blot analysis of the same extracts, as shown in Fig. 4(a). Two forms of UBF were detected at 0 h p.i., as well as up to 2 h p.i. (Fig. 4c, lanes 1–3). These two UBF-specific bands co-migrated with purified UBF (Fig. 4c, lane 6). At 3 h p.i., both bands were reduced, with a concomitant increase in one predominant, faster-migrating band with a molecular mass of approximately 78 kDa (Fig. 4c, lane 4). The intensity of UBF was further reduced at 4 h p.i., with the appearance of another faster-migrating band at ~52 kDa in addition to the 78 kDa polypeptide (Fig. 4c, lane 5). These results suggested that, although UBF is not cleaved by 3Cpro in vitro, it is modified in PV-infected cells.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PV inhibits transcription mediated by all three cellular RNA polymerases in the nuclei of infected cells, a phenomenon known as shut-off of host-cell transcription. We have presented evidence here that suggests that both pol I transcription factors, SL-1 and UBF, are modified and inactivated during infection of HeLa cells with PV. Neither purified SL-1 nor UBF was able to restore pol I transcription fully from an rDNA promoter in PV-infected extracts. However, both factors were necessary to restore transcription in infected extract to the level seen in mock-infected extracts (Fig. 1). The significant stimulation of rDNA transcription seen in mock-infected extract in the presence of both UBF and SL-1 appeared to be due mainly to SL-1 (Fig. 1). This suggested that SL-1, but not UBF, is limiting in mock-infected HeLa cells. In contrast, although both UBF and SL-1 appeared to stimulate pol I transcription individually in PV-infected cell extracts, complete restoration of transcription clearly required the presence of both factors.

Our results suggested that both UBF and SL-1 are almost completely depleted in PV-infected cells at 4 h p.i. (Fig. 4). Therefore, adding UBF or SL-1 alone into the PV-infected (4 h) transcription extract should not stimulate transcription. Results presented in Fig. 1(c), however, showed that the background transcription in PV-infected extract was somewhat stimulated by the addition of either UBF (Fig. 1c, lane 6) or SL-1 (Fig. 1c, lane 7) compared with that in mock-infected extract (Fig. 1c, lane 1). The precise reason for this is not known and could be due to differential sensitivity of the two assays – transcription and Western blotting – used here. Low levels of UBF and SL-1, not detectable by Western blotting, could still be present in the infected extracts, which could be responsible for the stimulation of transcription observed in the presence of exogenously added individual factors. The in vitro transcription assay, which uses incorporation of high-specific-activity [32P]-labelled UTP, is likely to be much more sensitive than the Western blot analysis. Additionally, the presence of limiting concentrations of SL-1 in mock-infected extract could also be partly responsible for this discrepancy.

The multi-subunit protein SL-1 consists of TBP and three TAFs (TAF110, TAF63 and TAF48). Results presented in Fig. 2 demonstrate clearly that TAF110 is the main target of PV 3Cpro in vitro. TAF110 was cleaved efficiently by 3Cpro into four fragments with molecular masses of 95, 65, 26 and 8 kDa. Two lines of evidence suggest that the 95 and 8 kDa products originate from cleavage of TAF110 at the Q805G806 site. First, the generation of the 95 and 8 kDa polypeptides after 30 min incubation with 3Cpro was consistent with an initial cleavage of TAF110 at the Q805G806 bond (Fig. 3a). Secondly, the Q805A mutation inhibited the formation of the 95 and 8 kDa products almost completely (Fig. 3c, lane 14). In addition, the Q805A mutation appeared to induce a conformational change in TAF110, possibly exposing other sites in the protein that could be cleaved by 3Cpro to generate two new polypeptides of 100 and 85 kDa (Fig. 3c, lanes 13–14 and 17–18). However, these two polypeptides were not seen in the 3Cpro cleavage assay with the wt TAF110 polypeptide and appeared to be due to the Q805A mutation-induced change in the protein structure.

The 26 kDa (predicted molecular mass, ~30 kDa) proteolysis product first appeared after 1 h incubation with 3Cpro (Fig. 3b, lane 4). The 26 kDa product was probably generated by 3Cpro-mediated cleavage at the Q265G266 bond, as the Q265A mutation almost completely blocked formation of the 26 kDa product (Fig. 3c, lane 10).

The 65 kDa polypeptide could be generated from full-length TAF110 by cleavage at the Q805G806 and Q265G266 sites. Alternatively, it could also result from cleavage of the 95 kDa polypeptide at the Q265G266 site (Fig. 3a). The appearance of the 65 kDa band did not correlate with a concomitant reduction in the intensity of the 95 kDa product, but did correlate with reduction in intensity of the full-length TAF110 polypeptide, suggesting that it was generated from TAF110 by cleavage at the Q805G806 and Q265G266 sites (Fig. 3b, lanes 4–6). Cleavage at the Q805G806 and Q265G266 sites in the same molecule of TAF110 should produce a polypeptide with an apparent molecular mass of 64·3 kDa. Consistent with this notion, the double mutant with Q805A and Q265A alterations failed to produce all four products following incubation with 3Cpro (Fig. 3c, lanes 17 and 18). However, although the intensity of the 65 kDa polypeptide was decreased significantly by the Q265G mutation, it was not eliminated (Fig. 3c, compare lanes 10 and 8), suggesting that cleavage at the Q265G266 site was not as efficient as that at the Q805G806 site. These results suggested that two cleavages, one at Q805G806 and a second at Q265G266, are probably responsible for the generation of the 95, 65, 26 and 8 kDa polypeptides in the in vitro 3Cpro cleavage assay.

PV 3Cpro has been shown to prefer glutamine at P1 and either glycine or alanine or serine at P1' for cleavage of viral precursor polypeptides (Dewalt et al., 1989; Kean et al., 1990). In addition, aliphatic amino acids (such as alanine, valine or isoleucine) at P4 are preferred for 3C/3CD-mediated cleavage between P1 and P1' (Blair & Semler, 1991; Harris et al., 1992). The observations that Q265G266 and Q805G806 were cleaved efficiently by 3Cpro are consistent with the presence of isoleucine and alanine, respectively, at P4 positions preceding these sites (Table 1). None of the other four QG sites (at positions 277, 414, 510 and 737) are preceded by aliphatic amino acids and are therefore not likely to be cleaved by 3Cpro.


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Table 1. Amino acid residues preceding the QG sites in TAF110

 
Previous studies from our laboratory have shown that inhibition of pol I transcription is first detected at 3 h p.i. with PV (Rubinstein & Dasgupta, 1989). Results presented in Fig. 4 suggest that this inhibition could be due to degradation of UBF (Fig. 4c). Both UBF-specific bands seen in the immunoblot appear to be cleaved at 3 h p.i. Our previous data have shown that both in vitro-translated and bacterially expressed UBF are totally resistant to cleavage by purified 3Cpro or 2Apro in vitro (Rubinstein et al., 1992). This suggests that the in vitro-translated/bacterially expressed UBF is not modified properly to undergo cleavage by the viral proteases. Alternatively, one or more host-cell factors may be required along with viral protease (3C or 2A) for UBF cleavage in vivo. PV-induced degradation of the tumour suppressor p53 requires both 3Cpro and host-cell proteins (Weidman et al., 2001). In contrast to UBF cleavage, the TAF110 polypeptide appeared to be degraded at 4 h p.i. (Fig. 4b). Despite repeated attempts, we were unable to detect specific proteolytic fragments of TAF110 in PV-infected cells. This could be due to rapid degradation of the fragments in infected cells. A similar phenomenon has previously been reported for p53 (Weidman et al., 2001). The p53 protein is degraded rapidly soon after infection of HeLa cells with PV. It is also possible that the antibody did not recognize the proteolytic fragments of TAF110, although this is unlikely as we used a polyclonal antibody to TAF110. Although the proteolytic fragments of TAF110 could not be detected in PV-infected cells, the fact that a mutant PV with a point mutation in the 3Cpro-coding sequence is unable to shut off pol I transcription efficiently in vivo (Weidman et al., 2003) suggests the involvement of 3Cpro in pol I transcription shut-off.

Recent results from our laboratory have shown that the viral 3Cpro enters the nucleus in PV-infected cells in the form of the polymerase–protease precursor 3CD (Sharma et al., 2004). We believe that autocatalysis of 3CD in the nucleus generates 3Cpro, which consequently cleaves SL-1/UBF, leading to pol I transcription shut-off. Alternatively, it is possible that 3CD, but not 3C, could be responsible for UBF cleavage in the nucleus. Future in vitro studies will be directed towards understanding the role of 3CD in UBF cleavage.

An intriguing observation in our studies was that TBP, a component of SL-1, was unable to restore pol I transcription in PV-infected cell extracts (data not shown). TBP plays a crucial role in the form of TFIID (TBP plus pol II TAFs) in RNA polymerase II-mediated transcription. Although TBP cleavage by 3Cpro is the major cause of inhibition of RNA pol II-mediated transcription in PV-infected cells (Clark et al., 1993), it appeared to have no significant effect on pol I transcription rescue in PV-infected extracts. This suggests that TBP in the SL-1 complex might be resistant to cleavage by 3Cpro. Perhaps the association of pol I TAFs with TBP somehow protects it from 3Cpro cleavage, whilst in TFIID, the pol II TAFs are unable to protect TBP from 3Cpro cleavage. Future studies on the susceptibility of TBP in the TFIID and SL-1 complexes to 3Cpro cleavage should shed light on the organization of these multiprotein complexes. Although TBP does not appear to rescue pol I transcription in PV-infected extracts, we cannot completely rule out the possibility that TBP is not involved in pol I shut-off, as recombinant TBP added to a cell extract may not incorporate into a functionally active SL-1 complex.

In summary, we have provided evidence suggesting that the pol I factors SL-1 and UBF are modified in PV-infected HeLa cells, leading to transcriptional inactivation of these factors. It is highly likely that the inactivation of these two factors contributes to the shut-off of cellular pol I transcription observed in PV-infected cells.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grant AI27451 to A. D. The authors would like to thank members of the Dasgupta and Comai laboratories for support and encouragement.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 9 December 2004; accepted 10 May 2005.



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