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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 34 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 34 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, 510 µCi (185370 kBq) [
32P]UTP [specific activity, 3000 Ci (111 TBq) mmol1], 100 µg
-amanitin ml1, 2·5 µg rDNA template pUCHU3 ml1 (Rubinstein & Dasgupta, 1989
) and 5060 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 EcoRIBamHI 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 heparinagarose 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
3Cpro-mediated cleavage at 265 and 805 QG sites of TAF110
Examination of the primary sequence of TAF110 (aa 1869) revealed that it contained six glutamineglycine (QG) sites at positions 265, 277, 414, 510, 737 and 805 (Fig. 3a; Comai et al., 1994
). Because PV 3Cpro preferentially cleaves glutamineglycine 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 16, 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 13). 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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1314 and 1718). 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 46). 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.
|
Recent results from our laboratory have shown that the viral 3Cpro enters the nucleus in PV-infected cells in the form of the polymeraseprotease 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blair, W. S. & Semler, B. L. (1991). Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase. J Virol 65, 61116123.[Medline]
Clark, M. E., Hämmerle, T., Wimmer, E. & Dasgupta, A. (1991). Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus. EMBO J 10, 29412947.[Abstract]
Clark, M. E., Lieberman, P. M., Berk, A. J. & Dasgupta, A. (1993). Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. Mol Cell Biol 13, 12321237.[Abstract]
Comai, L., Tanese, N. & Tjian, R. (1992). The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell 68, 965976.[CrossRef][Medline]
Comai, L., Zomerdijk, J. C., Beckmann, H., Zhou, S., Admon, A. & Tjian, R. (1994). Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits. Science 266, 19661972.[Medline]
Das, S. & Dasgupta, A. (1993). Identification of the cleavage site and determinants required for poliovirus 3Cpro-catalyzed cleavage of human TATA-binding transcription factor TBP. J Virol 67, 33263331.[Abstract]
Dasgupta, A., Yalamanchili, P., Clark, M. & 12 other authors (2003). Effects of picornavirus proteinases on host cell transcription. In Molecular Biology of Picornaviruses, pp. 321335. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Dewalt, P. G., Lawson, M. A., Colonno, R. J. & Semler, B. L. (1989). Chimeric picornavirus polyproteins demonstrate a common 3C proteinase substrate specificity. J Virol 63, 34443452.[Medline]
Grummt, I. (2003). Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev 17, 16911702.
Hämmerle, T., Hellen, C. U. T. & Wimmer, E. (1991). Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase. J Biol Chem 266, 54125416.
Harris, K. S., Reddigari, S. R., Nicklin, M. J. H., Hämmerle, T. & Wimmer, E. (1992). Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase. J Virol 66, 74817489.[Abstract]
Jantzen, H. M., Admon, A., Bell, S. P. & Tjian, R. (1990). Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature 344, 830836.[CrossRef][Medline]
Kean, K. M., Teterina, N. & Girard, M. (1990). Cleavage specificity of the poliovirus 3C protease is not restricted to GlnGly at the 3C/3D junction. J Gen Virol 71, 25532563.[Abstract]
Kitamura, N., Semler, B. L., Rothberg, P. G. & 9 other authors (1981). Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature 291, 547553.[CrossRef][Medline]
Krausslich, H. G. & Wimmer, E. (1988). Viral proteinases. Annu Rev Biochem 57, 701754.[CrossRef][Medline]
Kuhn, A. & Grummt, I. (1992). Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor. Proc Natl Acad Sci U S A 89, 73407344.
Kuhn, A., Stefanofsky, V. & Grummt, I. (1993). The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription. Nucleic Acids Res 21, 20572063.[Abstract]
Learned, R. M. & Tjian, R. (1982). In vitro transcription of human ribosomal RNA genes by RNA polymerase I. J Mol Appl Genet 1, 575584.[Medline]
Manley, J. L., Fire, A., Samuels, M. & Sharp, P. A. (1983). In vitro transcription: whole-cell extract. Methods Enzymol 101, 568582.[Medline]
Racaniello, V. R. & Baltimore, D. (1981). Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A 78, 48874891.
Rubinstein, S. J. & Dasgupta, A. (1989). Inhibition of rRNA synthesis by poliovirus: specific inactivation of transcription factors. J Virol 63, 46894696.[Medline]
Rubinstein, S. J., Hämmerle, T., Wimmer, E. & Dasgupta, A. (1992). Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter. J Virol 66, 30623068.[Abstract]
Sharma, R., Raychaudhuri, S. & Dasgupta, A. (2004). Nuclear entry of poliovirus protease-polymerase precursor 3CD: implications for host cell transcription shut-off. Virology 320, 195205.[CrossRef][Medline]
Tower, J., Culotta, V. C. & Sollner-Webb, B. (1986). Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex. Mol Cell Biol 6, 34513462.[Medline]
Weidman, M. K., Yalamanchili, P., Ng, B., Tsai, W. & Dasgupta, A. (2001). Poliovirus 3C protease-mediated degradation of transcriptional activator p53 requires a cellular activity. Virology 291, 260271.[CrossRef][Medline]
Weidman, M. K., Sharma, R., Raychaudhuri, S., Kundu, P., Tsai, W. & Dasgupta, A. (2003). The interaction of cytoplasmic RNA viruses with the nucleus. Virus Res 95, 7585.[CrossRef][Medline]
Yalamanchili, P., Harris, K., Wimmer, E. & Dasgupta, A. (1996). Inhibition of basal transcription by poliovirus: a virus-encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro. J Virol 70, 29222929.[Abstract]
Zhai, W., Tuan, J. A. & Comai, L. (1997). SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription. Genes Dev 11, 16051617.[Abstract]
Received 9 December 2004;
accepted 10 May 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |