The EMBO Journal vol.10 no.10 pp.2941 -2947, 1991
Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase Ill transcription by poliovirus Melody E.Clark, Thomas Hammerle1, Eckard Wimmer1 and Asim Dasgupta Department of Microbiology and Immunology, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024 and 'Department of Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Communicated by E.M.De Robertis
In HeLa cells, RNA polymerase III (pol H11)-mediated transcription is severely inhibited by poliovirus infection. This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol Ill genes. Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex 1) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex HI). We show here that a poliovirus with a point mutation in the proteinase 3C (3CPr) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild-type virus. Incubation of purified 3CPrO, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex 1) in vitro resulted in generation of the transcriptionally inactive complex Ill form of TFIIIC. In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3CPro almost completely inhibited pol HI transcription. Finally expression of the 3CPro gene in transfected HeLa cells resulted in significant inhibition of pol E-mediated transcription. The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3CPr° is a mechanism by which poliovirus inhibits host cell RNA pol III transcription. Key words: poliovirus proteinase 3C/polymerase III transcription/transcriptional inhibition/transcription factor IIIC
Introduction Infection of mammalian cells with poliovirus or other members of the picornavirus family leads to dramatic inhibition of host cell RNA and protein synthesis; an event referred to as host cell shut-off (Baltimore and Franklin, 1962; Bablanian, 1975; Kaariainen and Ranki, 1984). Host cell RNA transcription by all three polymerase systems [RNA polymerase (pol) I, II and III] is inhibited in HeLa cells infected with poliovirus (Zimmerman et al., 1963; Holland and Peterson, 1964; Crawford et al., 1981). Initially, the polymerases were investigated as a target for poliovirus-mediated inactivation of transcription; however, Oxford University Press
RNA pol I, II and III solubilized from infected cells were not impaired in their ability to synthesize RNA from a non-specific template (Schwartz et al., 1974). Also it was shown using an in vitro transcription assay that highly purified RNA pol II did not restore transcription of a pol II gene in poliovirus-infected extracts, but a chromatographic fraction containing pol II transcription factors did restore transcription (Crawford et al., 1981). More recent studies from our laboratory have shown that inhibition of transcription by poliovirus infection in all three polymerase systems is correlated with the inactivation of specific transcription factors; however, the mechanism(s) by which poliovirus inactivates these transcription factors is still not known (Fradkin et al., 1987; Kliewer and Dasgupta, 1988; Rubinstein and Dasgupta, 1989). In the pol III transcription system two transcription factors, TFIB and TFIIIC, are needed in addition to RNA pol III, to transcribe all tRNA-type pol Ill genes (reviewed in Geiduschek and Tocchini-Valentini, 1988). TFIIIC binds specifically to the B box element in pol III genes and this binding is the first event in the formation of a stable pre-initiation complex. In poliovirus-infected HeLa cells, inhibition of pol III transcription is correlated with a severe decrease in TFRIC activity, a slight decrease in TFIIIB activity, and no detectable change in RNA pol Ill activity (Fradkin et al., 1987). Although DNA footprint analyses show that poliovirus infection causes no significant change in the sequence specific DNA binding activity of TFIIIC, gel retardation assays reveal that changes in the DNA -protein complexes formed by TFIIC are induced by poliovirus infection (Clark and Dasgupta, 1990). Two forms of TFIIIC can be detected in mock-infected cell extracts by the gel retardation assay: a transcriptionally active form found in complex I and a transcriptionally inactive form found in complex II. In poliovirus-infected cell extracts, the transcriptionally active form of TFIIIC found in complex I is depleted, the form of TFIHC found in complex II is slightly more abundant, and a new transcriptionally inactive form of TFIIC appears in complex III. The appearance of the transcriptionally inactive form of TFIIIC found in complex Ill and the loss of the transcriptionally active form of TFIIIC found in complex I correlate kinetically with poliovirus-mediated inhibition of pol III transcription. Treatment of complexes I or II with chymotrypsin results in a complex of similar mobility to complex III, suggesting that complex III could be the result of limited proteolysis of complexes I or II (Clark and Dasgupta, 1990). Poliovirus encodes two proteinases, 2A and 3C, which process the viral polyprotein precursor into the capsid and non-capsid proteins (reviewed in Krausslich and Wimmer, 1988; Hellen et al., 1989). Proteinase 3C (3CPro) cleaves the polyprotein specifically at glutamine -glycine bonds and proteinase 2A (2APrO) cleaves at tyrosine -glycine bonds. These proteinases do not cleave every potential cleavage site 2941
M.E.Clark et al.
in the polyprotein, therefore other determinants such as accessibility and context of the cleavage site, are important. 2APro is involved in, but does not directly catalyze, the degradation of cap binding protein p220 (eIF-4F) which is correlated with the shut-off of host cell translation, and recently it has been proposed that 3CPrO, or a precursor of 3CPro, may have a role in viral RNA replication (Andino et al., 1990; Sonenberg, 1990). However, presently the only known substrate for the viral proteinases is the poliovirus polyprotein. In this report we show that poliovirus 3CPr0 is involved in cleavage of the transcriptionally active form of TFIIIC to a transcriptionally inactive form. Nuclear extracts obtained from cells infected with a poliovirus mutated in 3CP"° did not contain the transcriptionally inactive form of TFIIIC found in complex III, implying a role for 3CPrO in the formation of this transcriptionally inactive form of TFIIIC. The formation of complex III by 3CPro was confirmed since purified 3CPro, expressed in Escherichia coli, cleaved TFIIIC found in complexes I or II to give the complex III form of TFIIC in a gel retardation assay. Also, 3CP"° treatment of TFIIIC derived from complex I greatly decreased its transcriptional activity in an in vitro transcription assay. Finally, expression of the 3CP' gene in infected HeLa cells resulted in a decrease in pol III-mediated transcription. The results presented here suggest that poliovirus 3CPro-induced proteolysis of TFIIIC is a mechanism by which poliovirus inhibits pol Ill transcription.
Results
3CPrO mutant Se 13C-02 does not produce complex 11l Previous results from our laboratory showed that a new TFIIIC-containing gel retardation complex, complex Im, was formed in poliovirus-infected cells at the same time postinfection (4 h) that pol III transcription was first inhibited (Clark and Dasgupta, 1990). Comparison of the mobility of complex III with that of chymotrypsin-digested TFIIIC suggested that the TFIHC which forms complex III may be the result of limited proteolysis of TFIIIC from complexes I or II. Since poliovirus encodes its own proteinases, we tested to see if mutations in a viral proteinase would affect the formation of complex III. Poliovirus mutant Sel3C-02 (kindly provided by Dr Bert Semler) has a site-directed valine to alanine substitution at amino acid 54 of the poliovirus 3CPro gene (Dewalt and Semler, 1987). This virus produces less of the viral-encoded RNA dependent RNA polymerase (3DP°l) than wild-type poliovirus and very little 3CPro. The 3CPro that is produced has an altered mobility in an SDS gel. Although the mutant virus does not produce much polymerase it still grows to nearly wild-type titers (1 log/ml less than wild-type) since it is thought that poliovirus greatly overproduces 3CPro and 3DPm". In order to determine the effect of 3CPro on the formation of complex III, HeLa cells were infected with either Sel3C-02 or wild-type poliovirus for 1.5, 3.5, 4.5 or 5.5 h. At each time-point nuclear extracts were prepared and fractionated over phosphocellulose columns. The 0.10.6 M KCl eluate, which contains all the factors necessary for pol III transcription, was prepared for each time-point. Only a minimal level of purification was used to ensure that no complex Ill was lost during purification. Some purification of the nuclear extracts was necessary, however, 2942
since resolution of TFIIIC-containing complexes directly from nuclear extract is poor. These fractions were used in a gel retardation assay with an end-labeled fragment of the adenovirus VAI gene which contained the B box promoter region. Complexes I, II and III are the only gel retardation complexes which contain TFLIC since they were specifically competed with an excess of B box oligonucleotide but not with HTLV oligonucleotide (Clark and Dasgupta, 1990; Figure IA, lanes 11-14). No complex III was observed at 1.5 or 3.5 h post-infection for the wild-type or Sel3C-02infected cell extracts as expected (lanes 3-6). Complex III was observed in the wild-type-infected cell extract at 4.5 and 5.5 h post-infection (lanes 8 and 10); however, no complex IH was formed in Sel3C-02-infected cell extracts at the same times post-infection (lanes 7 and 9). Thus these results with the 3CPro mutant virus provided initial genetic evidence that 3CPro was involved in the formation of the complex HI form of TFIIIC. In vitro transcription analysis of poliovirus mutant Se 13C-02 To determine if Sel3C-02 infection had any effect on pol III transcription the same phosphocellulose fractions were tested for their ability to transcribe the VAI gene in vitro. Inhibition of pol Ill transcription in wild-type poliovirusinfected cell extracts began at 4.5 h post-infection and was complete at 5.5 h post-infection (Figure 2, lanes 6 and 8). In contrast transcription was not inhibited at 4.5 h postinfection in Sel3C-02-infected cell extracts but did begin to show some inhibition at 5.5 h post-infection (lanes 7 and 9). The inhibition of transcription seen at this time-point was surprising since in the gel retardation assay the transcriptionally active form of TFIIIC in complex I appeared intact and no transcriptionally inactive TFIHC in complex III was seen (Figure IA, lane 9). A titration of the Sel3C-02 and wild-type 5.5 h infected cell extracts showed more clearly that complex I remained after Sel3C-02 infection but was absent after wild-type infection (Figure iB). Although the free probe has been run off the gel in Figure IB, gel retardation experiments using an excess of probe also show that complex I is depleted in wild-type poliovirus-infected cell extracts (data not shown). Since in a wild-type poliovirus infection the activity of TFHB is also affected at later times post-infection (Fradkin et al., 1987), we tested whether the inhibition of transcription seen after 5.5 h of Sel3C-02 infection was the result of TFIIIB inactivation. As seen in Figure 3, addition of partially purified TFIIIB restored transcription to the Sel3C-02infected cell extract whereas addition of the complex I form of TFIIIC did not restore transcription (Figure 3, lanes 5 and 7). At 5.5 h post wild-type infection both TFIIIC and TFIIIB activities were inhibited as expected and thus transcription could not be restored by addition of either transcription factor alone (lanes 6 and 8). The TFIIIB and TFIIC fractions used in this experiment were well separated and transcriptionally active since only when added together did they result in VAI transcription (lanes 9, 10 and 11). While Sel3C-02-infected cell extracts looked the same as mock-infected cell extracts in the gel retardation assay (see Figure IA), the decrease in pol III transcription seen at 5.5 h post-infection with the Sel3C-02 virus shows that the cells were productively infected with the mutant virus. Interestingly, since TFIIIB activity is reduced in Sel3C-02-
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Fig. 1. Gel retardation analysis of B box binding activity of extracts from HeLa cells infected for 1.5-5.5 h with the 3CP"O mutant virus Se13C-02, wild-type poliovirus, or mock-infected. (A) Equal amounts (3 gg) of a 0.1-0.6 M KCl phosphocellulose fraction prepared from mock-infected (lanes 1 and 2), wild-type poliovirus-infected (lanes 4, 6, 8 and 10-14) and 3CP'° mutant virus Sel3C-02-infected (lanes 3, 5, 7 and 9) cells at 1.5, 3.5, 4.5 and 5.5 h post-infection were used in the gel retardation assay. Competitions were performed with a 100-fold molar excess of specific B box oligonucleotides (lanes 11 and 12) or non-specific HTLV oligonucleotide (lanes 13 and 14). TFHIC-containing complexes I, II and III are labeled along with the free probe (FP). (B) Increasing amounts (4, 8 and 12 tLg) of the 5.5 h Se13C-02 and wild-type poliovirus-infected phosphocellulose fraction were used in the gel retardation assay. The free probe was run off the gel and is not shown.
infected cell extracts that implies that while 3CPrO is involved in TFIIIC inactivation, it is probably not involved in TFIIIB inactivation. Poliovirus 3CP')' can cleave TFIIIC-containing complexes in vitro The mutant virus experiment implied that 3CPrO was somehow involved in the formation of the transcriptionally inactive complex III form of TFIIIC. To determine if direct treatment of TFIIIC with the 3CPrO protein could result in the generation of complex Ill, 3CP"' overexpressed and purified from E. coli was added to TFIIIC fractions in vitro and then gel retardation reactions were performed. The three different forms of TFIIIC, complexes I, II and III, were separated by FPLC phosphocellulose gradient purification of mock- and poliovirus-infected cell extracts as previously described (Clark and Dasgupta, 1990) and incubated with either purified 3CPrO or a 3CP"° mutant, 3C C147S. The 3Cpr, mutant 3C C 147S has a cysteine to serine substitution at amino acid 147 which inactivates the proteinase (Hammerle et al., 1991). A synthetic peptide with a 3Cpro cleavage site was used to test for activity, or lack of activity, of the proteinases (data not shown). Both proteinases were overexpressed in the same strain of bacteria and purified in the same manner to >98 % purity (Nicklin et al., 1988; Hammerle et al., 1991). When 3Cpr0 was incubated with fractions containing either the complex I or complex H forms of TFIIIC in vitro and then added to a gel retardation reaction, complex III was formed (Figure 4, lanes 2, 5
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Fig. 2. In vitro transcriptional analysis of cell extracts prepared at different times post-infection. Equal amounts (10 ug) of the 0.1-0.6 M phosphocellulose fraction prepared from mock-infected (lanes 1 and 10), wild-type poliovirus-infected (lanes 2, 4, 6 and 8) and 3CPr" mutant virus Sel3C-02-infected (lanes 3, 5, 7 and 9) cells at 1.5, 3.5, 4.5 and 5.5 h post-infection were used in an in vitro transcription assay. The correctly initiated VAI transcript position is indicated.
and 8). Complex III purified from poliovirus-infected cell extracts was not affected by 3CPro treatment (lane 11). The mutant 3C C 147S had no effect on any of the TFIIIC
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M.E.Clark et al.
complexes (lanes 3, 6, 9 and 12). This shows that the formation of complex III was indeed caused by 3CPro and not by a different proteinase that possibly copurified with 3CPr'. The gel retardation assay in Figure 4 shows that complexes I and II are cleaved by 3CPro to form complex III, but since the complex I fraction could contain proteins in addition to TFIIIC, we cannot say that TFIIIC is directly cleaved by 3CP'°. TFIIIC must first be cloned before this can be tested directly. The arrow in Figure 4 shows a complex of slower mobility than the complex III found in lanes where complexes I and II were treated with 3CPFO (Figure 4, lanes 2 and 5). This complex has been detected previously in poliovirus-infected cell extracts and was proposed to be an intermediate in the formation in complex III (Clark and Dasgupta, 1990). In order to test whether this complex was indeed an intermediate in the formation of complex Im, a TFHIC fraction containing complex I (and a small amount of complex II) was incubated with varying amounts of 3CPrO. At low amounts of 3CPrO only the intermediate complex was formed (Figure 5, lanes 3 and 4). At higher concentrations both the intermediate
Fig. 3. In vitro transcription analysis of infected cell extracts with the addition of TFIIIB and TFIIIC. Partially purified TFIIIB or TFIIIC (complex I) was added to the wild-type and Sel3C-02 mutant 5.5 h infected cell extract fractions used in Figure 2 (lanes 5-9). The amount of transcription from cell extract fractions without added TFIIIB or TFIIIC is shown in lanes 3 and 4. Transcription from 1.5 h infected cell extract fractions is shown in lanes 1 and 2. Transcription in the presence of TFIIIB alone, TFIIIC alone and TFIIIB plus TFIIIC is shown (lanes 9-1I1).
complex and complex III were formed (lanes 5 and 6). At the highest concentration tested only complex III was formed (lane 7). A similar result was seen using a fraction which contained the complex II form of TFIIIC (data not shown). Thus the complex indicated with an arrow was an intermediate complex, formed in vitro as well as in poliovirus-infected cells, which was the result of incomplete cleavage of TFIIIC from complexes I or II by 3CPKM. Complete cleavage of TFIIIC resulted in complex III formation.
3CPr0 treatment of TFIIIC and transfection of 3CPIO gene in HeLa cells decreases pol 111 transcription in
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Fig. 4. Gel retardation analysis of TFIIIC-containing complexes I, II and III treated with 3CPrO or 3CPr0 mutant 3C C147S. A gel retardation assay was performed using TFIIIC-containing complexes I, II and III isolated from mock- or poliovirus-infected cells using gradient elution of a FPLC phosphocellulose column. These fractions were either treated with 10 yg of 3CP'° (lanes 2, 5, 8 and 11), 10 ltg of 3CPrK mutant 3C C 147S (lanes 3, 6, 9 and 12) or left untreated (lanes 1, 4, 7 and 10). Complexes I, II and Ill are labeled as is the free probe (FP) and an intermediate complex is indicated with an arrow.
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vitro Previous results from our laboratory showed that fractions containing the complex III form of TFIIIC purified from poliovirus-infected HeLa cells were transcriptionally inactive in an in vitro transcription assay (Clark and Dasgupta, 1990). Since 3CPrO treatment of transcriptionally active complex I resulted in formation of complex III in the gel retardation assay (Figure 4), it was predicted that 3CPro treatment of complex I would result in decreased pol III transcription in vitro when the other factors necessary for transcription (pol III and TFIIIB) were added. As seen in Figure 6A, 3CPro treatment of complex I resulted in total inhibition of pol III transcription while treatment with the 3CPro mutant 3C C 147S did not affect pol III transcription in vitro. We have tried using inhibitors of 3CPro to show that the proteinase does not cleave other components of the transcriptional machinery but unfortunately these inhibitors themselves abolish transcription (data not shown). However, we do not believe that 3CPro affects the TFIIIB component of the transcriptional apparatus because in the 3CPro mutant virus Se13C-02, TFIIIB activity is still affected (Figure 3). Since direct treatment of TFIIIC with 3CPro in vitro resulted in transcriptional inhibition (Figure 6A), we next asked whether the 3CPro gene transiently transfected into HeLa cells could cause inhibition of pol III transcription. Two plasmids, pRSV3CPro and pRSV3CProrev, were constructed which contained the 3CPro gene downstream of the RSV promoter in the forward and reverse orientations, respectively. Cells were transfected with either pRSV3CPrO,
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Fig. 5. Titration of 3Cpro with the FPLC-purified complex I form of TFIIIC. The complex I-containing fraction untreated (lane 1), or treated with the indicated amount of 3CPro for 2 h (lanes 3-7), was used in the gel retardation assay. Lane 2 contains complex I treated with 3CPro for only 30 min. The intermediate complex is again indicated with an arrow.
pRSV3CProrev, the parental plasmid pUC 18 or with no DNA. Cells transfected with pRSV3CPro were harvested after 8, 12 and 24 h of transfection while cells transfected with pRSV3CProrev, pUC18 or no DNA were harvested after 12 h of transfection. No morphological differences were seen in cells transfected with the proteinase gene compared with cells transfected with no DNA, indicating that the proteinase gene did not produce a general toxic effect on the cells within 24 h (data not shown). Small-scale nuclear extracts were prepared at each time-point of transfection and used in pol III in vitro transcription reactions. Extracts from cells transfected with the pRSV3CPro for 8 h were not affected in their ability to transcribe the pol III template whereas extracts from cells transfected for 12 and 24 h with the proteinase gene were significantly reduced in their ability to transcribe the VAI gene (Figure 6B, lanes 1-4). Extracts from cells transfected with pRSV3CProrev, pUC 18 or no DNA for 12 h were not inhibited in the pol III transcription assay (lanes 1, 5 and 6). Also no inhibition was observed when these same plasmids were transfected for 24 h (data not shown). We did not expect to see total inhibition of transcription in cells transfected with pRSV3CPro since not all of the cells used to make the extract would incorporate and express the 3CP" gene construct. Thus the transfection efficiency in each experiment was important to the level of inhibition observed. Typically our transfection efficiency was -10-20% as measured by X-gal staining of cells transfected with a 3-gal construct (data not shown). The decrease in transcription from cells transfected with pRSV3CPro could be due to inactivation of TFIIIC or to some non-specific inhibition of the transcription machinery. In order to test these possibilities, a TFIIIC fraction (complex I) was added back to the extract prepared from cells
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Fig. 6. In vitro transcriptional analysis of TFIIIC treated directly with 3CP"° or nuclear extracts from cells transfected with the 3CPrT gene. (A) The complex I form of TFIIIC from mock-infected cells was added to a partially purified fraction containing TFIIIB and pol III (lane 1). In lane 2 the TFIIIC fraction was first incubated with 10 yg of 3CPrK and in lane 3 it was incubated with 10 Ag of the 3CP'0 mutant 3C C147S. (B) Nuclear extracts (9.0 Ag) from cells which were transfected with no DNA for 12 h (lane 1), transfected with the 3Cpr0 gene (pRSV3CPro) for 8, 12 and 24 h (lanes 2, 3 and 4), transfected with the 3CPr" gene in the reverse orientation (pRSV3CPrOrev) for 12 h (lane 5) or transfected with the parental plasmid pUC18 for 12 h (lane 6) were used in an in vitro transcription assay. A fraction containing the complex I form of TFIIIC was added to the extract from cells transfected for 24 h with pRSV3CPro in the in vitro transcription assay (lane 7).
transfected for 24 h with pRSV3CPro. Addition of TFIIIC greatly stimulated transcription showing that the extract was not generally inhibited in its transcriptional ability, and that these extracts were deficient in TFIIIC activity (lane 7). These results strongly suggest that expression of the 3CPro gene, without other viral genes, can cause the decrease in TFIIIC activity and inhibition of pol III transcription seen in poliovirus-infected cells.
Discussion We have shown in this report that poliovirus 3Cpro proteolyzes a TFIIIC-containing gel retardation complex in vivo and in vitro to form the transcriptionally inactive complex III form of TFIIIC. In vivo, complex III was formed in wild-type-infected HeLa cells whereas no complex III was formed in cells infected with a poliovirus containing a point mutation in the 3CP"° gene. In vitro, TFIIICcontaining complexes I and II were cleaved by cloned, purified 3CP"° to form complex EII. This cleavage of TFHIIC can account for the inhibition of pol III transcription observed in poliovirus-infected cells since treatment of the transcriptionally active complex I form of TFIIIC with 3Cpro resulted in inhibition of pol III transcription in an in vitro transcription assay. Finally, the 3CPr° gene transiently transfected into HeLa cells resulted in inhibition of pol III transcription. These data strongly suggest that 3CPro-induced cleavage of TFIIIC is a mechanism by
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which poliovirus infection results in inhibition of pol III transcription.
Although poliovirus encodes at least two proteinases, poliovirus infection does not result in large-scale or random proteolysis of viral or cellular proteins. When two-dimensional gel analysis is performed on mock versus poliovirus-infected cell extracts < 10 proteins are shown to be altered by poliovirus infection (Urzainqui and Carrasco, 1989). While it cannot be determined until TFHIC is cloned whether it is a direct substrate of 3CPro, 3CP° does induce a change in a TFIIIC-containing complex which converts it from a transcriptionally active to a transcriptionally inactive form. The lack of large-scale modification of cellular proteins in poliovirus-infected cells argues that the conversion of TFIIIC by 3CPrO is a specific event and is not due to generalized proteolysis in the infected cell. Poliovirus replicates in the cytoplasm of infected cells, but in order to cleave TFIIIC in the nucleus, 3CP' must be able to enter the nucleus. There is immunohistochemical evidence that a precursor of 3CPro, 3CD, enters the nucleus after infection (Fernandez-Tomas, 1982). Also since 3CPr' is a small protein (20 kDa), it could enter the nucleus through diffusion. One might expect that if 3CP'O were in the nucleus that it may be active in nuclear extracts made from infected or transfected cells. However, 3Cpro in nuclear extracts is inactive, since when poliovirus-infected cell extracts are mixed with mock-infected cell extracts, pol III transcription is not inhibited in vitro (Fradkin et al., 1987). Also 3CPr° did not remain active in extracts made from cells transfected with the 3CPro gene since when TFIIIC was added to these extracts transcription was restored (Figure 6B). It is not unexpected that 3CPro could lose activity quickly in these extracts since it is prone to autocleavage
(Hammerle et al., 1991). The proteolyzed complex III form of TFIIIC still binds to the B box DNA binding site in the VAI gene but is inactive in an in vitro transcription assay in the presence of added TFIIIB and pol III (Clark and Dasgupta, 1990). We propose that this proteolyzed form of TFIHC can no longer interact with TFIIHIB or pol III and thus cannot form the pre-initiation complex necessary for transcription. Interestingly the complex II form (as well as the complex I form) of TFIIIC is cleaved by 3CPrO in vitro (see Figure 4) but in poliovirusinfected cells the complex II form of TFIIIC is intact (see Figure 1). Although it is not clear how the virus cleaves TFIIIC in complex I and not in complex II, this is advantageous to the virus since the TFIIIC in complex II is already transcriptionally inactive. It is possible that complex I is more accessible for cleavage in vivo because of its conformation in vivo or its sub-nuclear localization. It is also possible that the complex I form of TFIIIC is first made into the complex II form and then the complex II form is cleaved to produce the complex III form. It has been shown previously that the complex II form of TFIIIC can be formed in vitro by acid phosphatase treatment of the complex I form of TFIIIC (Hoeffler et al., 1988; Clark and Dasgupta, 1990). Titration experiments rule out the complex II form of TFIIC being generated directly from the complex I form by 3CPrO cleavage (Figure 5), although it is still possible that the proteinase has an indirect role, possibly by proteolyzing a kinase which normally converts complex II to complex I. The homologous 3CPrO from another picornavirus, foot and mouth disease virus, also inhibits gene expression when transiently transfected into BHK cells (Tesar and Marquardt,
2946
1990). The inhibition is believed to be caused by cleavage of histone H3 although this has not been shown directly (Falk et al., 1990). Degradation of histone H3 is not observed in poliovirus-infected cells; thus, although poliovirus and foot and mouth disease virus are in the same virus family, they have different mechanisms for transcriptional inhibition. Interestingly, foot and mouth disease virus 3CPrO and poliovirus 3CPrK have different cleavage site specificities (Krausslich and Wimmer, 1988). Although the shut-off of host cell functions by poliovirus has been known for many years no mechanisms have been identified to elucidate these events. We have proposed here that 3Cpro_mediated cleavage of a complex which contains a transcriptionally active form of TFJIC to a transcriptionally inactive form is a mechanism by which poliovirus inhibits pol III transcription. Future studies on the mechanism of pol III transcriptional inhibition by poliovirus will require cloning of TFIIIC and determining if it is cleaved directly by cloned 3CKMT. Future studies will also be aimed at further determining the role of 3CPrO in the inhibition of pol I and pol II transcription in poliovirus-infected cells.
Materials and methods Cells and viruses HeLa cells were grown in spinner culture with minimal essential media (GIBCO Laboratories) supplemented with 1 gll glucose and 6% newborn calf serum. Cells were infected with poliovirus type 1 (Mahoney) or poliovirus mutant Sel3C-02 (Dewalt and Semler, 1987) at a multiplicity of infection (m.o.i.) of 20 as previously described (Dasgupta, 1983). HeLa cell monolayers were grown in Dulbecco's modified Eagle's medium (high glucose) (GIBCO) supplemented with 10% fetal calf serum.
Extract preparation and fractionation Nuclear extracts were prepared from mock- and poliovirus-infected cells as previously described (Dignam et al., 1983). Small-scale nuclear extracts were prepared from transfected cells using a modified Dignam procedure (Lee et al., 1988). Nuclear extracts were fractionated by chromatography on phosphocellulose (Whatman P11) using buffer A [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.9), 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride] as previously described (Segall et al., 1980). One step elution of 0.10-0.60 M KCl was used for some gel retardation and in vitro transcription reactions. Separation of complexes I, II and III with a 70 ml 0.1 -1 M KCI gradient elution of an FPLC phosphocellulose column was performed as described previously (Clark and Dasgupta, 1990).
Gel retardation and in vitro transcription analysis Gel retardation reactions were performed as described (Clark and Dasgupta, 1990). Briefly, each reaction (15 ltl) contained 7.5 Al of protein (1-3 pg) plus buffer A. The total salt concentration was adjusted to 75 mM KCl per reaction. The reactions were started by addition of a premix containing (per reaction) 2 jg of poly(dI-dC), 1.5 ,ul of binding buffer (100 mM Tris, pH 7.6, 5 mM EDTA, 30 mM MgCI2, 50 mM DTT, 500 mM KCl) and 20 000 c.p.m. of the -y-ATP end-labeled Sal I-Bst El fragment of the pVAI gene. Complete gel retardation reactions were incubated at room temperature for 30 min, loaded, with current on, into a 4% polyacrylamide gel in x TBE. Reactions containing the 0.6 M KCI phosphocellulose fraction were loaded onto a 4% polyacrylamide gel in 0.5 x TBE. In vitro transcription reactions were performed as previously described (Fradkin et al., 1987). Essentially transcription reaction mixtures (40 al) contained 30 mM HEPES, pH 7.9, 3 mM DTT, 7.5 mM MgCI,, 8.25 jsg/ml creatine phosphokinase, 500 mM each ATP, CTP and UTP, and 25 pM GTP with 2 pCi of [cz-32P]GTP. pVAI template DNA was added to a concentration of 6.25 Ag/ml. Reactions were stopped after 90 min of incubation at 30°C and processed for electrophoresis in an 8% acrylamide-8 M urea sequencing gel. Proteinase purification and treatment 3CPr" and 3CPr" mutant 3C C147S were overexpressed in E.coli and purified as described (Nicklin et al., 1988; Hammerle et al., 1991). The indicated amount of 3CPrO was added directly to various amounts of TFIIIC
Inhibition of pol III transcription by poliovirus and incubated at 30°C for 2 h, after which salts and reaction mixtures for gel retardation or in titro transcription were added.
Transfections and constructs Two constructs, pRSV3CPro and pRSV3CProrev, were constructed from the RSV expression vector pDEX which contains the RSV promoter and the SV40 polyadenylation site cloned into pUC 18 (Garcia et al., 1988). The XbaI-HindlIl fragment (nucleotides 34-623) encoding the entire 3Cpro gene from pMN35 (Nicklin et al., 1988) was subcloned into pGEM3. The 3CPKm gene was isolated by cutting with BamHI and HinduI and cloned into pDEX which had been cut with HindlIl and BglII giving pRSV3CProrev. pRSV3CP"° was constructed by cutting pDEX with ScaI and HindlIl and ScaI and BglII. These two fragments along with the HindIII-BglII piece of the 3CPro gene were ligated together. Monolayer cultures of HeLa cells (80% confluent) were transfected using Lipofectin reagent (BRL) according to the manufacturer's instructions.
Urzainqui,A. and Carrasco,L. (1989) J. Virol., 63, 4729-4735. Zimmerman,E.R., Hecter,M. and Darnell,J.E. (1963) Virology, 19, 400-408.
Received on MaY 21, 1991; revised on June 28, 1991
Acknowledgements We wish to thank Bert Semler for the Sel3C-02 virus, Dave Harrich and Richard Gaynor for the pDEX expression vector, and Joe Garcia for cloning advice. We are indebted to Steve Kliewer, Arnie Berk and members of the Dasgupta and Berk laboratories for helpful discussions throughout the course of this work. This work was supported by Public Health Service grants Al 27451 to A.D., and Al 15122 and CA 28146 to E.W. from the National Institutes of Health. A.D. is a member of the Molecular Biology Institute, UCLA. M.E.C. is supported by Public Health Service National Research Award Al 07323 from the National Institutes of Health and T.H. is a recipient of a fellowship from Deutsche Forschungsgemeinschaft, Bonn, FRG.
References Andino,R., Rieckhof,G.E. and Baltimore,D. (1990) Cell, 63, 369-380. Bablanian,R. (1975) Virology, 19, 54-66. Baltimore,D. and Franklin,R.M. (1962) Proc. Natl. Acad. Sci. USA, 48, 1383-1390. Clark,M.E. and Dasgupta,A. (1990) Mol. Cell. Biol., 10, 5106-5113. Crawford,N., Fire,A., Samuels,M., Sharp,P.A. and Baltimore,D. (1981) Cell, 27, 555-561. Dasgupta,A. (1983) Virology, 128, 245-251. Dewalt,P.G. and Semler,B.L. (1987) J. Virol., 61, 2162-2170. Dignam,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Nucleic Acids Res., 11, 1475-1489. Falk,M.M., Griegera,P.R., Bergmann,I.E., Zibert,A., Multhaup,G. and Beck,E. (1990) J. Virol., 64, 748-756. Fernandez-Tomras,C. (1982) Virology, 116, 629-634. Fradkin,L.G., Yoshinaga,S.K., Berk,A.J. and Dasgupta,A. (1987) Mol. Cell. Biol., 7, 3880-3887. Garcia,J., Harrich,D., Pearson,L., Mitsuyasu,R. and Gaynor,R.B. (1988) EMBOJ., 7, 3143-3147. Geiduschek,E.P. and Tocchini-Valentini,G.P. (1988) Annu. Rev. Biochem., 57, 873-914. Hammerle,T., Hellen,C.U.T. and Wimmer,E. (1981) J. Biol. Chem., 266, 5412-5416. Hellen,C.U.T., Krausslich,H. and Wimmer,E. (1989) Biochemistry, 28, 9981 -9890. Hoeffler,W.K., Kovelman,R. and Roeder,R.G. (1988) Cell, 53, 907-920. Holland,J.J. and Peterson,J.A. (1964) J. Mol. Biol., 8, 556-573. Kaariainen,L. and Ranki,M. (1984) Annu. Rev. Microbiol., 38, 91 - 109. Kliewer,S. and Dasgupta,A. (1988) Mol. Cell. Biol., 8, 3175-3182. Krausslich,H. and Wimmer,E. (1988) Annu. Rev. Biochem., 57, 710-754. Lee,K.A.W., Bindereif,A. and Green,M. (1988) Gene Anal. Techn., 5, 22-34. Nicklin,M.J.H., Harris,K.S., Pallai,P.V. and Wimmer,E. (1988) J. Virol., 62, 4586-4593. Rubinstein,S.J. and Dasgupta,A. (1989) J. Virol., 63, 4689-4696. Schwartz,L.B., Lawrence.C., Thach.R.E. and Roeder,R.G. (1974)J. Virol., 14, 611-622. Segall,J.T., Matsui,T. and Roeder,R.G. (1980) J. Biol. Chem., 255, 11986-11991. Sonenberg,N. (1990) Curr. Top. Microbiol. Inmnunol., 161, 23-47. Tesar,M. and Marquardt,O. (1990) Virology, 174, 364-374.
2947
Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase Ill transcription by poliovirus Melody E.Clark, Thomas Hammerle1, Eckard Wimmer1 and Asim Dasgupta Department of Microbiology and Immunology, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024 and 'Department of Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Communicated by E.M.De Robertis
In HeLa cells, RNA polymerase III (pol H11)-mediated transcription is severely inhibited by poliovirus infection. This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol Ill genes. Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex 1) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex HI). We show here that a poliovirus with a point mutation in the proteinase 3C (3CPr) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild-type virus. Incubation of purified 3CPrO, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex 1) in vitro resulted in generation of the transcriptionally inactive complex Ill form of TFIIIC. In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3CPro almost completely inhibited pol HI transcription. Finally expression of the 3CPro gene in transfected HeLa cells resulted in significant inhibition of pol E-mediated transcription. The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3CPr° is a mechanism by which poliovirus inhibits host cell RNA pol III transcription. Key words: poliovirus proteinase 3C/polymerase III transcription/transcriptional inhibition/transcription factor IIIC
Introduction Infection of mammalian cells with poliovirus or other members of the picornavirus family leads to dramatic inhibition of host cell RNA and protein synthesis; an event referred to as host cell shut-off (Baltimore and Franklin, 1962; Bablanian, 1975; Kaariainen and Ranki, 1984). Host cell RNA transcription by all three polymerase systems [RNA polymerase (pol) I, II and III] is inhibited in HeLa cells infected with poliovirus (Zimmerman et al., 1963; Holland and Peterson, 1964; Crawford et al., 1981). Initially, the polymerases were investigated as a target for poliovirus-mediated inactivation of transcription; however, Oxford University Press
RNA pol I, II and III solubilized from infected cells were not impaired in their ability to synthesize RNA from a non-specific template (Schwartz et al., 1974). Also it was shown using an in vitro transcription assay that highly purified RNA pol II did not restore transcription of a pol II gene in poliovirus-infected extracts, but a chromatographic fraction containing pol II transcription factors did restore transcription (Crawford et al., 1981). More recent studies from our laboratory have shown that inhibition of transcription by poliovirus infection in all three polymerase systems is correlated with the inactivation of specific transcription factors; however, the mechanism(s) by which poliovirus inactivates these transcription factors is still not known (Fradkin et al., 1987; Kliewer and Dasgupta, 1988; Rubinstein and Dasgupta, 1989). In the pol III transcription system two transcription factors, TFIB and TFIIIC, are needed in addition to RNA pol III, to transcribe all tRNA-type pol Ill genes (reviewed in Geiduschek and Tocchini-Valentini, 1988). TFIIIC binds specifically to the B box element in pol III genes and this binding is the first event in the formation of a stable pre-initiation complex. In poliovirus-infected HeLa cells, inhibition of pol III transcription is correlated with a severe decrease in TFRIC activity, a slight decrease in TFIIIB activity, and no detectable change in RNA pol Ill activity (Fradkin et al., 1987). Although DNA footprint analyses show that poliovirus infection causes no significant change in the sequence specific DNA binding activity of TFIIIC, gel retardation assays reveal that changes in the DNA -protein complexes formed by TFIIC are induced by poliovirus infection (Clark and Dasgupta, 1990). Two forms of TFIIIC can be detected in mock-infected cell extracts by the gel retardation assay: a transcriptionally active form found in complex I and a transcriptionally inactive form found in complex II. In poliovirus-infected cell extracts, the transcriptionally active form of TFIIIC found in complex I is depleted, the form of TFIHC found in complex II is slightly more abundant, and a new transcriptionally inactive form of TFIIC appears in complex III. The appearance of the transcriptionally inactive form of TFIIIC found in complex Ill and the loss of the transcriptionally active form of TFIIIC found in complex I correlate kinetically with poliovirus-mediated inhibition of pol III transcription. Treatment of complexes I or II with chymotrypsin results in a complex of similar mobility to complex III, suggesting that complex III could be the result of limited proteolysis of complexes I or II (Clark and Dasgupta, 1990). Poliovirus encodes two proteinases, 2A and 3C, which process the viral polyprotein precursor into the capsid and non-capsid proteins (reviewed in Krausslich and Wimmer, 1988; Hellen et al., 1989). Proteinase 3C (3CPro) cleaves the polyprotein specifically at glutamine -glycine bonds and proteinase 2A (2APrO) cleaves at tyrosine -glycine bonds. These proteinases do not cleave every potential cleavage site 2941
M.E.Clark et al.
in the polyprotein, therefore other determinants such as accessibility and context of the cleavage site, are important. 2APro is involved in, but does not directly catalyze, the degradation of cap binding protein p220 (eIF-4F) which is correlated with the shut-off of host cell translation, and recently it has been proposed that 3CPrO, or a precursor of 3CPro, may have a role in viral RNA replication (Andino et al., 1990; Sonenberg, 1990). However, presently the only known substrate for the viral proteinases is the poliovirus polyprotein. In this report we show that poliovirus 3CPr0 is involved in cleavage of the transcriptionally active form of TFIIIC to a transcriptionally inactive form. Nuclear extracts obtained from cells infected with a poliovirus mutated in 3CP"° did not contain the transcriptionally inactive form of TFIIIC found in complex III, implying a role for 3CPrO in the formation of this transcriptionally inactive form of TFIIIC. The formation of complex III by 3CPro was confirmed since purified 3CPro, expressed in Escherichia coli, cleaved TFIIIC found in complexes I or II to give the complex III form of TFIIC in a gel retardation assay. Also, 3CP"° treatment of TFIIIC derived from complex I greatly decreased its transcriptional activity in an in vitro transcription assay. Finally, expression of the 3CP' gene in infected HeLa cells resulted in a decrease in pol III-mediated transcription. The results presented here suggest that poliovirus 3CPro-induced proteolysis of TFIIIC is a mechanism by which poliovirus inhibits pol Ill transcription.
Results
3CPrO mutant Se 13C-02 does not produce complex 11l Previous results from our laboratory showed that a new TFIIIC-containing gel retardation complex, complex Im, was formed in poliovirus-infected cells at the same time postinfection (4 h) that pol III transcription was first inhibited (Clark and Dasgupta, 1990). Comparison of the mobility of complex III with that of chymotrypsin-digested TFIIIC suggested that the TFIHC which forms complex III may be the result of limited proteolysis of TFIIIC from complexes I or II. Since poliovirus encodes its own proteinases, we tested to see if mutations in a viral proteinase would affect the formation of complex III. Poliovirus mutant Sel3C-02 (kindly provided by Dr Bert Semler) has a site-directed valine to alanine substitution at amino acid 54 of the poliovirus 3CPro gene (Dewalt and Semler, 1987). This virus produces less of the viral-encoded RNA dependent RNA polymerase (3DP°l) than wild-type poliovirus and very little 3CPro. The 3CPro that is produced has an altered mobility in an SDS gel. Although the mutant virus does not produce much polymerase it still grows to nearly wild-type titers (1 log/ml less than wild-type) since it is thought that poliovirus greatly overproduces 3CPro and 3DPm". In order to determine the effect of 3CPro on the formation of complex III, HeLa cells were infected with either Sel3C-02 or wild-type poliovirus for 1.5, 3.5, 4.5 or 5.5 h. At each time-point nuclear extracts were prepared and fractionated over phosphocellulose columns. The 0.10.6 M KCl eluate, which contains all the factors necessary for pol III transcription, was prepared for each time-point. Only a minimal level of purification was used to ensure that no complex Ill was lost during purification. Some purification of the nuclear extracts was necessary, however, 2942
since resolution of TFIIIC-containing complexes directly from nuclear extract is poor. These fractions were used in a gel retardation assay with an end-labeled fragment of the adenovirus VAI gene which contained the B box promoter region. Complexes I, II and III are the only gel retardation complexes which contain TFLIC since they were specifically competed with an excess of B box oligonucleotide but not with HTLV oligonucleotide (Clark and Dasgupta, 1990; Figure IA, lanes 11-14). No complex III was observed at 1.5 or 3.5 h post-infection for the wild-type or Sel3C-02infected cell extracts as expected (lanes 3-6). Complex III was observed in the wild-type-infected cell extract at 4.5 and 5.5 h post-infection (lanes 8 and 10); however, no complex IH was formed in Sel3C-02-infected cell extracts at the same times post-infection (lanes 7 and 9). Thus these results with the 3CPro mutant virus provided initial genetic evidence that 3CPro was involved in the formation of the complex HI form of TFIIIC. In vitro transcription analysis of poliovirus mutant Se 13C-02 To determine if Sel3C-02 infection had any effect on pol III transcription the same phosphocellulose fractions were tested for their ability to transcribe the VAI gene in vitro. Inhibition of pol Ill transcription in wild-type poliovirusinfected cell extracts began at 4.5 h post-infection and was complete at 5.5 h post-infection (Figure 2, lanes 6 and 8). In contrast transcription was not inhibited at 4.5 h postinfection in Sel3C-02-infected cell extracts but did begin to show some inhibition at 5.5 h post-infection (lanes 7 and 9). The inhibition of transcription seen at this time-point was surprising since in the gel retardation assay the transcriptionally active form of TFIIIC in complex I appeared intact and no transcriptionally inactive TFIHC in complex III was seen (Figure IA, lane 9). A titration of the Sel3C-02 and wild-type 5.5 h infected cell extracts showed more clearly that complex I remained after Sel3C-02 infection but was absent after wild-type infection (Figure iB). Although the free probe has been run off the gel in Figure IB, gel retardation experiments using an excess of probe also show that complex I is depleted in wild-type poliovirus-infected cell extracts (data not shown). Since in a wild-type poliovirus infection the activity of TFHB is also affected at later times post-infection (Fradkin et al., 1987), we tested whether the inhibition of transcription seen after 5.5 h of Sel3C-02 infection was the result of TFIIIB inactivation. As seen in Figure 3, addition of partially purified TFIIIB restored transcription to the Sel3C-02infected cell extract whereas addition of the complex I form of TFIIIC did not restore transcription (Figure 3, lanes 5 and 7). At 5.5 h post wild-type infection both TFIIIC and TFIIIB activities were inhibited as expected and thus transcription could not be restored by addition of either transcription factor alone (lanes 6 and 8). The TFIIIB and TFIIC fractions used in this experiment were well separated and transcriptionally active since only when added together did they result in VAI transcription (lanes 9, 10 and 11). While Sel3C-02-infected cell extracts looked the same as mock-infected cell extracts in the gel retardation assay (see Figure IA), the decrease in pol III transcription seen at 5.5 h post-infection with the Sel3C-02 virus shows that the cells were productively infected with the mutant virus. Interestingly, since TFIIIB activity is reduced in Sel3C-02-
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Fig. 1. Gel retardation analysis of B box binding activity of extracts from HeLa cells infected for 1.5-5.5 h with the 3CP"O mutant virus Se13C-02, wild-type poliovirus, or mock-infected. (A) Equal amounts (3 gg) of a 0.1-0.6 M KCl phosphocellulose fraction prepared from mock-infected (lanes 1 and 2), wild-type poliovirus-infected (lanes 4, 6, 8 and 10-14) and 3CP'° mutant virus Sel3C-02-infected (lanes 3, 5, 7 and 9) cells at 1.5, 3.5, 4.5 and 5.5 h post-infection were used in the gel retardation assay. Competitions were performed with a 100-fold molar excess of specific B box oligonucleotides (lanes 11 and 12) or non-specific HTLV oligonucleotide (lanes 13 and 14). TFHIC-containing complexes I, II and III are labeled along with the free probe (FP). (B) Increasing amounts (4, 8 and 12 tLg) of the 5.5 h Se13C-02 and wild-type poliovirus-infected phosphocellulose fraction were used in the gel retardation assay. The free probe was run off the gel and is not shown.
infected cell extracts that implies that while 3CPrO is involved in TFIIIC inactivation, it is probably not involved in TFIIIB inactivation. Poliovirus 3CP')' can cleave TFIIIC-containing complexes in vitro The mutant virus experiment implied that 3CPrO was somehow involved in the formation of the transcriptionally inactive complex III form of TFIIIC. To determine if direct treatment of TFIIIC with the 3CPrO protein could result in the generation of complex Ill, 3CP"' overexpressed and purified from E. coli was added to TFIIIC fractions in vitro and then gel retardation reactions were performed. The three different forms of TFIIIC, complexes I, II and III, were separated by FPLC phosphocellulose gradient purification of mock- and poliovirus-infected cell extracts as previously described (Clark and Dasgupta, 1990) and incubated with either purified 3CPrO or a 3CP"° mutant, 3C C147S. The 3Cpr, mutant 3C C 147S has a cysteine to serine substitution at amino acid 147 which inactivates the proteinase (Hammerle et al., 1991). A synthetic peptide with a 3Cpro cleavage site was used to test for activity, or lack of activity, of the proteinases (data not shown). Both proteinases were overexpressed in the same strain of bacteria and purified in the same manner to >98 % purity (Nicklin et al., 1988; Hammerle et al., 1991). When 3Cpr0 was incubated with fractions containing either the complex I or complex H forms of TFIIIC in vitro and then added to a gel retardation reaction, complex III was formed (Figure 4, lanes 2, 5
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Fig. 2. In vitro transcriptional analysis of cell extracts prepared at different times post-infection. Equal amounts (10 ug) of the 0.1-0.6 M phosphocellulose fraction prepared from mock-infected (lanes 1 and 10), wild-type poliovirus-infected (lanes 2, 4, 6 and 8) and 3CPr" mutant virus Sel3C-02-infected (lanes 3, 5, 7 and 9) cells at 1.5, 3.5, 4.5 and 5.5 h post-infection were used in an in vitro transcription assay. The correctly initiated VAI transcript position is indicated.
and 8). Complex III purified from poliovirus-infected cell extracts was not affected by 3CPro treatment (lane 11). The mutant 3C C 147S had no effect on any of the TFIIIC
2943
M.E.Clark et al.
complexes (lanes 3, 6, 9 and 12). This shows that the formation of complex III was indeed caused by 3CPro and not by a different proteinase that possibly copurified with 3CPr'. The gel retardation assay in Figure 4 shows that complexes I and II are cleaved by 3CPro to form complex III, but since the complex I fraction could contain proteins in addition to TFIIIC, we cannot say that TFIIIC is directly cleaved by 3CP'°. TFIIIC must first be cloned before this can be tested directly. The arrow in Figure 4 shows a complex of slower mobility than the complex III found in lanes where complexes I and II were treated with 3CPFO (Figure 4, lanes 2 and 5). This complex has been detected previously in poliovirus-infected cell extracts and was proposed to be an intermediate in the formation in complex III (Clark and Dasgupta, 1990). In order to test whether this complex was indeed an intermediate in the formation of complex Im, a TFHIC fraction containing complex I (and a small amount of complex II) was incubated with varying amounts of 3CPrO. At low amounts of 3CPrO only the intermediate complex was formed (Figure 5, lanes 3 and 4). At higher concentrations both the intermediate
Fig. 3. In vitro transcription analysis of infected cell extracts with the addition of TFIIIB and TFIIIC. Partially purified TFIIIB or TFIIIC (complex I) was added to the wild-type and Sel3C-02 mutant 5.5 h infected cell extract fractions used in Figure 2 (lanes 5-9). The amount of transcription from cell extract fractions without added TFIIIB or TFIIIC is shown in lanes 3 and 4. Transcription from 1.5 h infected cell extract fractions is shown in lanes 1 and 2. Transcription in the presence of TFIIIB alone, TFIIIC alone and TFIIIB plus TFIIIC is shown (lanes 9-1I1).
complex and complex III were formed (lanes 5 and 6). At the highest concentration tested only complex III was formed (lane 7). A similar result was seen using a fraction which contained the complex II form of TFIIIC (data not shown). Thus the complex indicated with an arrow was an intermediate complex, formed in vitro as well as in poliovirus-infected cells, which was the result of incomplete cleavage of TFIIIC from complexes I or II by 3CPKM. Complete cleavage of TFIIIC resulted in complex III formation.
3CPr0 treatment of TFIIIC and transfection of 3CPIO gene in HeLa cells decreases pol 111 transcription in
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Fig. 4. Gel retardation analysis of TFIIIC-containing complexes I, II and III treated with 3CPrO or 3CPr0 mutant 3C C147S. A gel retardation assay was performed using TFIIIC-containing complexes I, II and III isolated from mock- or poliovirus-infected cells using gradient elution of a FPLC phosphocellulose column. These fractions were either treated with 10 yg of 3CP'° (lanes 2, 5, 8 and 11), 10 ltg of 3CPrK mutant 3C C 147S (lanes 3, 6, 9 and 12) or left untreated (lanes 1, 4, 7 and 10). Complexes I, II and Ill are labeled as is the free probe (FP) and an intermediate complex is indicated with an arrow.
2944
vitro Previous results from our laboratory showed that fractions containing the complex III form of TFIIIC purified from poliovirus-infected HeLa cells were transcriptionally inactive in an in vitro transcription assay (Clark and Dasgupta, 1990). Since 3CPrO treatment of transcriptionally active complex I resulted in formation of complex III in the gel retardation assay (Figure 4), it was predicted that 3CPro treatment of complex I would result in decreased pol III transcription in vitro when the other factors necessary for transcription (pol III and TFIIIB) were added. As seen in Figure 6A, 3CPro treatment of complex I resulted in total inhibition of pol III transcription while treatment with the 3CPro mutant 3C C 147S did not affect pol III transcription in vitro. We have tried using inhibitors of 3CPro to show that the proteinase does not cleave other components of the transcriptional machinery but unfortunately these inhibitors themselves abolish transcription (data not shown). However, we do not believe that 3CPro affects the TFIIIB component of the transcriptional apparatus because in the 3CPro mutant virus Se13C-02, TFIIIB activity is still affected (Figure 3). Since direct treatment of TFIIIC with 3CPro in vitro resulted in transcriptional inhibition (Figure 6A), we next asked whether the 3CPro gene transiently transfected into HeLa cells could cause inhibition of pol III transcription. Two plasmids, pRSV3CPro and pRSV3CProrev, were constructed which contained the 3CPro gene downstream of the RSV promoter in the forward and reverse orientations, respectively. Cells were transfected with either pRSV3CPrO,
Inhibition of pol III transcription by poliovirus
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Fig. 5. Titration of 3Cpro with the FPLC-purified complex I form of TFIIIC. The complex I-containing fraction untreated (lane 1), or treated with the indicated amount of 3CPro for 2 h (lanes 3-7), was used in the gel retardation assay. Lane 2 contains complex I treated with 3CPro for only 30 min. The intermediate complex is again indicated with an arrow.
pRSV3CProrev, the parental plasmid pUC 18 or with no DNA. Cells transfected with pRSV3CPro were harvested after 8, 12 and 24 h of transfection while cells transfected with pRSV3CProrev, pUC18 or no DNA were harvested after 12 h of transfection. No morphological differences were seen in cells transfected with the proteinase gene compared with cells transfected with no DNA, indicating that the proteinase gene did not produce a general toxic effect on the cells within 24 h (data not shown). Small-scale nuclear extracts were prepared at each time-point of transfection and used in pol III in vitro transcription reactions. Extracts from cells transfected with the pRSV3CPro for 8 h were not affected in their ability to transcribe the pol III template whereas extracts from cells transfected for 12 and 24 h with the proteinase gene were significantly reduced in their ability to transcribe the VAI gene (Figure 6B, lanes 1-4). Extracts from cells transfected with pRSV3CProrev, pUC 18 or no DNA for 12 h were not inhibited in the pol III transcription assay (lanes 1, 5 and 6). Also no inhibition was observed when these same plasmids were transfected for 24 h (data not shown). We did not expect to see total inhibition of transcription in cells transfected with pRSV3CPro since not all of the cells used to make the extract would incorporate and express the 3CP" gene construct. Thus the transfection efficiency in each experiment was important to the level of inhibition observed. Typically our transfection efficiency was -10-20% as measured by X-gal staining of cells transfected with a 3-gal construct (data not shown). The decrease in transcription from cells transfected with pRSV3CPro could be due to inactivation of TFIIIC or to some non-specific inhibition of the transcription machinery. In order to test these possibilities, a TFIIIC fraction (complex I) was added back to the extract prepared from cells
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Fig. 6. In vitro transcriptional analysis of TFIIIC treated directly with 3CP"° or nuclear extracts from cells transfected with the 3CPrT gene. (A) The complex I form of TFIIIC from mock-infected cells was added to a partially purified fraction containing TFIIIB and pol III (lane 1). In lane 2 the TFIIIC fraction was first incubated with 10 yg of 3CPrK and in lane 3 it was incubated with 10 Ag of the 3CP'0 mutant 3C C147S. (B) Nuclear extracts (9.0 Ag) from cells which were transfected with no DNA for 12 h (lane 1), transfected with the 3Cpr0 gene (pRSV3CPro) for 8, 12 and 24 h (lanes 2, 3 and 4), transfected with the 3CPr" gene in the reverse orientation (pRSV3CPrOrev) for 12 h (lane 5) or transfected with the parental plasmid pUC18 for 12 h (lane 6) were used in an in vitro transcription assay. A fraction containing the complex I form of TFIIIC was added to the extract from cells transfected for 24 h with pRSV3CPro in the in vitro transcription assay (lane 7).
transfected for 24 h with pRSV3CPro. Addition of TFIIIC greatly stimulated transcription showing that the extract was not generally inhibited in its transcriptional ability, and that these extracts were deficient in TFIIIC activity (lane 7). These results strongly suggest that expression of the 3CPro gene, without other viral genes, can cause the decrease in TFIIIC activity and inhibition of pol III transcription seen in poliovirus-infected cells.
Discussion We have shown in this report that poliovirus 3Cpro proteolyzes a TFIIIC-containing gel retardation complex in vivo and in vitro to form the transcriptionally inactive complex III form of TFIIIC. In vivo, complex III was formed in wild-type-infected HeLa cells whereas no complex III was formed in cells infected with a poliovirus containing a point mutation in the 3CP"° gene. In vitro, TFIIICcontaining complexes I and II were cleaved by cloned, purified 3CP"° to form complex EII. This cleavage of TFHIIC can account for the inhibition of pol III transcription observed in poliovirus-infected cells since treatment of the transcriptionally active complex I form of TFIIIC with 3Cpro resulted in inhibition of pol III transcription in an in vitro transcription assay. Finally, the 3CPr° gene transiently transfected into HeLa cells resulted in inhibition of pol III transcription. These data strongly suggest that 3CPro-induced cleavage of TFIIIC is a mechanism by
2945
M.E.Clark et al.
which poliovirus infection results in inhibition of pol III transcription.
Although poliovirus encodes at least two proteinases, poliovirus infection does not result in large-scale or random proteolysis of viral or cellular proteins. When two-dimensional gel analysis is performed on mock versus poliovirus-infected cell extracts < 10 proteins are shown to be altered by poliovirus infection (Urzainqui and Carrasco, 1989). While it cannot be determined until TFHIC is cloned whether it is a direct substrate of 3CPro, 3CP° does induce a change in a TFIIIC-containing complex which converts it from a transcriptionally active to a transcriptionally inactive form. The lack of large-scale modification of cellular proteins in poliovirus-infected cells argues that the conversion of TFIIIC by 3CPrO is a specific event and is not due to generalized proteolysis in the infected cell. Poliovirus replicates in the cytoplasm of infected cells, but in order to cleave TFIIIC in the nucleus, 3CP' must be able to enter the nucleus. There is immunohistochemical evidence that a precursor of 3CPro, 3CD, enters the nucleus after infection (Fernandez-Tomas, 1982). Also since 3CPr' is a small protein (20 kDa), it could enter the nucleus through diffusion. One might expect that if 3CP'O were in the nucleus that it may be active in nuclear extracts made from infected or transfected cells. However, 3Cpro in nuclear extracts is inactive, since when poliovirus-infected cell extracts are mixed with mock-infected cell extracts, pol III transcription is not inhibited in vitro (Fradkin et al., 1987). Also 3CPr° did not remain active in extracts made from cells transfected with the 3CPro gene since when TFIIIC was added to these extracts transcription was restored (Figure 6B). It is not unexpected that 3CPro could lose activity quickly in these extracts since it is prone to autocleavage
(Hammerle et al., 1991). The proteolyzed complex III form of TFIIIC still binds to the B box DNA binding site in the VAI gene but is inactive in an in vitro transcription assay in the presence of added TFIIIB and pol III (Clark and Dasgupta, 1990). We propose that this proteolyzed form of TFIHC can no longer interact with TFIIHIB or pol III and thus cannot form the pre-initiation complex necessary for transcription. Interestingly the complex II form (as well as the complex I form) of TFIIIC is cleaved by 3CPrO in vitro (see Figure 4) but in poliovirusinfected cells the complex II form of TFIIIC is intact (see Figure 1). Although it is not clear how the virus cleaves TFIIIC in complex I and not in complex II, this is advantageous to the virus since the TFIIIC in complex II is already transcriptionally inactive. It is possible that complex I is more accessible for cleavage in vivo because of its conformation in vivo or its sub-nuclear localization. It is also possible that the complex I form of TFIIIC is first made into the complex II form and then the complex II form is cleaved to produce the complex III form. It has been shown previously that the complex II form of TFIIIC can be formed in vitro by acid phosphatase treatment of the complex I form of TFIIIC (Hoeffler et al., 1988; Clark and Dasgupta, 1990). Titration experiments rule out the complex II form of TFIIC being generated directly from the complex I form by 3CPrO cleavage (Figure 5), although it is still possible that the proteinase has an indirect role, possibly by proteolyzing a kinase which normally converts complex II to complex I. The homologous 3CPrO from another picornavirus, foot and mouth disease virus, also inhibits gene expression when transiently transfected into BHK cells (Tesar and Marquardt,
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1990). The inhibition is believed to be caused by cleavage of histone H3 although this has not been shown directly (Falk et al., 1990). Degradation of histone H3 is not observed in poliovirus-infected cells; thus, although poliovirus and foot and mouth disease virus are in the same virus family, they have different mechanisms for transcriptional inhibition. Interestingly, foot and mouth disease virus 3CPrO and poliovirus 3CPrK have different cleavage site specificities (Krausslich and Wimmer, 1988). Although the shut-off of host cell functions by poliovirus has been known for many years no mechanisms have been identified to elucidate these events. We have proposed here that 3Cpro_mediated cleavage of a complex which contains a transcriptionally active form of TFJIC to a transcriptionally inactive form is a mechanism by which poliovirus inhibits pol III transcription. Future studies on the mechanism of pol III transcriptional inhibition by poliovirus will require cloning of TFIIIC and determining if it is cleaved directly by cloned 3CKMT. Future studies will also be aimed at further determining the role of 3CPrO in the inhibition of pol I and pol II transcription in poliovirus-infected cells.
Materials and methods Cells and viruses HeLa cells were grown in spinner culture with minimal essential media (GIBCO Laboratories) supplemented with 1 gll glucose and 6% newborn calf serum. Cells were infected with poliovirus type 1 (Mahoney) or poliovirus mutant Sel3C-02 (Dewalt and Semler, 1987) at a multiplicity of infection (m.o.i.) of 20 as previously described (Dasgupta, 1983). HeLa cell monolayers were grown in Dulbecco's modified Eagle's medium (high glucose) (GIBCO) supplemented with 10% fetal calf serum.
Extract preparation and fractionation Nuclear extracts were prepared from mock- and poliovirus-infected cells as previously described (Dignam et al., 1983). Small-scale nuclear extracts were prepared from transfected cells using a modified Dignam procedure (Lee et al., 1988). Nuclear extracts were fractionated by chromatography on phosphocellulose (Whatman P11) using buffer A [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.9), 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride] as previously described (Segall et al., 1980). One step elution of 0.10-0.60 M KCl was used for some gel retardation and in vitro transcription reactions. Separation of complexes I, II and III with a 70 ml 0.1 -1 M KCI gradient elution of an FPLC phosphocellulose column was performed as described previously (Clark and Dasgupta, 1990).
Gel retardation and in vitro transcription analysis Gel retardation reactions were performed as described (Clark and Dasgupta, 1990). Briefly, each reaction (15 ltl) contained 7.5 Al of protein (1-3 pg) plus buffer A. The total salt concentration was adjusted to 75 mM KCl per reaction. The reactions were started by addition of a premix containing (per reaction) 2 jg of poly(dI-dC), 1.5 ,ul of binding buffer (100 mM Tris, pH 7.6, 5 mM EDTA, 30 mM MgCI2, 50 mM DTT, 500 mM KCl) and 20 000 c.p.m. of the -y-ATP end-labeled Sal I-Bst El fragment of the pVAI gene. Complete gel retardation reactions were incubated at room temperature for 30 min, loaded, with current on, into a 4% polyacrylamide gel in x TBE. Reactions containing the 0.6 M KCI phosphocellulose fraction were loaded onto a 4% polyacrylamide gel in 0.5 x TBE. In vitro transcription reactions were performed as previously described (Fradkin et al., 1987). Essentially transcription reaction mixtures (40 al) contained 30 mM HEPES, pH 7.9, 3 mM DTT, 7.5 mM MgCI,, 8.25 jsg/ml creatine phosphokinase, 500 mM each ATP, CTP and UTP, and 25 pM GTP with 2 pCi of [cz-32P]GTP. pVAI template DNA was added to a concentration of 6.25 Ag/ml. Reactions were stopped after 90 min of incubation at 30°C and processed for electrophoresis in an 8% acrylamide-8 M urea sequencing gel. Proteinase purification and treatment 3CPr" and 3CPr" mutant 3C C147S were overexpressed in E.coli and purified as described (Nicklin et al., 1988; Hammerle et al., 1991). The indicated amount of 3CPrO was added directly to various amounts of TFIIIC
Inhibition of pol III transcription by poliovirus and incubated at 30°C for 2 h, after which salts and reaction mixtures for gel retardation or in titro transcription were added.
Transfections and constructs Two constructs, pRSV3CPro and pRSV3CProrev, were constructed from the RSV expression vector pDEX which contains the RSV promoter and the SV40 polyadenylation site cloned into pUC 18 (Garcia et al., 1988). The XbaI-HindlIl fragment (nucleotides 34-623) encoding the entire 3Cpro gene from pMN35 (Nicklin et al., 1988) was subcloned into pGEM3. The 3CPKm gene was isolated by cutting with BamHI and HinduI and cloned into pDEX which had been cut with HindlIl and BglII giving pRSV3CProrev. pRSV3CP"° was constructed by cutting pDEX with ScaI and HindlIl and ScaI and BglII. These two fragments along with the HindIII-BglII piece of the 3CPro gene were ligated together. Monolayer cultures of HeLa cells (80% confluent) were transfected using Lipofectin reagent (BRL) according to the manufacturer's instructions.
Urzainqui,A. and Carrasco,L. (1989) J. Virol., 63, 4729-4735. Zimmerman,E.R., Hecter,M. and Darnell,J.E. (1963) Virology, 19, 400-408.
Received on MaY 21, 1991; revised on June 28, 1991
Acknowledgements We wish to thank Bert Semler for the Sel3C-02 virus, Dave Harrich and Richard Gaynor for the pDEX expression vector, and Joe Garcia for cloning advice. We are indebted to Steve Kliewer, Arnie Berk and members of the Dasgupta and Berk laboratories for helpful discussions throughout the course of this work. This work was supported by Public Health Service grants Al 27451 to A.D., and Al 15122 and CA 28146 to E.W. from the National Institutes of Health. A.D. is a member of the Molecular Biology Institute, UCLA. M.E.C. is supported by Public Health Service National Research Award Al 07323 from the National Institutes of Health and T.H. is a recipient of a fellowship from Deutsche Forschungsgemeinschaft, Bonn, FRG.
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