Communication Vol. 263 No. 36 Issue of December 25 pp. 19267-19269 1988 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1988 by The American’Societ;for Biochemistry and Molecular Biolok. Inc. Printed in U.S.A.

Poliovirus-induced Modification of Host Cell RNA Polymerase I10 Is Prevented by Cycloheximide and Zinc*

(Received for publication, June 16, 1988, and in revised form, October 21, 1988)

Luz Maria Rangel$, Carlos Ferniindez-Tomas$, Michael E. Dahmuss, and Patricio GariglioS From the $Departamento de Genitica y Biologia Moleculnr, Centro de Investigacwn y de Estudios Avanzados-IPN, Apartado Postal 14-740, 07000 Mkxico, D.F., Mkxico and the §Department of Biochemistry and Biophysics, University of California, Davis, California 95616

Infection of HeLa cells with poliovirus results in a decrease in the level of RNA polymerase 110, the tran- scriptionally active form of the enzyme, and a shut- down of host transcription (Rangel, L. M., Fernindez- Tomas, C., Dahmus, M. E., and Gariglio, P. (1987) J. Virol. 61,1002-1006). The effect of cycloheximide on poliovirus-induced modification of host RNA polym- erase I10 was investigated. The inhibition of protein synthesis, at sequential stages during viral replication, prevents the modification of both total and chromatin- bound RNA polymerase 110. Furthermore, the inclu- sion of zinc at a concentration that inhibits the prote- olytic post-translational processing of viral polypro- tein also prevents the modification of RNA polymerase 110. These results suggest that host cell enzyme modi- fication depends on the synthesis and processing of protein(s) encoded by the viral genome.

Poliovirus infection of HeLa cells is followed by a reduction in the number of transcriptionally active DNA-bound RNA polymerase I1 molecules (1). The reduction in enzyme appears to be due to the modification of RNA polymerase I10 (2), the transcriptionally active form of the enzyme (3).

RNA polymerase I1 is responsible for the synthesis of pre- mRNA and consists of three distinct subspecies, 110, IIA, and IIB, that differ in the apparent M, of their largest subunit (110,240,000; IIa, 214,000; and IIb, 180,000, for the calf thymus enzyme). Subspecies IIA and IIB constitute the bulk of most purified preparations of the enzyme and appear to have the same specific activity in nonselective transcription assays (4). Sequence analysis of the gene encoding the largest RNA polymerase I1 subunit indicates that the C-terminal domain

American States (PRDCyT) and Grant 139/768.87 from Consejo del * This research was supported by a grant from the Organization of

Sistema Nacional de Educaci6n Tecnolbgica, M6xico (to C. F-T.), Grants PCSABNA-030914 and PCSACNA-050185 from Consejo Na- cional de Ciencia y Tecnologia and Eli Lilly y Cia. de MBxico (to P. G.), and Research Grant GM33300 from the National Institutes of Health (to M. E. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

of subunit IIa consists of tandem repeats of a heptapeptide consensus sequence (5-7). This domain is not contained in subunit IIb and is extensively phosphorylated in subunit 110 (3,6). Recent results suggest that RNA polymerase I10 is the predominant “in uiuo” form and that it is involved in tran- scription (3,8--10).

Since experimental evidence suggests that viral proteids) might be responsible for the shut-off of host transcription (11-13) it was of interest to investigate the requirements of mature viral proteins for the poliovirus-induced modification of RNA polymerase I10 (2). In an effort to determine if a temporal correlation exists between viral-induced modifying activity and the synthesis and processing of poliovirus pro- teins, we have analyzed the content of both total and chro- matin-bound RNA polymerase I10 in poliovirus-infected HeLa cells under conditions that inhibit protein synthesis or that affect viral protein processing.

EXPERIMENTAL PROCEDURES

Poliovirus infection of HeLa cells and the analysis of RNA polym- erase I10 was carried out as previously described (2).

RESULTS AND DISCUSSION

Infected and mock-infected HeLa cells (2, 14) were treated with cycloheximide (150 rg/ml) at different times after infec- tion (see legend to Fig. 1). At 3 h 30 min postinfection (p.i.)’ cells were harvested and the relative content of RNA polym- erase I10 determined by immunoblotting. As previously re- ported (2) whole cell extracts of poliovirus-infected HeLa cells contain reduced amounts of subunit 110 (Fig. 1, lane l+), which is in keeping with the kinetics of inhibition of host cell RNA synthesis. In contrast, if cycloheximide is present throughout the infection period (Fig. 1, lune 2+) the content of subunit 110 remains almost unchanged up to 3.5 h p.i. (90% of control) (see below). Thus the poliovirus-induced modifi- cation of RNA polymerase I10 is prevented if protein synthe- sis is interrupted during infection.

Kinetic experiments of host and viral protein synthesis in poliovirus-infected HeLa cells (14) show that at a multiplicity of infection of 100, host protein synthesis shut-off begins at 60 min p.i., whereas poliovirus-encoded proteins began to be detectable around 2 h p.i. In order to analyze the time of appearance of the poliovirus-induced modifying activity, in- fected HeLa cells were treated with cycloheximide during the exponential period of viral mRNA translation. The data in lanes 3 and 4 of Fig. 1 are from an experiment in which cycloheximide was added at 2 h 30 min and 2 h 45 min p.i., respectively, and the level of subunit 110 determined at 3 h 30 min (as for lanes 1 and 2). It can be seen that addition of cycloheximide at 2 h 30 min after infection did not prevent the modification of subunit 110. In an effort to quantify these differences, the autoradiogram (Fig. 1) was scanned. As can be seen in Fig. 2, the addition of cycloheximide 2 h 30 min and 2 h 45 min after poliovirus infection resulted in a partial stabilization of subunit 110 (45 and 35%, respectively, of the level in mock-infected cells).

The effect of cycloheximide treatment on chromatin-bound RNA polymerase I10 during poliovirus infection was also examined (Fig. 3). Again, the presence of cycloheximide, from

The abbreviation used is: p.i., postinfection.

19267

19268

240Kd-

RNA Polymerase 110 Modification

1 2 3 4 - + - + - + - +

FIG. 1. Effect of cycloheximide on the level of whole cell RNA polymerase I10 in poliovirus-infected HeLa cells. Spin- ner cultures of HeLa cells were mock or polio-infected at a multiplicity of infection of 100. A t different times after infection, aliquots (4 X lo6 cells) of both cultures were removed and supplemented with cycloheximide (150 pg/ml). A t 3.5 h p.i. cells were harvested and the RNA polymerase I10 (240 kDa) in the whole cell extract was deter- mined by imrn*moblotting (1 X lo6 cells/lane). Lanes 1, cells received no cycloheximide; lanes 2, cells received cycloheximide at zero time, whereas in lanes 3 and 4 cells received cycloheximide at 2 h 30 min and 2 h 45 min p.i., respectively. -, mock-infected cells; +, poliovirus infected cells.

v) x

cycloheximide lrealrnenl ( m i n )

FIG. 2. Quantitation of RNA polymerase subunit IIo in mock and poliovirus-infected cells grown in the presence of cycloheximide. Different lanes of the autoradiogram in Fig. 1 were scanned at 550 nm. The percent of subunit 110 relative to uninfected cells was plotted as a function of time of treatment with cycloheximide in a total period of infection of 3.5 h. 0-"0, mock-infected cells; O " 0 , poliovirus-infected cells. Protein synthesis in poliovirus- infected HeLa cells was measured by [%3]methionine incorporation into acid-insoluble material by a continuous-label experiment from zero to 3.5 h p.i. in the absence and in the presence of the inhibitor.

zero to 3.5 h p.i., prevents the disappearance of the 110 subunit (compare lanes I+ and 2+) in agreement with the results obtained from whole cell extracts (Fig. 1). Thus, these exper- iments demonstrate that the modification of the RNA polym- erase I 1 0 can be bkcked by cycloheximide. Since cyclohexi- mide, a t a concentration of 150 pglml, interrupts poliovirus protein synthesis within 1 min after its addition (15), the above results indicate that the RNA polymerase I 1 0 modifi- cation activity is present at the initiation of the exponential period of poliovirus protein synthesis and that accumulation of viral protein(s) is required for the modification.

240 2 14 180

3 4

1 2 - + - +

4 4

4

+

FIG. 3. Effect of cycloheximide on the level of chromatin- bound RNA polymerase I10 in poliovirus-infected cells. The experiment was carried out as described in Fig. 1, except that the chromatin-bound population of RNA polymerase was analyzed on immunoblots reacted with peroxidase conjugated antibody. Lanes 1 , infection in the presence of cycloheximide; lones 2, infection with no cycloheximide. -, mock-infected cells; +, poliovirus-infected cells.

Zinc chloride has been shown to inhibit the growth of several different types of picornavirus (for references, see Ref. 16). The mechanism of antiviral action is to inhibit the normal cleavage by which the viral polypeptides are processed (17). In poliovirus-infected HeLa cells the three primary transla- tion products (16, 17) become predominant when maturation cleavages are inhibited by 0.8 mM ZnC12 (16). Recently (18), it has been shown that zinc exerts a significant inhibitory effect on the polio 2A sulfhydryl protease activity. We inves- tigated if poliovirus-cleaved proteins are involved in the mod- ification of RNA polymerase 110, taking advantage of the reported ability of zinc to prevent poliovirus maturation cleav- ages. Because the precursor polypeptides are rapidly processed in infected cells, ZnC12 was supplemented (0.8 mM final con- centration) at the time of infection and maintained through- out the 3.5-h infection period. For comparison, a similar culture was maintained in parallel but in the presence of cycloheximide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of poliovirus proteins produced in the presence of 0.8 mM ZnC12 showed the predominance of the primary large translation products as has been previously reported (16) (data not shown) and resulted in 40% inhibition of protein synthesis (see below). Analysis of the RNA polymer- ase subunit 110 content in the whole cell lysate, prepared from poliovirus-infected HeLa cells in the presence or absence of cycloheximide or ZnC12, is shown in Fig. 4, A and B, respec- tively. As can be seen, zinc also prevents the poliovirus- induced modification of the RNA polymerase subunit 110. These results suggest that the processing of viral proteins may be required for the modification of RNA polymerase 110.

At this point it is not possible to rule out the possibility that the effect of zinc on RNA polymerase I 1 0 involves activities other than the blockage of poliovirus protein proc- essing (for review see Refs. 19 and 20). For instance, it is clear from the data in Table I that zinc is less effective in the stabilization of subunit 110 than is cycloheximide (52 uersus 91%, respectively), even though ZnC12 also inhibits protein synthesis. This could be in part due to zinc-dependent pro-

RNA Polymerase 110 Modification 19269

uu FIG. 4. Effect of ZnClz on the level of whole cell RNA po-

lymerase I10 in poliovirus-infected cells. HeLa cells (1 x lo') were infected with poliovirus, and at zero time the culture was divided into three portions; one received ZnClp (0.8 mM final concentration), the second was supplemented with cycloheximide (as described in Fig. l), and the third was allowed to continue in the absence of inhibitor. Whole cell extracts were prepared at 3.5 h p.i. and analyzed for RNA polymerase subunit 110 by immunoblotting (1 X lo6 cells/ lane). Lanes 1, uninfected HeLa cells used as markers. Panel A : lanes 2 and 3, poliovirus-infected cells in the absence or presence of cyclo- heximide, respectively. Panel B: same as panel A but in the absence (lane 2 ) or presence ( l a n e 3 ) of ZnClp, respectively.

TABLE I Poliovirus-induced modification of RNA polymerase IIO in the

presence of cycloheximide and ZnCh Additions during Subunit IIoa Conkntb Source of

cellular lysate infection

arbitrary units %

HeLa cells None 9.1 100 Polio-infected None 2.3 25 Polio-infected Cycloheximide 8.3 91 Polio-infected ZnCln 4.8 52

'The data were obtained from the scanning at 550 nm of the lanes

' Percent subunit 110 remaining after poliovirus infection. in the autoradiogram described in Fig. 4.

teases, such as carboxypeptidases (10, 21)) that may be in- volved in the conversion of subunit 110 to IIb. It is known that high levels of chelator stabilize RNA polymerase I10 (10). The ability of zinc and cycloheximide to prevent the disappearance of RNA polymerase I10 is in agreement with the idea that one or more of the viral-cleaved proteins are a t least in part responsible for the modification of subunit 110.

The poliovirus-induced disappearance of RNA polymerase I10 could result from: (a) dephosphorylation of the C-terminal domain of subunit 110 (3), resulting in the formation of subunit IIa; ( 6 ) limited proteolysis involving the loss of the C-terminal domain (6), resulting in the formation of subunit I b , or ( c ) a combination of the two. Due to the fact that the transfer efficiency and immunoreactivity of subunits 110, IIa, and IIb differ, there is not a simple relationship between the intensity of a band in an immunoblot and the concentration of a given subunit (10). Consequently, the disappearance of subunit I10 is not paralleled by an apparent increase in subunit IIa and/or IIb.

The fact that (a) monoclonal antibody directed against the C-terminal domain inhibits promoter-dependent transcrip- tion (22); ( b ) this domain is conserved in RNA polymerase from yeast to mammals (5 , 6); and ( c ) mutagenesis in the region of IIa gene encoding the C-terminal domain is lethal (23-25) provide strong support for the idea that this domain plays an essential role in transcription. Consequently, the

extensive modification of this domain, such as detected during poliovirus infection, could result in major changes in host cell transcription.

Crawford et al. (26) have shown that addition of a partially purified SlOO extract from mock-infected cells restores activ- ity to a transcriptionally deficient poliovirus-infected HeLa cell extract. The modification of a transcription factor during poliovirus infection has been reported also in RNA polymer- ase 111-mediated transcription (27). The fact that the inhibi- tion of both viral protein synthesis and the post-translational processing of viral proteins results in the stabilization of RNA polymerase I10 does not, however, establish that these pro- teins are directly responsible for the loss of the largest subunit of RNA polymerase I10 following infection. The possibility that the disappearance of RNA polymerase I10 is the result of the shutoff of host transcription, rather than the cause, cannot be eliminated and needs further study.

REFERENCES 1. Flores-Otero, G., Fernindez-Tomas, C., and Gariglio, P. (1982)

2. Rangel, L. M., Fernindez-Tomas, C., Dahmus, M. E., and Gar-

3. Cadena. D. L.. and Dahmus. M. E. (1987) J. Bwl. Chem. 262,

Virology 116,619-628

iglio, P. (1987) J. Virol. 61, 1002-1006

4.

5.

6.

7.

8.

9.

10.

11.

12.

13. 14.

15.

16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

12468-12474

290 Kedinger, C., and Chambon, P. (1972) Eur. J. Biochem. 28,283-

Lori, A. A., Matthew, M., Shales, M., and Ingles, C. J. (1985) Cell

Corden, J. L., Cadena, D. L., Ahearn, J. M., and Dahmus, M. E.

Nonet, M., Sweetser, D., and Young, Y. A. (1987) Cell 50,909-

Bartolomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Bwl.

Garcia-Carranca, A., Miguel, F., Dahmus, M. E., and Gariglio, P.

Kim, W. Y., and Dahmus, M. E. (1986) J. Biol. Chem. 261,

Fernandez-Tomas, C. (1987) in Mechanisms of Viral Toxicity in Animo1 Cells (Carrasco, L., ed) pp. 21-58, CRC Press, Inc., Boca Raton, FL

Kaariainen, L., and Ranki, M. (1984) Annu. Rev. MicrobwL 38, 91-109

Fernindez-Tomas, C. (1982) Virology 116,629-634 Cantero-Aguilar, L., Sanchez-Trujillo, A., and Fernindez-Tomas,

Baltimore, D., Girard, M., and Darnell, J. E. (1966) Virobgy 29,

Semler, B. L., Anderson, C. W., Kitamura, N., Rothberg, P. G., Wishart, W. L., and Wimmer, E. (1981) Proc. Natl. Acad. Sci.

Nicklin, M. J. H., Toyoda, H., Murray, G., and Wimmer, E.

Konig, H., and Rosenwirth, B. (1988) J. Virol. 62,1243-1250 Bond, J. S., and Butler, P. E. (1987) Annu. Rev. Biochem. 65,

Leonard, A., Gerber, G. B., and Leonard, F. (1986) Mutat. Res.

Ivonne, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977)

Dahmus, M. E., and Kedinger, C. (1983) J. BWL Chem. 258,

Allison, L. A., Wong, J. K. C., Danial, V., Moyle, M., and Ingles, C. J. (1988) Mol. Cell. Bwl. 8,321-329

Bartolomei, M. S., Halden, N. F., Ruta, C., and Corden, J. L. (1988) Mol. Cell. Biol. 8, 330-339

Zehring, W. A., Lee, J. M., Weeks, J. R., Jokerst, R. S., and Greenleaf, A. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,3698- 3702

Crawford, N., Fire, A., Samuels, S., Sharp, P., and Baltimore, D. (1981) Cell 27,555-561

Fradkin, L. G., Yoshinaga, S. K., Berk, A. J., and Dasguptz, A. (1987) Mol. Cell. Biol. 7, 3880-3887

42,599-610

(1985) Proc. Natl. Acad. Sci. U. S. A. 82,7934-7938

915

Chem. 26,14226-14231

(1986) Arch. Biochem. Biophys. 251, 232-238

14219-14225

C. (1987) Virology 156,259-267

179-189

U. S. A. 78,3464-3468

(1986) Biotechnology 4,33-42

333-364

168,343-353

J. Bwl. Chem. 252,7610-7616

2303-2307