Abstract
2A protease (2Apro) catalyzes the initial cleavage of the poliovirus polyprotein which separates the P1 structural protein precursor from the P2-P3 nonstructural protein precursor. In addition, 2Apro indirectly induces cleavage of the p220 component of eukaryotic initiation factor 4F, which is thought to contribute to the specific inhibition of host cell protein synthesis observed in virus-infected HeLa cells. However, it is unclear whether the trans function of 2Apro which induces host cell shutoff is essential or merely facilitates efficient poliovirus replication. In this study, three point mutations in 2Apro (D38E, Y88L, and Y89L [S. F. Yu and R. E. Lloyd, Virology 182:615-625, 1991]) which cause specific loss of trans but not cis cleavage function were independently introduced into the full-length poliovirus cDNA. In addition, mutations which caused only partial loss of both cis and trans cleavage activities (Y88S) or resulted in a wild-type phenotype (Y88F) were individually introduced. When each of these mutant poliovirus cDNAs was transcribed and translated in vitro, normal proteolytic processing of the viral polyprotein was observed, and p220 was not cleaved in those reactions containing proteases defective in trans function, as expected. Surprisingly, Northern (RNA) blot analysis and reverse transcriptase-PCRs performed after transfection of COS-7 or HeLa cells with these viral RNAs revealed that Y88S and Y88L RNAs replicated at only very low levels. RNA replication could not be detected at all in cells transfected with D38E and Y89L RNAs. Taken together, the results suggest a correlation between the function of 2Apro and productive poliovirus RNA replication in vivo that may be independent of the ability to cause p220 cleavage.
Full Text
The Full Text of this article is available as a PDF (694.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Andino R., Rieckhof G. E., Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. doi: 10.1016/0092-8674(90)90170-j. [DOI] [PubMed] [Google Scholar]
- Bernstein H. D., Sonenberg N., Baltimore D. Poliovirus mutant that does not selectively inhibit host cell protein synthesis. Mol Cell Biol. 1985 Nov;5(11):2913–2923. doi: 10.1128/mcb.5.11.2913. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Charini W. A., Burns C. C., Ehrenfeld E., Semler B. L. trans rescue of a mutant poliovirus RNA polymerase function. J Virol. 1991 May;65(5):2655–2665. doi: 10.1128/jvi.65.5.2655-2665.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Clark M. E., Dasgupta A. A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells. Mol Cell Biol. 1990 Oct;10(10):5106–5113. doi: 10.1128/mcb.10.10.5106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clark M. E., Lieberman P. M., Berk A. J., Dasgupta A. Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. Mol Cell Biol. 1993 Feb;13(2):1232–1237. doi: 10.1128/mcb.13.2.1232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Das S., Dasgupta A. Identification of the cleavage site and determinants required for poliovirus 3CPro-catalyzed cleavage of human TATA-binding transcription factor TBP. J Virol. 1993 Jun;67(6):3326–3331. doi: 10.1128/jvi.67.6.3326-3331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorbalenya A. E., Chumakov K. M., Agol V. I. RNA-binding properties of nonstructural polypeptide G of encephalomyocarditis virus. Virology. 1978 Jul 1;88(1):183–185. doi: 10.1016/0042-6822(78)90122-8. [DOI] [PubMed] [Google Scholar]
- Hambidge S. J., Sarnow P. Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10272–10276. doi: 10.1073/pnas.89.21.10272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hellen C. U., Fäcke M., Kräusslich H. G., Lee C. K., Wimmer E. Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220. J Virol. 1991 Aug;65(8):4226–4231. doi: 10.1128/jvi.65.8.4226-4231.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jore J., De Geus B., Jackson R. J., Pouwels P. H., Enger-Valk B. E. Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. J Gen Virol. 1988 Jul;69(Pt 7):1627–1636. doi: 10.1099/0022-1317-69-7-1627. [DOI] [PubMed] [Google Scholar]
- Kliewer S., Muchardt C., Gaynor R., Dasgupta A. Loss of a phosphorylated form of transcription factor CREB/ATF in poliovirus-infected cells. J Virol. 1990 Sep;64(9):4507–4515. doi: 10.1128/jvi.64.9.4507-4515.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kräusslich H. G., Nicklin M. J., Toyoda H., Etchison D., Wimmer E. Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220. J Virol. 1987 Sep;61(9):2711–2718. doi: 10.1128/jvi.61.9.2711-2718.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
- Lee C. K., Wimmer E. Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis. Virology. 1988 Oct;166(2):405–414. doi: 10.1016/0042-6822(88)90511-9. [DOI] [PubMed] [Google Scholar]
- Liebig H. D., Ziegler E., Yan R., Hartmuth K., Klump H., Kowalski H., Blaas D., Sommergruber W., Frasel L., Lamphear B. Purification of two picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on in vitro translation. Biochemistry. 1993 Jul 27;32(29):7581–7588. doi: 10.1021/bi00080a033. [DOI] [PubMed] [Google Scholar]
- Lloyd R. E., Bovee M. Persistent infection of human erythroblastoid cells by poliovirus. Virology. 1993 May;194(1):200–209. doi: 10.1006/viro.1993.1250. [DOI] [PubMed] [Google Scholar]
- Lloyd R. E., Grubman M. J., Ehrenfeld E. Relationship of p220 cleavage during picornavirus infection to 2A proteinase sequencing. J Virol. 1988 Nov;62(11):4216–4223. doi: 10.1128/jvi.62.11.4216-4223.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lloyd R. E., Jense H. G., Ehrenfeld E. Restriction of translation of capped mRNA in vitro as a model for poliovirus-induced inhibition of host cell protein synthesis: relationship to p220 cleavage. J Virol. 1987 Aug;61(8):2480–2488. doi: 10.1128/jvi.61.8.2480-2488.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Macadam A. J., Ferguson G., Fleming T., Stone D. M., Almond J. W., Minor P. D. Role for poliovirus protease 2A in cap independent translation. EMBO J. 1994 Feb 15;13(4):924–927. doi: 10.1002/j.1460-2075.1994.tb06336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Molla A., Paul A. V., Schmid M., Jang S. K., Wimmer E. Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication. Virology. 1993 Oct;196(2):739–747. doi: 10.1006/viro.1993.1531. [DOI] [PubMed] [Google Scholar]
- Morley S. J., Buhl W. J., Jackson R. J. A rabbit reticulocyte factor which stimulates protein synthesis in several mammalian cell-free systems. Biochim Biophys Acta. 1985 May 24;825(1):57–69. doi: 10.1016/0167-4781(85)90079-x. [DOI] [PubMed] [Google Scholar]
- Nicklin M. J., Kräusslich H. G., Toyoda H., Dunn J. J., Wimmer E. Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4002–4006. doi: 10.1073/pnas.84.12.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Neill R. E., Racaniello V. R. Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. J Virol. 1989 Dec;63(12):5069–5075. doi: 10.1128/jvi.63.12.5069-5075.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pelletier I., Couderc T., Borzakian S., Wyckoff E., Crainic R., Ehrenfeld E., Colbere-Garapin F. Characterization of persistent poliovirus mutants selected in human neuroblastoma cells. Virology. 1991 Feb;180(2):729–737. doi: 10.1016/0042-6822(91)90086-q. [DOI] [PubMed] [Google Scholar]
- Percy N., Belsham G. J., Brangwyn J. K., Sullivan M., Stone D. M., Almond J. W. Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis. J Virol. 1992 Mar;66(3):1695–1701. doi: 10.1128/jvi.66.3.1695-1701.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sommergruber W., Ahorn H., Klump H., Seipelt J., Zoephel A., Fessl F., Krystek E., Blaas D., Kuechler E., Liebig H. D. 2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif. Virology. 1994 Feb;198(2):741–745. doi: 10.1006/viro.1994.1089. [DOI] [PubMed] [Google Scholar]
- Sun X. H., Baltimore D. Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2143–2146. doi: 10.1073/pnas.86.7.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toyoda H., Nicklin M. J., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell. 1986 Jun 6;45(5):761–770. doi: 10.1016/0092-8674(86)90790-7. [DOI] [PubMed] [Google Scholar]
- Wyckoff E. E., Hershey J. W., Ehrenfeld E. Eukaryotic initiation factor 3 is required for poliovirus 2A protease-induced cleavage of the p220 component of eukaryotic initiation factor 4F. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9529–9533. doi: 10.1073/pnas.87.24.9529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wyckoff E. E., Lloyd R. E., Ehrenfeld E. Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity. J Virol. 1992 May;66(5):2943–2951. doi: 10.1128/jvi.66.5.2943-2951.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]
- Ypma-Wong M. F., Semler B. L. In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase. Nucleic Acids Res. 1987 Mar 11;15(5):2069–2088. doi: 10.1093/nar/15.5.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu S. F., Lloyd R. E. Characterization of the roles of conserved cysteine and histidine residues in poliovirus 2A protease. Virology. 1992 Feb;186(2):725–735. doi: 10.1016/0042-6822(92)90039-r. [DOI] [PubMed] [Google Scholar]
- Yu S. F., Lloyd R. E. Identification of essential amino acid residues in the functional activity of poliovirus 2A protease. Virology. 1991 Jun;182(2):615–625. doi: 10.1016/0042-6822(91)90602-8. [DOI] [PubMed] [Google Scholar]