Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Oct;69(10):6280–6288. doi: 10.1128/jvi.69.10.6280-6288.1995

A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

I Ventoso 1, L Carrasco 1
PMCID: PMC189526  PMID: 7666528

Abstract

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.

Full Text

The Full Text of this article is available as a PDF (579.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aldabe R., Carrasco L. Induction of membrane proliferation by poliovirus proteins 2C and 2BC. Biochem Biophys Res Commun. 1995 Jan 5;206(1):64–76. doi: 10.1006/bbrc.1995.1010. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bonneau A. M., Sonenberg N. Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection. J Virol. 1987 Apr;61(4):986–991. doi: 10.1128/jvi.61.4.986-991.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrasco L. Picornavirus inhibitors. Pharmacol Ther. 1994;64(2):215–290. doi: 10.1016/0163-7258(94)90040-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. D'Agostino D. M., Felber B. K., Harrison J. E., Pavlakis G. N. The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Mol Cell Biol. 1992 Mar;12(3):1375–1386. doi: 10.1128/mcb.12.3.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davies M. V., Pelletier J., Meerovitch K., Sonenberg N., Kaufman R. J. The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation. J Biol Chem. 1991 Aug 5;266(22):14714–14720. [PubMed] [Google Scholar]
  7. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Etchison D., Milburn S. C., Edery I., Sonenberg N., Hershey J. W. Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem. 1982 Dec 25;257(24):14806–14810. [PubMed] [Google Scholar]
  9. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hagino-Yamagishi K., Nomoto A. In vitro construction of poliovirus defective interfering particles. J Virol. 1989 Dec;63(12):5386–5392. doi: 10.1128/jvi.63.12.5386-5392.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  15. Irurzun A., Pérez L., Carrasco L. Brefeldin A blocks protein glycosylation and RNA replication of vesicular stomatitis virus. FEBS Lett. 1993 Dec 28;336(3):496–500. doi: 10.1016/0014-5793(93)80863-p. [DOI] [PubMed] [Google Scholar]
  16. Lacal J. C., Carrasco L. Modification of membrane permeability in poliovirus-infected HeLa cells: effect of guanidine. J Gen Virol. 1983 Apr;64(Pt 4):787–793. doi: 10.1099/0022-1317-64-4-787. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Malim M. H., Cullen B. R. HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell. 1991 Apr 19;65(2):241–248. doi: 10.1016/0092-8674(91)90158-u. [DOI] [PubMed] [Google Scholar]
  21. Martínez-Abarca F., Alonso M. A., Carrasco L. High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro. J Gen Virol. 1993 Dec;74(Pt 12):2645–2652. doi: 10.1099/0022-1317-74-12-2645. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Novoa I., Feduchi E., Carrasco L. Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro. FEBS Lett. 1994 Nov 21;355(1):45–48. doi: 10.1016/0014-5793(94)01157-5. [DOI] [PubMed] [Google Scholar]
  24. Pal-Ghosh R., Morrow C. D. A poliovirus minireplicon containing an inactive 2A proteinase is expressed in vaccinia virus-infected cells. J Virol. 1993 Aug;67(8):4621–4629. doi: 10.1128/jvi.67.8.4621-4629.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pallansch M. A., Kew O. M., Semler B. L., Omilianowski D. R., Anderson C. W., Wimmer E., Rueckert R. R. Protein processing map of poliovirus. J Virol. 1984 Mar;49(3):873–880. doi: 10.1128/jvi.49.3.873-880.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Palmenberg A. C. Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol. 1990;44:603–623. doi: 10.1146/annurev.mi.44.100190.003131. [DOI] [PubMed] [Google Scholar]
  27. Pérez L., Carrasco L. Lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection. Virology. 1992 Jul;189(1):178–186. doi: 10.1016/0042-6822(92)90693-j. [DOI] [PubMed] [Google Scholar]
  28. Qian X. Y., Alonso-Caplen F., Krug R. M. Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA. J Virol. 1994 Apr;68(4):2433–2441. doi: 10.1128/jvi.68.4.2433-2441.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rodriguez P. L., Carrasco L. Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol. J Virol. 1992 Apr;66(4):1971–1976. doi: 10.1128/jvi.66.4.1971-1976.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sommergruber W., Ahorn H., Zöphel A., Maurer-Fogy I., Fessl F., Schnorrenberg G., Liebig H. D., Blaas D., Kuechler E., Skern T. Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A. J Biol Chem. 1992 Nov 5;267(31):22639–22644. [PubMed] [Google Scholar]
  32. Sonenberg N. Poliovirus translation. Curr Top Microbiol Immunol. 1990;161:23–47. doi: 10.1007/978-3-642-75602-3_2. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Urzainqui A., Carrasco L. Degradation of cellular proteins during poliovirus infection: studies by two-dimensional gel electrophoresis. J Virol. 1989 Nov;63(11):4729–4735. doi: 10.1128/jvi.63.11.4729-4735.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Yu S. F., Benton P., Bovee M., Sessions J., Lloyd R. E. Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity. J Virol. 1995 Jan;69(1):247–252. doi: 10.1128/jvi.69.1.247-252.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2330–2334. doi: 10.1073/pnas.83.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES