Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Mar 11;15(5):2069–2088. doi: 10.1093/nar/15.5.2069

In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase.

M F Ypma-Wong, B L Semler
PMCID: PMC340618  PMID: 3031587

Abstract

We describe a completely in vitro system for generating defined poliovirus proteinase mutations and subsequently assaying the phenotypic expression of such mutations. A complete cDNA copy of the entire poliovirus genome has been inserted into a bacteriophage T7 transcription vector. We have introduced proteinase and/or cleavage site mutations into this cDNA. Mutant RNA is transcribed from the altered cDNA template and is subsequently translated in vitro. Employing such a system, we provide direct evidence for the bimolecular cleavage events carried out by the 3C proteinase. We show that specific genetically-altered precursor polypeptides containing authentic Q-G cleavage sites will not act as substrates for 3C either in cis or in trans. We also provide evidence that almost the entire P3 region is required to generate 3C proteinase activity capable of cleaving the P1 precursor to capsid proteins. However, only the 3C portion of P3 is required to generate 3C proteinase activity capable of cleaving P2 and its processing products.

Full text

PDF
2069

Images in this article

2076Image
on p.2076
2078Image
on p.2078
2082Image
on p.2082

Selected References

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

  1. Adhya S., Gottesman M. Control of transcription termination. Annu Rev Biochem. 1978;47:967–996. doi: 10.1146/annurev.bi.47.070178.004535. [DOI] [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Brown B. A., Ehrenfeld E. Translation of poliovirus RNA in vitro: changes in cleavage pattern and initiation sites by ribosomal salt wash. Virology. 1979 Sep;97(2):396–405. doi: 10.1016/0042-6822(79)90350-7. [DOI] [PubMed] [Google Scholar]
  4. Butterworth B. E., Korant B. D. Characterization of the large picornaviral polypeptides produced in the presence of zinc ion. J Virol. 1974 Aug;14(2):282–291. doi: 10.1128/jvi.14.2.282-291.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dorner A. J., Dorner L. F., Larsen G. R., Wimmer E., Anderson C. W. Identification of the initiation site of poliovirus polyprotein synthesis. J Virol. 1982 Jun;42(3):1017–1028. doi: 10.1128/jvi.42.3.1017-1028.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dorner A. J., Semler B. L., Jackson R. J., Hanecak R., Duprey E., Wimmer E. In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate. J Virol. 1984 May;50(2):507–514. doi: 10.1128/jvi.50.2.507-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Golini F., Semler B. L., Dorner A. J., Wimmer E. Protein-linked RNA of poliovirus is competent to form an initiation complex of translation in vitro. Nature. 1980 Oct 16;287(5783):600–603. doi: 10.1038/287600a0. [DOI] [PubMed] [Google Scholar]
  8. Hanecak R., Semler B. L., Anderson C. W., Wimmer E. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3973–3977. doi: 10.1073/pnas.79.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hanecak R., Semler B. L., Ariga H., Anderson C. W., Wimmer E. Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase. Cell. 1984 Jul;37(3):1063–1073. doi: 10.1016/0092-8674(84)90441-0. [DOI] [PubMed] [Google Scholar]
  10. Kaplan G., Lubinski J., Dasgupta A., Racaniello V. R. In vitro synthesis of infectious poliovirus RNA. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8424–8428. doi: 10.1073/pnas.82.24.8424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., van der Werf S. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  14. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Omata T., Kohara M., Sakai Y., Kameda A., Imura N., Nomoto A. Cloned infectious complementary DNA of the poliovirus Sabin 1 genome: biochemical and biological properties of the recovered virus. Gene. 1984 Dec;32(1-2):1–10. doi: 10.1016/0378-1119(84)90026-x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Palmenberg A. C., Rueckert R. R. Evidence for intramolecular self-cleavage of picornaviral replicase precursors. J Virol. 1982 Jan;41(1):244–249. doi: 10.1128/jvi.41.1.244-249.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  20. Phillips B. A., Emmert A. Modulation of the expression of poliovirus proteins in reticulocyte lysates. Virology. 1986 Jan 30;148(2):255–267. doi: 10.1016/0042-6822(86)90323-5. [DOI] [PubMed] [Google Scholar]
  21. Putnak J. R., Phillips B. A. Picornaviral structure and assembly. Microbiol Rev. 1981 Jun;45(2):287–315. doi: 10.1128/mr.45.2.287-315.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Racaniello V. R., Baltimore D. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science. 1981 Nov 20;214(4523):916–919. doi: 10.1126/science.6272391. [DOI] [PubMed] [Google Scholar]
  23. Semler B. L., Anderson C. W., Kitamura N., Rothberg P. G., Wishart W. L., Wimmer E. Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3464–3468. doi: 10.1073/pnas.78.6.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Semler B. L., Dorner A. J., Wimmer E. Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals. Nucleic Acids Res. 1984 Jun 25;12(12):5123–5141. doi: 10.1093/nar/12.12.5123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shih D. S., Shih C. T., Kew O., Pallansch M., Rueckert R., Kaesberg P. Cell-free synthesis and processing of the proteins of poliovirus. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5807–5811. doi: 10.1073/pnas.75.12.5807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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 Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES