Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1991 Nov;65(11):6111–6123. doi: 10.1128/jvi.65.11.6111-6123.1991

Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase.

W S Blair 1, B L Semler 1
PMCID: PMC250289  PMID: 1656088

Abstract

Amino acid insertions or substitutions were introduced into the poliovirus P1 capsid precursor at locations proximal to the two known Q-G cleavage sites to examine the role of the P4 residue in substrate processing by proteinase 3CD. Analysis of the processing profile of P1 precursors containing four-amino-acid insertions into the carboxy terminus of VP3 or a single-amino-acid substitution at the P4 position of the VP3-VP1 cleavage site demonstrates that substitution of the alanine residue in the P4 position of the VP3-VP1 cleavage site significantly affects cleavage at that site by proteinase 3CD. A single-amino-acid substitution at the P4 position of the VP0-VP3 cleavage site, on the other hand, has only a slight effect on 3CD-mediated processing at this cleavage site. Finally, analysis of six amino acid insertion mutations containing Q-G amino acid pairs demonstrates that the in vitro and in vivo selection of a cleavage site from two adjacent Q-G amino acid pairs depends on the presence of an alanine in the P4 position of the cleaved site. Our data provide genetic and biochemical evidence that the alanine residue in the P4 position of the VP3-VP1 cleavage site is a required substrate determinant for the recognition and cleavage of that site by proteinase 3CD and suggest that the P4 alanine residue may be specifically recognized by proteinase 3CD.

Full text

PDF
6111

Images in this article

6114Image
on p.6114
6115Image
on p.6115
6116Image
on p.6116
6118Image
on p.6118
6119Image
on p.6119
6120Image
on p.6120

Selected References

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

  1. Blair W. S., Hwang S. S., Ypma-Wong M. F., Semler B. L. A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation. J Virol. 1990 Apr;64(4):1784–1793. doi: 10.1128/jvi.64.4.1784-1793.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Callahan P. L., Mizutani S., Colonno R. J. Molecular cloning and complete sequence determination of RNA genome of human rhinovirus type 14. Proc Natl Acad Sci U S A. 1985 Feb;82(3):732–736. doi: 10.1073/pnas.82.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Campos R., Villarreal L. P. An SV40 deletion mutant accumulates late transcripts in a paranuclear extract. Virology. 1982 May;119(1):1–11. doi: 10.1016/0042-6822(82)90059-9. [DOI] [PubMed] [Google Scholar]
  4. Clarke B. E., Sangar D. V. Processing and assembly of foot-and-mouth disease virus proteins using subgenomic RNA. J Gen Virol. 1988 Sep;69(Pt 9):2313–2325. doi: 10.1099/0022-1317-69-9-2313. [DOI] [PubMed] [Google Scholar]
  5. Cordingley M. G., Callahan P. L., Sardana V. V., Garsky V. M., Colonno R. J. Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J Biol Chem. 1990 Jun 5;265(16):9062–9065. [PubMed] [Google Scholar]
  6. Dewalt P. G., Semler B. L. Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase. J Virol. 1987 Jul;61(7):2162–2170. doi: 10.1128/jvi.61.7.2162-2170.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Emini E. A., Elzinga M., Wimmer E. Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites. J Virol. 1982 Apr;42(1):194–199. doi: 10.1128/jvi.42.1.194-199.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamlyn P. H., Browniee G. G., Cheng C. C., Gait M. J., Milstein C. Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing. Cell. 1978 Nov;15(3):1067–1075. doi: 10.1016/0092-8674(78)90290-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hogle J. M., Chow M., Filman D. J. Three-dimensional structure of poliovirus at 2.9 A resolution. Science. 1985 Sep 27;229(4720):1358–1365. doi: 10.1126/science.2994218. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Kirkegaard K. Mutations in VP1 of poliovirus specifically affect both encapsidation and release of viral RNA. J Virol. 1990 Jan;64(1):195–206. doi: 10.1128/jvi.64.1.195-206.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kirkegaard K., Nelsen B. Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA. J Virol. 1990 Jan;64(1):185–194. doi: 10.1128/jvi.64.1.185-194.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kuhn R. J., Wimmer E., Semler B. L. Expression of the poliovirus genome from infectious cDNA is dependent upon arrangements of eukaryotic and prokaryotic sequences in recombinant plasmids. Virology. 1987 Apr;157(2):560–564. doi: 10.1016/0042-6822(87)90302-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Larsen G. R., Anderson C. W., Dorner A. J., Semler B. L., Wimmer E. Cleavage sites within the poliovirus capsid protein precursors. J Virol. 1982 Jan;41(1):340–344. doi: 10.1128/jvi.41.1.340-344.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lawson M. A., Dasmahapatra B., Semler B. L. Species-specific substrate interaction of picornavirus 3C proteinase suballelic exchange mutants. J Biol Chem. 1990 Sep 15;265(26):15920–15931. [PubMed] [Google Scholar]
  18. Lawson M. A., Semler B. L. Picornavirus protein processing--enzymes, substrates, and genetic regulation. Curr Top Microbiol Immunol. 1990;161:49–87. [PubMed] [Google Scholar]
  19. Lindberg A. M., Stålhandske P. O., Pettersson U. Genome of coxsackievirus B3. Virology. 1987 Jan;156(1):50–63. doi: 10.1016/0042-6822(87)90435-1. [DOI] [PubMed] [Google Scholar]
  20. Nicklin M. J., Harris K. S., Pallai P. V., Wimmer E. Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro. J Virol. 1988 Dec;62(12):4586–4593. doi: 10.1128/jvi.62.12.4586-4593.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pallai P. V., Burkhardt F., Skoog M., Schreiner K., Bax P., Cohen K. A., Hansen G., Palladino D. E., Harris K. S., Nicklin M. J. Cleavage of synthetic peptides by purified poliovirus 3C proteinase. J Biol Chem. 1989 Jun 15;264(17):9738–9741. [PubMed] [Google Scholar]
  22. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988 Mar;8(3):1103–1112. doi: 10.1128/mcb.8.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Semler B. L., Anderson C. W., Hanecak R., Dorner L. F., Wimmer E. A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide. Cell. 1982 Feb;28(2):405–412. doi: 10.1016/0092-8674(82)90358-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  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. Trono D., Pelletier J., Sonenberg N., Baltimore D. Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region. Science. 1988 Jul 22;241(4864):445–448. doi: 10.1126/science.2839901. [DOI] [PubMed] [Google Scholar]
  28. Vaheri A., Pagano J. S. Infectious poliovirus RNA: a sensitive method of assay. Virology. 1965 Nov;27(3):434–436. doi: 10.1016/0042-6822(65)90126-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Ypma-Wong M. F., Filman D. J., Hogle J. M., Semler B. L. Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs. J Biol Chem. 1988 Nov 25;263(33):17846–17856. [PubMed] [Google Scholar]
  31. 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]
  32. Ypma-Wong M. F., Semler B. L. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. J Virol. 1987 Oct;61(10):3181–3189. doi: 10.1128/jvi.61.10.3181-3189.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES