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. 1986 Aug;83(15):5392–5396. doi: 10.1073/pnas.83.15.5392

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

L A Ivanoff, T Towatari, J Ray, B D Korant, S R Petteway Jr
PMCID: PMC386292  PMID: 3016701

Abstract

We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.

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Selected References

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

  1. Argos P., Kamer G., Nicklin M. J., Wimmer E. Similarity in gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families. Nucleic Acids Res. 1984 Sep 25;12(18):7251–7267. doi: 10.1093/nar/12.18.7251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes W. M., Bevan M. Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition. Nucleic Acids Res. 1983 Jan 25;11(2):349–368. doi: 10.1093/nar/11.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Cesareni G., Muesing M. A., Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6313–6317. doi: 10.1073/pnas.79.20.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christie G. E., Platt T. A functional hybrid ribosome binding site in tryptophan operon messenger RNA of Escherichia coli. J Mol Biol. 1980 Nov 5;143(3):335–341. doi: 10.1016/0022-2836(80)90195-3. [DOI] [PubMed] [Google Scholar]
  6. Franssen H., Leunissen J., Goldbach R., Lomonossoff G., Zimmern D. Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. EMBO J. 1984 Apr;3(4):855–861. doi: 10.1002/j.1460-2075.1984.tb01896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [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. 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]
  11. Jacobson M. F., Asso J., Baltimore D. Further evidence on the formation of poliovirus proteins. J Mol Biol. 1970 May 14;49(3):657–669. doi: 10.1016/0022-2836(70)90289-5. [DOI] [PubMed] [Google Scholar]
  12. Klump W., Marquardt O., Hofschneider P. H. Biologically active protease of foot and mouth disease virus is expressed from cloned viral cDNA in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3351–3355. doi: 10.1073/pnas.81.11.3351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Korant B. D. Cleavage of poliovirus-specific polypeptide aggregates. J Virol. 1973 Sep;12(3):556–563. doi: 10.1128/jvi.12.3.556-563.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Nichols B. P., Yanofsky C. Plasmids containing the trp promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes. Methods Enzymol. 1983;101:155–164. doi: 10.1016/0076-6879(83)01011-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Pelham H. R. Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme. Eur J Biochem. 1978 Apr 17;85(2):457–462. doi: 10.1111/j.1432-1033.1978.tb12260.x. [DOI] [PubMed] [Google Scholar]
  19. Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rueckert R. R., Wimmer E. Systematic nomenclature of picornavirus proteins. J Virol. 1984 Jun;50(3):957–959. doi: 10.1128/jvi.50.3.957-959.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Stanway G., Hughes P. J., Mountford R. C., Minor P. D., Almond J. W. The complete nucleotide sequence of a common cold virus: human rhinovirus 14. Nucleic Acids Res. 1984 Oct 25;12(20):7859–7875. doi: 10.1093/nar/12.20.7859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Villafranca J. E., Howell E. E., Voet D. H., Strobel M. S., Ogden R. C., Abelson J. N., Kraut J. Directed mutagenesis of dihydrofolate reductase. Science. 1983 Nov 18;222(4625):782–788. doi: 10.1126/science.6356360. [DOI] [PubMed] [Google Scholar]
  25. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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