Home LiteratureArticle Details
PMID: 2846872 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.

Journal of virology ·Vol. 62 ·No. 12 ·1988-12-00 ·Pages 4586-93

Nicklin MJ, Harris KS, Pallai PV, Wimmer E

Abstract

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.

MeSH Terms
3C Viral Proteases Capsid/metabolism Cysteine Endopeptidases/biosynthesis,genetics,isolation & purification,metabolism Electrophoresis, Polyacrylamide Gel Enzyme Precursors/metabolism Escherichia coli/genetics Gene Expression Regulation HeLa Cells Humans Peptides/metabolism Plasmids Poliovirus/enzymology,genetics Protein Precursors/metabolism Recombinant Proteins/biosynthesis,genetics,isolation & purification,metabolism Viral Proteins
Chemicals
Enzyme Precursors Peptides Protein Precursors Recombinant Proteins Viral Proteins Cysteine Endopeptidases 3C Viral Proteases 3C proteases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Nicklin M J
Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Harris K S
Pallai P V
Wimmer E
References (31)
31 references, click to expand
  1. Evidence for histidine in the active site of papain.
    Biochem J. 1968 Aug;108(5):855-9 PMID: 5673530
  2. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor.
    Virology. 1988 Sep;166(1):265-70 PMID: 2842953
  3. Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.
    Eur J Biochem. 1978 Apr 17;85(2):457-62 PMID: 206439
  4. Biochemical evidence for intertypic genetic recombination of polioviruses.
    FEBS Lett. 1980 Aug 25;118(1):109-12 PMID: 6250903
  5. Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.
    J Virol. 1980 Sep;35(3):732-40 PMID: 6252335
  6. Primary structure, gene organization and polypeptide expression of poliovirus RNA.
    Nature. 1981 Jun 18;291(5816):547-53 PMID: 6264310
  7. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.
    Biochem J. 1982 Jan 1;201(1):189-98 PMID: 7044372
  8. Protein processing map of poliovirus.
    J Virol. 1984 Mar;49(3):873-80 PMID: 6321771
  9. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants.
    J Mol Biol. 1984 Apr 25;174(4):561-85 PMID: 6202874
  10. Systematic nomenclature of picornavirus proteins.
    J Virol. 1984 Jun;50(3):957-9 PMID: 6726891
  11. Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase.
    Cell. 1984 Jul;37(3):1063-73 PMID: 6331675
  12. 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):7251-67 PMID: 6384934
  13. Association of poliovirus proteins with the endoplasmic reticulum.
    J Virol. 1984 Dec;52(3):777-83 PMID: 6092710
  14. Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase.
    J Biol Chem. 1985 May 10;260(9):5382-91 PMID: 2985590
  15. Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families.
    FEBS Lett. 1986 Jan 6;194(2):253-7 PMID: 3000829
  16. A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products.
    Virology. 1986 Feb;149(1):114-27 PMID: 3004023
  17. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein.
    Cell. 1986 Jun 6;45(5):761-70 PMID: 3011278
  18. Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.
    Proc Natl Acad Sci U S A. 1986 Aug;83(15):5392-6 PMID: 3016701
  19. Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region.
    J Virol. 1986 Nov;60(2):376-84 PMID: 3021972
  20. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  21. Implications of the picornavirus capsid structure for polyprotein processing.
    Proc Natl Acad Sci U S A. 1987 Jan;84(1):21-5 PMID: 3467351
  22. Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.
    J Bacteriol. 1987 Feb;169(2):751-7 PMID: 3027045
  23. 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):2069-88 PMID: 3031587
  24. Myristylation of picornavirus capsid protein VP4 and its structural significance.
    Nature. 1987 Jun 11-17;327(6122):482-6 PMID: 3035380
  25. 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):4002-6 PMID: 3035560
  26. Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220.
    J Virol. 1987 Sep;61(9):2711-8 PMID: 3039165
  27. Vectors for selective expression of cloned DNAs by T7 RNA polymerase.
    Gene. 1987;56(1):125-35 PMID: 3315856
  28. Capsid protein VP4 of poliovirus is N-myristoylated.
    Proc Natl Acad Sci U S A. 1987 Nov;84(22):7827-31 PMID: 2825164
  29. Purification and partial characterization of poliovirus protease 2A by means of a functional assay.
    J Virol. 1988 Apr;62(4):1243-50 PMID: 2831385
  30. Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro.
    J Gen Virol. 1988 Jul;69 ( Pt 7):1627-36 PMID: 2839599
  31. Strategy and tactics in protein chemistry.
    Biochem J. 1970 Oct;119(5):805-22 PMID: 4923920
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.62.12.4586-4593.1988
Published
1988-12-00
Pages
4586-93
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC254243
Subset
IM
Grants
NIAID NIH HHS · AI15122 · United States
NCI NIH HHS · CA28146 · United States
NCRR NIH HHS · RR02427 · United States
External Links
PubMed source DOI: 10.1128/JVI.62.12.4586-4593.1988 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2