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J Virol. Dec 1992; 66(12): 7481–7489.
PMCID: PMC240456

Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase.


A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.

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  • Andino R, Rieckhof GE, Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. [PubMed]
  • Andino R, Rieckhof GE, Trono D, Baltimore D. Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region. J Virol. 1990 Feb;64(2):607–612. [PMC free article] [PubMed]
  • Berger A, Schechter I. Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):249–264. [PubMed]
  • Blair WS, Semler BL. Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase. J Virol. 1991 Nov;65(11):6111–6123. [PMC free article] [PubMed]
  • Burns CC, Lawson MA, Semler BL, Ehrenfeld E. Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage. J Virol. 1989 Nov;63(11):4866–4874. [PMC free article] [PubMed]
  • Charini WA, Burns CC, Ehrenfeld E, Semler BL. trans rescue of a mutant poliovirus RNA polymerase function. J Virol. 1991 May;65(5):2655–2665. [PMC free article] [PubMed]
  • Cordingley MG, Callahan PL, Sardana VV, Garsky VM, Colonno RJ. Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J Biol Chem. 1990 Jun 5;265(16):9062–9065. [PubMed]
  • Cordingley MG, Register RB, Callahan PL, Garsky VM, Colonno RJ. Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli. J Virol. 1989 Dec;63(12):5037–5045. [PMC free article] [PubMed]
  • Fairclough GF, Jr, Fruton JS. Peptide-protein interaction as studied by gel filtration. Biochemistry. 1966 Feb;5(2):673–683. [PubMed]
  • Flanegan JB, Baltimore D. Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proc Natl Acad Sci U S A. 1977 Sep;74(9):3677–3680. [PMC free article] [PubMed]
  • Flanegan JB, Baltimore D. Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. J Virol. 1979 Jan;29(1):352–360. [PMC free article] [PubMed]
  • Hämmerle T, Hellen CU, Wimmer E. Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase. J Biol Chem. 1991 Mar 25;266(9):5412–5416. [PubMed]
  • Hellen CU, Fäcke M, Kräusslich HG, Lee CK, 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. [PMC free article] [PubMed]
  • HUMMEL JP, DREYER WJ. Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta. 1962 Oct 8;63:530–532. [PubMed]
  • Jore J, De Geus B, Jackson RJ, Pouwels PH, Enger-Valk BE. 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. [PubMed]
  • Kean KM, Teterina N, Girard M. Cleavage specificity of the poliovirus 3C protease is not restricted to Gln-Gly at the 3C/3D junction. J Gen Virol. 1990 Nov;71(Pt 11):2553–2563. [PubMed]
  • Kräusslich HG, Hölscher C, Reuer Q, Harber J, Wimmer E. Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol. 1990 May;64(5):2433–2436. [PMC free article] [PubMed]
  • Kuhn RJ, Tada H, Ypma-Wong MF, Semler BL, Wimmer E. Mutational analysis of the genome-linked protein VPg of poliovirus. J Virol. 1988 Nov;62(11):4207–4215. [PMC free article] [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Molla A, Paul AV, Wimmer E. Cell-free, de novo synthesis of poliovirus. Science. 1991 Dec 13;254(5038):1647–1651. [PubMed]
  • Nicklin MJ, Harris KS, Pallai PV, 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. [PMC free article] [PubMed]
  • Nicklin MJ, Kräusslich HG, Toyoda H, Dunn JJ, Wimmer E. 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):4002–4006. [PMC free article] [PubMed]
  • Orr DC, Long AC, Kay J, Dunn BM, Cameron JM. Hydrolysis of a series of synthetic peptide substrates by the human rhinovirus 14 3C proteinase, cloned and expressed in Escherichia coli. J Gen Virol. 1989 Nov;70(Pt 11):2931–2942. [PubMed]
  • Pallai PV, Burkhardt F, Skoog M, Schreiner K, Bax P, Cohen KA, Hansen G, Palladino DE, Harris KS, Nicklin MJ, et al. Cleavage of synthetic peptides by purified poliovirus 3C proteinase. J Biol Chem. 1989 Jun 15;264(17):9738–9741. [PubMed]
  • Palmenberg AC, Pallansch MA, Rueckert RR. Protease required for processing picornaviral coat protein resides in the viral replicase gene. J Virol. 1979 Dec;32(3):770–778. [PMC free article] [PubMed]
  • Rueckert RR, Wimmer E. Systematic nomenclature of picornavirus proteins. J Virol. 1984 Jun;50(3):957–959. [PMC free article] [PubMed]
  • Semler BL, Johnson VH, Dewalt PG, Ypma-Wong MF. Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase. J Cell Biochem. 1987 Jan;33(1):39–51. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Ypma-Wong MF, Dewalt PG, Johnson VH, Lamb JG, Semler BL. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. [PubMed]
  • Ypma-Wong MF, Filman DJ, Hogle JM, Semler BL. 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]

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