Proteolytic Processing of the MHV Polymerase Polyprotein

Identification of the P28 Cleavage Site and the Adjacent Protein, P65
  • Shanghong Dong
  • Hong-Qiang Gao
  • Susan C. Baker
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 380)


The polymerase gene of Mouse Hepatitis Virus strain JHM (MHV-JHM) encodes a polyprotein larger than 750 kilodaltons. This polyprotein is proposed to be processed by several viral proteinases into functional subunits. The amino-terminal subunit is p28, which is cleaved by the first viral papain-like proteinase domain. In this study, we identified the cleavage site of this papain-like cysteine proteinase by amino acid sequencing of radiolabeled polypeptide adjacent to p28. Proteolysis occurs between the glycine-247 and valine-248 dipeptide bond. To determine which amino acid residues are critical for proteolysis, we preformed site-directed mutagenesis on the coding sequences surrounding the cleavage site and assayed for the efficiency of cleavage of p28 in an in vitro transcription and translation system. We report that glycine-247 and arginine-246 are the most critical residues for efficient processing of p28.


Cleavage Site Tobacco Etch Virus Sindbis Virus Mouse Hepatitis Virus Equine Arteritis Virus 
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  1. 1.
    Lee, H.-J.,Shieh, C.-K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J.,Bagdzhadzhyan, A., andLai, M.M.C. The complete sequence (22 kilobases) of murine coronavirus gene 1encoding the putative proteases and RNApolymerases. Virology 1991; 180: 567–582.PubMedCrossRefGoogle Scholar
  2. 2.
    Pachuk, C. J.,Bredenbeek, P.J., Zoltick, P.W., Spaan, W.J.M.,and Weiss, S.R. Molecularcloning of the gene encoding the putative polymerase of mouse hepatitiscoronavirus, strain A59. Virology 1989; 171: 141–148.PubMedCrossRefGoogle Scholar
  3. 3.
    Baker, S. C.,Shieh, C.-K., Soe, L. H., Chang, M. F., Vannier, D. M., and Lai, M. M. C. Identification of a domain required forautoproteolytic cleavage of murine coronavirus gene A polyprotein. J Virol.1989; 63: 3693–3699.PubMedGoogle Scholar
  4. 4.
    Baker, S. C.,Yokomori, K., Dong, S., Carlisle, R., Gorbalenya, A. E., Koonin, E. V., andLai, M.M.C. Identificationof the catalytic sites of a papain-like cysteine proteinase of murinecoronavirus. J. Virol. 1993; 67:6056–6063.PubMedGoogle Scholar
  5. 5.
    Matsudaira,P. Sequence from picomole quantities of proteins electroblotted ontopolyvinylidene di-fluoride membranes. J.Biol. Chem. 1987; 262: 10.035-10.038.Google Scholar
  6. 6.
    Soe, L. H.,Shieh, C.-K., Baker, S. C., Chang, M.-F., and Lai, M.M.C. Sequence andtranslation of the murinecoronavirus 5’-end genomic RNA reveals theN-terminal structure of the putative RNA polym-erase. J. Virol. 1989; 61: 3968–3976.Google Scholar
  7. 7.
    Dong, S. and Baker,S.C. Determinants of the p28 cleavage site recognized by the first papain-likecysteine proteinase of murine coronavirus.Virology 1994; 204: 541–549.PubMedCrossRefGoogle Scholar
  8. 8.
    Hutchison, C.A., Phillips, S., Edgell, M.H., Gilliam, S., Jahnke, P., and Smith, M.Mutagenesis at a specific position in a DNA sequence.J. Biol. Chem. 1978; 253: 6551–6560.PubMedGoogle Scholar
  9. 9.
    Lewis, M.K.and Thompson, D.V. Efficient site directed in vitro mutagenesis usingampicillin selection. Nucleic Acids Res.1990; 18,3439–3443.PubMedCrossRefGoogle Scholar
  10. 10.
    Snijder, E.J., Wassenaar, A.L.M. and Spaan, W.J.M. The 5’-end of the equine arteritisvirus replicase gene encodes a papainlikecysteine protease. J. Virol. 1992; 66: 7040–7048.PubMedGoogle Scholar
  11. 11.
    Carrington,J.C., Cary, S.M., Parks, T.D. and Dougherty, W.G. A second proteinase encodedby a plant potyvirus genome. EMBO J. 1989;8: 365–370.PubMedGoogle Scholar
  12. 12.
    Carrington,J.C. and Herndon,K.L.Characterization of the potyviral HC-Pro autoproteolytic cleavage site. Virology 1992; 187: 308–315.PubMedCrossRefGoogle Scholar
  13. 13.
    Choi, G. H.,Shapira, R. andNuss, D.L. Cotranslational autoproteolysis involved in geneexpression from adouble-stranded RNA genetic element associated with hypovirulence of thechestnut blight fungus. Proc. Natl. Acad. Sci. USA1991a; 88: 1167–1171.PubMedCrossRefGoogle Scholar
  14. 14.
    Choi, G.H.,Pawlyk, D.M. and Nuss, D.L. The autocatalytic proteinase p29 encoded by ahypovirulenceassociatedvirus of the chestnut blight fungus resembles the potyvirus-encoded proteinaseHC-Pro. Virology 1991b; 183: 747–752.PubMedCrossRefGoogle Scholar
  15. 15.
    Gorbalenya,A.E., Koonin, E.V., Donchenko, A.P. and Blinov, V.M. Coronavirus genome:prediction of putativefunctional domains in the non-structural polyprotein by comparative amino acidsequence analysis. Nuclic Acids Res. 1989;17: 4847–4861.CrossRefGoogle Scholar
  16. 16.
    De Groot, R., Hardy, W.R., Shirako,Y. and Strauss, J.H. Cleavage-site preferences of Sindbis virus polyproteinscontaining the non-structural proteinase. Evidence for temporal regulation ofpolyprotein processing in vivo. EMBO J.1990; 9: 2631–2638.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Shanghong Dong
    • 1
  • Hong-Qiang Gao
    • 1
  • Susan C. Baker
    • 1
  1. 1.Department of Microbiology and ImmunologyLoyola University Medical CenterMaywoodUSA

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