Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Feb 11;189(1):274–284. doi: 10.1016/0042-6822(92)90703-R

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

Mark R Denison ∗,1, Philip W Zoltick †,2, Scott A Hughes ∗,3, Bernadette Giangreco ∗, Ann L Olson ‡, Stanley Perlman ‡, Julian L Leibowitz §, Susan R Weiss †
PMCID: PMC7130892  PMID: 1318604

Abstract

Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusionn roteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of >320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

References

  1. Appleyard G., Tisdale M. Inhibition of the growth of human coronavirus 229E by leupeptin. J. Gen. Virol. 1985;66:363–366. doi: 10.1099/0022-1317-66-2-363. [DOI] [PubMed] [Google Scholar]
  2. Baker S.C., Shieh C.-K., Soe L.H., Chang M.-F., Vannier D.M., Lai M.M.C. Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. J. Virol. 1989;63:3693–3699. doi: 10.1128/jvi.63.9.3693-3699.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boursnell M.E.G., Brown T.D.K., Foulds I.J., Green P.F., Tomley F.M., Binns M.M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J. Gen. Virol. 1987;68:57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  4. Breedenbeek P.J., Pachuk C.J., Noten A.F.H., Charite J., Luytjes W., Weiss S.R., Spaan W.J.M. The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59: A highly conserved polymerase is expressed by an efficient ribosomal frame-shifting mechanism. Nucleic Acids Res. 1990;18:1825–1832. doi: 10.1093/nar/18.7.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brierley I., Boursnell M.E.G., Binns M.M., Billimoria B., Blok V.C., Brown T.D.K., Inglis S.C. An efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus IBV. EMBO J. 1987;6:3779–3785. doi: 10.1002/j.1460-2075.1987.tb02713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. de Groot R.J., Hardy W.R., Strauss J.H. Cleavage-site preferences of Sindbis virus polyproteins containing the non-tructural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J. 1990;9:2631–2638. doi: 10.1002/j.1460-2075.1990.tb07445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Denison M., Perlman S. Identification of a putative polymerase gene product in cells infected with murine coronavirus A59. Virology. 1987;157:565–568. doi: 10.1016/0042-6822(87)90303-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denison M.R., Perlman S. Translation and processing of mouse hepatitis virus virion RNA in a cell-free system. J. Virol. 1986;60:12–18. doi: 10.1128/jvi.60.1.12-18.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denison M.R., Zoltick P.W., Leibowitz J.L., Pachuk C.J., Weiss S.R. Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59. J. Virol. 1991;65:3076–3082. doi: 10.1128/jvi.65.6.3076-3082.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorner A.J., Semler B.L., Jackson R.J., Hanecak R., Duprey E., Wimmer E. In vitro translation of poliovirus RNA: Utilization of internal initiation sites in reticulocyte lysate. J. Virol. 1984;50:507–514. doi: 10.1128/jvi.50.2.507-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorbalenya A.E., Koonin E.V., Donchenko A.P., Blinov V.M. Coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Res. 1989;17:4847–4861. doi: 10.1093/nar/17.12.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gordon P.B., Seglen P.O. In: Proteolytic Enzymes: A Practical Approach. Benyon R.J., Bond J.S., editors. IRL Press; Oxford: 1989. pp. 201–210. [Google Scholar]
  13. Krausslich H.-G., Wimmer E. Viral proteinases. Annu. Rev. Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lamblin P. Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal. Biochem. 1978;85:114–125. doi: 10.1016/0003-2697(78)90281-6. [DOI] [PubMed] [Google Scholar]
  16. Lamblin P., Fine J.M. Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents. Anal. Biochem. 1979;98:160–168. doi: 10.1016/0003-2697(79)90721-8. [DOI] [PubMed] [Google Scholar]
  17. Lamblin P., Rochu D., Fine J.M. A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel. Anal. Biochem. 1976;74:567–575. doi: 10.1016/0003-2697(76)90239-6. [DOI] [PubMed] [Google Scholar]
  18. Lee H.-J., Shieh C.-K., Gorbalenya A.E., Koonin E.V., LaMonica N., Tuler J., Bagdzhadhzyan A., Lai M.M.C. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991;180:567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leibowitz J.L., Weiss S.R., Paavola E., Bond C.W. Cell-free translation of murine coronavirus RNA. J. Virol. 1982;43:903–913. doi: 10.1128/jvi.43.3.905-913.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pachuk C.J., Breedenbeek P.J., Zoltick P.W., Spaan W.J.M., Weiss S.R. Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59. Virology. 1989;171:141–148. doi: 10.1016/0042-6822(89)90520-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Palmenburg A.C. Proteolytic processing of picornaviral polyprotein. Annu. Rev. Microbiol. 1990;44:603–623. doi: 10.1146/annurev.mi.44.100190.003131. [DOI] [PubMed] [Google Scholar]
  22. Perlman S., Reese D., Bolger E., Chang L.J., Stoltzfus C.M. MHV nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand MHV RNA. Virus Res. 1987;6:261–272. doi: 10.1016/0168-1702(86)90074-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Saborio J.L., Pong S.-S., Koch G. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 1974;85:195–211. doi: 10.1016/0022-2836(74)90360-x. [DOI] [PubMed] [Google Scholar]
  24. Sawicki D.L., Sawicki S.G. Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis. J. Virol. 1986;57:328–334. doi: 10.1128/jvi.57.1.328-334.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Soe L.H., Shieh C.-K., Baker S.C., Chang M.-F., Lai M.M.C. Sequence and translation of the murine coronavirus 5′-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J. Virol. 1987;61:3968–3976. doi: 10.1128/jvi.61.12.3968-3976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zoltick P.W., Leibowitz J.L., De Vries J.R., Weinstock G.M., Weiss S.R. A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene. Gene. 1989;85:413–420. doi: 10.1016/0378-1119(89)90434-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES