Murine Coronavirus Gene 1 Polyprotein Contains an Autoproteolytic Activity

  • Susan C. Baker
  • Nicola La Monica
  • Chien-Kou Shieh
  • Michael M. C. Lai
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 276)


The 5′ most gene of the murine Coronavirus genome, gene 1, is presumed to encode the viral RNA-dependent RNA polymerase. cDNA clones representing this gene encompass more than 22 kilobases, suggesting that this region may encode multifunctional polyprotein(s). It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To identify possible functional domains of gene 1 and further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5′-most 5.3 kilobases of the murine Coronavirus mouse hepatitis virus strain JHM were subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. Although p28 is encoded from the first 1 kilobase at the 5′-end of the genome, translation of in vitro transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3 kilobase region was synthesized. This result suggests that the region close to 5.3 kilobases from the 5′-end of the genomic RNA is essential for the proteolytic cleavage and may contain an autoproteolytic activity. Addition of the protease inhibitor ZnCl2 blocked cleavage of the p28 protein. Site-directed mutagenesis of Cys residue 1137 significantly reduced the cleavage of the p28 protein, indicating that this residue, probably in conjuction with a downstream domain, plays an essential role in the cleavage of p28. This Cys residue may be part of a papain-like autoprotease encoded by gene 1.


Rabbit Reticulocyte Lysate Mouse Hepatitis Virus Translation Reaction Primary Translation Product Avian Infectious Bronchitis Virus 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Boursnell, M. E. G., T. D. K. Brown, I. J. Foulds, P. F. Green, F. M. Tomley, and M. M. Binns. 1987. Completion of the sequence of the genome of the Coronavirus avian infectious bronchitis virus. J. Gen.Virol. 68:57–77.PubMedCrossRefGoogle Scholar
  2. 2.
    Brayton, P. R., M. M. C. Lai, C. D. Patton, and S. A. Stohlman. 1982. Characterization of two RNA polymerase activities induced by mouse hepatitis virus. J. Virol. 42:847–853.PubMedGoogle Scholar
  3. 3.
    Brayton, P. R., S. A. Stohlman, and M. M. C. Lai. 1984. Further characterization of mouse hepatitis virus RNA dependent RNA polymerases. Virology 133:197–201.PubMedCrossRefGoogle Scholar
  4. 4.
    Brierly, I., M.E.G. Boursnell, M.M. Binns, B. Bilimoria, V.C. Blok, T.D.K. Brown and S.C. Inglis. 1987. An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the Coronavirus IBV. EMBO 6:3779–3785.Google Scholar
  5. 5.
    Brierley, I. P. Digard and S. C. Inglis. Characterization of an efficient Coronavirus ribosomal frameshift signal: requirement for an RNA pseudoknot. Cell 57: 537–547.Google Scholar
  6. 6.
    Brown, T. D. K., M. E. G. Boursnell, M. M. Binns, and F. M. Tomley. 1986. Cloning and sequencing of 5’ terminal sequences from avian infectious bronchitis virus genomic RNA. J. Gen. Virol. 67:221–228.PubMedCrossRefGoogle Scholar
  7. 7.
    Denison, M. R., and S. Perlman. 1986. Translation and processing of mouse hepatitis virus virion RNA in a cell-free system. J. Virol. 60:12–18.PubMedGoogle Scholar
  8. 8.
    Denison, M. R., and S. Perlman. 1987. Identification of a putative polymerase gene product in cells infected with murine Coronavirus A59. Virology 157:565–568.PubMedCrossRefGoogle Scholar
  9. 9.
    Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko, and V. M. Blinov. 1989. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nuc. Acids Res. 17:4847–4861.CrossRefGoogle Scholar
  10. 10.
    Higuchi, R., B. Krummel, and R. K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nuc. Acids Res. 16: 7351–7367.CrossRefGoogle Scholar
  11. 11.
    Kessler, S. W. (1981). Use of protein A bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. In “Methods in Enzymology” (J.J. Longone and H. Van Vunakis, Eds.), Vol. 73, pp. 442–459. Academic Press, New York.Google Scholar
  12. 12.
    Lai, M. M. C. 1988. Replication of Coronavirus RNA. In “RNA Genetics.” (E. Domingo, J.J. Holland, and P. Ahlquist, Eds.), Vol. I, pp. 115–136. CRC Press, Inc., Boca Raton, Florida.Google Scholar
  13. 13.
    Lai, M. M. C, R. S. Baric, P. R. Brayton, and S. A. Stohlman. 1984. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus-a cytoplasmic RNA virus. Proc. Natl. Acad. Sci. USA 81:3626–3630.PubMedCrossRefGoogle Scholar
  14. 14.
    Lai, M. M. C, P. R. Brayton, R. C. Armen, C. D. Patton, C. Pugh, and S. A. Stohlman. 1981. Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3. J. Virol. 39:823–834.PubMedGoogle Scholar
  15. 15.
    Lai, M. M. C, and S. A. Stohlman. 1978. RNA of mouse hepatitis virus. J. Virol. 26:236–242.PubMedGoogle Scholar
  16. 16.
    Leibowitz, J. L., J. R. DeVries, and M.V. Haspel. 1982. Genetic analysis of murine hepatitis virus strain JHM. J. Virol. 42:1080–1088.PubMedGoogle Scholar
  17. 17.
    Leibowitz, J. L., S. R. Weiss, E. Paavola, and C. W. Bond. 1982. Cell-free translation of murine Coronavirus RNA. J. Virol. 43:905–913.PubMedGoogle Scholar
  18. 18.
    Maizel, J. 1971. Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5:176–246.Google Scholar
  19. 19.
    Makino, S., S. A. Stohlman, and M. M. C. Lai. 1986. Leader sequences of murine Coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. Proc. Natl. Acad. Sci. USA 83:4204–4208.PubMedCrossRefGoogle Scholar
  20. 20.
    Semler, B. L., R. J. Kuhn, and E. Wimmer. 1988. Replication of the poliovirus genome. In “RNA Genetics.” (E. Domingo, J J. Holland, and P. Ahlquist, Eds.), Vol. I, pp. 23–48. CRC Press, Inc., Boca Raton, Florida.Google Scholar
  21. 21.
    Shieh, C.-K., L.H. Soe, S. Makino, M.-F. Chang, S. A. Stohlman, and M. M. C. Lai. 1987. The 5’-end sequence of the murine Coronavirus genome: implications for multiple fusion sites in leader-primed transcription. Virology 156:321–330.PubMedCrossRefGoogle Scholar
  22. 22.
    Siddell, S. G. 1983. Coronavirus JHM: coding assignments of subgenomic mRNAs. J. Gen. Virol. 64:113–125.PubMedCrossRefGoogle Scholar
  23. 23.
    Soe, L. H., C.-K. Shieh, S. C. Baker, M.-F. Chang, and M. M. C. Lai. 1987. Sequence and translation of the murine Coronavirus 5’-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J. Virol. 61:3968–3976.PubMedGoogle Scholar
  24. 24.
    Spaan, W. J. M., H. Delius, M. Skinner, J. Armstrong, P. Rottier, S. Smeekens, B. A. M. van der Zeijst, and S. G. Siddell. 1983. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J. 2:1839–1844.PubMedGoogle Scholar
  25. 25.
    Strauss, J. H. and E. G. Strauss. 1988. Replication of the RNAs of alphaviruses and flaviviruses. In “RNA Genetics.” (E. Domingo, J.J. Holland, and P. Ahlquist, Eds.), Vol. I, pp. 71–90. CRC Press, Inc., Boca Raton, Florida.Google Scholar
  26. 26.
    Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymcrase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074–1078.PubMedCrossRefGoogle Scholar
  27. 27.
    Wege, H., A. MUller, and V. ter Meulen. 1978. Genomic RNA of the murine Coronavirus JHM. J. Gen. Virol. 41:217–227.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Susan C. Baker
    • 1
  • Nicola La Monica
    • 1
  • Chien-Kou Shieh
    • 1
  • Michael M. C. Lai
    • 1
  1. 1.Department of MicrobiologyUniversity of Southern California, School of MedicineLos AngelesUSA

Personalised recommendations