Analysis of Coronavirus Transcription Regulation

  • Myungsoo Joo
  • Shinji Makino
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 380)

Abstract

Insertion of an intergenic region from murine coronavirus mouse hepatitis virus (MHV) into an MHV defective interfering (DI) RNA led to transcription of subgenomic DI RNA in helper virus-infected cells. Using this system we studied how two intergenic regions positioned in close proximity affected subgenomic RNA synthesis. When two intergenic regions were separated by more than 100 nt, slightly less of the larger subgenomic DI RNA (synthesized from the upstream intergenic region) was made; this difference was significant when the intergenic region separation was less than about 35 nucleotides. Deletion of sequences flanking the two intergenic regions inserted in close proximity did not affect transcription. No significant change in the ratio of the two subgenomic DI RNAs was observed when the sequence between the two intergenic regions was altered. Removal of the downstream intergenic region restored transcription of the larger subgenomic DI RNA. These results demonstrated the downstream intergenic sequence was suppressing subgenomic DI RNA synthesis from the upstream intergenic region.

Keywords

Hepatitis Urea Agarose Electrophoresis Dimethyl 

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References

  1. 1.
    Lai, M. M. C., and Stohlman, S. A. RNA of mouse hepatitis virus. J Virol 1978;26: 236–242.PubMedGoogle Scholar
  2. 2.
    Lee, H.-J., Shieh, C.-K., Gorbalenya, A. E., Eugene, E. V, La Monica, N., Tuler, J., Bagdzhadzhyan, A., and 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.PubMedCrossRefGoogle Scholar
  3. 3.
    Pachuk, C. J., Bredenbeek, P. J. Zoltick, P. W., Spaan, W. J. M., and Weiss, S. R. Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis virus, strain A59. Virology 1989; 171:141–148.PubMedCrossRefGoogle Scholar
  4. 4.
    Lai, M. M. C., P. R. Brayton, P. R., Armen, R. C, Patton, C. D., Pugh, C., and Stohlman, S. A. Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3. J Virol 1981;39:823–834.PubMedGoogle Scholar
  5. 5.
    Leibowitz, J. L., Wilhelmsen, K. C, and Bond, C. W. The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM. Virology 1981;114:39–51.PubMedCrossRefGoogle Scholar
  6. 6.
    Lai, M. M. C., Baric, R. S., Brayton, P. R., and Stohlman, S. A. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc Natl Acad Sci USA 1984;81:3626–3630.PubMedCrossRefGoogle Scholar
  7. 7.
    Spaan, W., Delius, H., Skinner, M., Armstrong, J., Rottier, P., Smeekens, S., van der Zeijst, B. A. M., and Siddell,S. G. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J 1983;2:1939–1944.Google Scholar
  8. 8.
    Shieh, C.-K., Soe, L. H., Makino, S., Chang, M.-R, Stohlman, S. A., and Lai, M. M. C. The 5’-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription. Virology 1987;156:321–330.PubMedCrossRefGoogle Scholar
  9. 9.
    Makino, S., Joo, M., and Makino, J. K. A System for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J Virol 1991;65:6031–6041.PubMedGoogle Scholar
  10. 10.
    Makino, S., and Joo, M. Effect of intergenic consensus sequence flanking sequences on coronavirus transcription. J Virol 1993;67:3304–3311.PubMedGoogle Scholar
  11. 11.
    Taguchi, F., Ikeda, T., Makino, S., and Yoshikura, H. A murine coronavirus MHV-S isolate from persistently infected cells has a leader and two consensus sequences between the M and N genes. Virology 1994;198:355–359.PubMedCrossRefGoogle Scholar
  12. 12.
    Hofmann, M. A., Chang, R.-Y., Ku, S., and Brian, D. A. Leader-mRNA junction sequences are unique for each subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection. Virology 1993; 196: 163–171.PubMedCrossRefGoogle Scholar
  13. 13.
    Higuchi, R. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. (eds) PCR protocols. Acadiemic Press, San Diego 1990 pp 177–183.Google Scholar
  14. 14.
    Makino, S., and Lai, M. M. C. High-frequency leader sequence switching during coronavirus defective interfering RNA replication J Virol 1989;63:5285–5292.PubMedGoogle Scholar
  15. 15.
    Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989Google Scholar
  16. 16.
    Makino, S., Shieh, C.-K., Soe, L. H., Baker, S. C, and Lai, M. M. C. Primary structure and translation of a defective interfering RNA of murine coronavirus. Virology 1988;166:550–560.PubMedCrossRefGoogle Scholar
  17. 17.
    Winship, P. R. An improved method for directly sequencing PCR material using dimethyl sulfoxide. Nucleic Acids Res 1989; 17:1266.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Myungsoo Joo
    • 1
  • Shinji Makino
    • 1
  1. 1.Department of MicrobiologyThe University of TexasAustinUSA

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