Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Aug;68(8):4738–4746. doi: 10.1128/jvi.68.8.4738-4746.1994

Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis.

X Zhang 1, C L Liao 1, M M Lai 1
PMCID: PMC236413  PMID: 8035476

Abstract

Mouse hepatitis virus (MHV), a coronavirus, utilizes a discontinuous transcription mechanism for subgenomic mRNA synthesis. Previous studies (C.-L. Liao and M. C. C. Lai, J. Virol. 68:4727-4737, 1994) have demonstrated that an upstream cis-acting leader sequence serves as a transcriptional enhancer, but the mechanism of transcriptional regulation is not clear. In this study, we constructed a series of defective interfering (DI) RNAs containing the chloramphenicol acetyltransferase (CAT) gene behind a differentially expressed transcription initiation (intergenic) sequence (for mRNA2-1). These DI RNAs had different copy numbers of the UCUAA pentanucleotide sequence at the 3' end of the leader. Transfection of these DI RNA constructs into cells infected with a helper MHV, which contains either two or three UCUAA copies at the 3' end of the leader, resulted in differential expression of CAT activities. We demonstrated that the copy number of UCUAA repeats in the leaders of both helper viral and DI RNAs affected the level of CAT activity, suggesting that MHV leader RNA could regulate both in trans and in cis the transcription of subgenomic mRNAs. The leader RNA of subgenomic mRNAs was derived from either the trans- or the cis-acting leader. Furthermore, insertion of a UA-rich sequence (UUUAUAAAC) immediately downstream of the leader in DI RNA, to match the sequence of helper viral RNA, enhanced the CAT activity by threefold, suggesting that this nine-nucleotide sequence is a cis-acting element. Interestingly, when the nine-nucleotide sequence was absent in DI RNA, the leaders of subgenomic mRNAs were exclusively derived from the helper virus. In contrast, when the nine-nucleotide sequence was present in DI RNA, the leaders were derived from both helper viral and DI RNAs. These results suggest that the nine-nucleotide sequence either is required for the leader RNA to initiate mRNA synthesis or, alternatively, serves as a transcription terminator for the leader RNA synthesis. However, when a constitutively expressed intergenic sequence (for mRNA7) was used, no difference in transcription efficiency was noted, regardless of the copy number of UCUAA in the DI RNA and helper virus. This study thus indicates that MHV subgenomic RNA transcription requires the interaction among the intergenic (promoter) sequence, a trans-acting leader, and a cis-acting leader sequence. A novel model of transcriptional regulation of coronavirus subgenomic mRNAs is presented.

Full text

PDF
4738

Images in this article

4742Image
on p.4742
4743Image
on p.4743

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker S. C., Lai M. M. An in vitro system for the leader-primed transcription of coronavirus mRNAs. EMBO J. 1990 Dec;9(12):4173–4179. doi: 10.1002/j.1460-2075.1990.tb07641.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Furuya T., Lai M. M. Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J Virol. 1993 Dec;67(12):7215–7222. doi: 10.1128/jvi.67.12.7215-7222.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hirano N., Fujiwara K., Hino S., Matumoto M. Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture. Arch Gesamte Virusforsch. 1974;44(3):298–302. doi: 10.1007/BF01240618. [DOI] [PubMed] [Google Scholar]
  4. Jeong Y. S., Makino S. Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication. J Virol. 1992 Jun;66(6):3339–3346. doi: 10.1128/jvi.66.6.3339-3346.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Joo M., Makino S. Mutagenic analysis of the coronavirus intergenic consensus sequence. J Virol. 1992 Nov;66(11):6330–6337. doi: 10.1128/jvi.66.11.6330-6337.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. La Monica N., Yokomori K., Lai M. M. Coronavirus mRNA synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences. Virology. 1992 May;188(1):402–407. doi: 10.1016/0042-6822(92)90774-J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lai M. M., Baric R. S., Brayton P. R., 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 U S A. 1984 Jun;81(12):3626–3630. doi: 10.1073/pnas.81.12.3626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lai M. M., Brayton P. R., Armen R. C., Patton C. D., Pugh C., Stohlman S. A. Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3. J Virol. 1981 Sep;39(3):823–834. doi: 10.1128/jvi.39.3.823-834.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lai M. M. Coronavirus: organization, replication and expression of genome. Annu Rev Microbiol. 1990;44:303–333. doi: 10.1146/annurev.mi.44.100190.001511. [DOI] [PubMed] [Google Scholar]
  10. Lai M. M., Makino S., Soe L. H., Shieh C. K., Keck J. G., Fleming J. O. Coronavirus: a jumping RNA transcription. Cold Spring Harb Symp Quant Biol. 1987;52:359–365. doi: 10.1101/sqb.1987.052.01.041. [DOI] [PubMed] [Google Scholar]
  11. Lai M. M., Patton C. D., Baric R. S., Stohlman S. A. Presence of leader sequences in the mRNA of mouse hepatitis virus. J Virol. 1983 Jun;46(3):1027–1033. doi: 10.1128/jvi.46.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee H. J., Shieh C. K., Gorbalenya A. E., Koonin E. V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M. M. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991 Feb;180(2):567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leibowitz J. L., Wilhelmsen K. C., Bond C. W. The virus-specific intracellular RNA species of two murine coronaviruses: MHV-a59 and MHV-JHM. Virology. 1981 Oct 15;114(1):39–51. doi: 10.1016/0042-6822(81)90250-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liao C. L., Lai M. M. Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription. J Virol. 1994 Aug;68(8):4727–4737. doi: 10.1128/jvi.68.8.4727-4737.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Makino S., Fujioka N., Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol. 1985 May;54(2):329–336. doi: 10.1128/jvi.54.2.329-336.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Makino S., Joo M. Effect of intergenic consensus sequence flanking sequences on coronavirus transcription. J Virol. 1993 Jun;67(6):3304–3311. doi: 10.1128/jvi.67.6.3304-3311.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Makino S., Joo M., 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 Nov;65(11):6031–6041. doi: 10.1128/jvi.65.11.6031-6041.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Makino S., Keck J. G., Stohlman S. A., Lai M. M. High-frequency RNA recombination of murine coronaviruses. J Virol. 1986 Mar;57(3):729–737. doi: 10.1128/jvi.57.3.729-737.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makino S., Lai M. M. Evolution of the 5'-end of genomic RNA of murine coronaviruses during passages in vitro. Virology. 1989 Mar;169(1):227–232. doi: 10.1016/0042-6822(89)90060-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Makino S., Lai M. M. High-frequency leader sequence switching during coronavirus defective interfering RNA replication. J Virol. 1989 Dec;63(12):5285–5292. doi: 10.1128/jvi.63.12.5285-5292.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Makino S., Soe L. H., Shieh C. K., Lai M. M. Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs. J Virol. 1988 Oct;62(10):3870–3873. doi: 10.1128/jvi.62.10.3870-3873.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Makino S., Stohlman S. A., Lai M. M. Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4204–4208. doi: 10.1073/pnas.83.12.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sawicki S. G., Sawicki D. L. Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis. J Virol. 1990 Mar;64(3):1050–1056. doi: 10.1128/jvi.64.3.1050-1056.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shieh C. K., Lee H. J., Yokomori K., La Monica N., Makino S., Lai M. M. Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome. J Virol. 1989 Sep;63(9):3729–3736. doi: 10.1128/jvi.63.9.3729-3736.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shieh C. K., Soe L. H., Makino S., Chang M. F., Stohlman S. A., Lai M. M. The 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription. Virology. 1987 Feb;156(2):321–330. doi: 10.1016/0042-6822(87)90412-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spaan W., Delius H., Skinner M., Armstrong J., Rottier P., Smeekens S., van der Zeijst B. A., Siddell S. G. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J. 1983;2(10):1839–1844. doi: 10.1002/j.1460-2075.1983.tb01667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yokomori K., Banner L. R., Lai M. M. Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses. Virology. 1991 Aug;183(2):647–657. doi: 10.1016/0042-6822(91)90994-M. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES