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. 1995 Mar;69(3):1637–1644. doi: 10.1128/jvi.69.3.1637-1644.1995

Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed.

X Zhang 1, M M Lai 1
PMCID: PMC188761  PMID: 7853499

Abstract

Previous studies suggested that coronavirus RNA transcription involves interaction between leader RNA and the intergenic (IG) sequences, probably via protein-RNA interactions (X. M. Zhang, C.-L. Liao, and M. M. C. Lai, J. Virol., 68:4738-4746, 1994; X. M. Zhang and M. M. C. Lai, J. Virol., 68:6626-6633, 1994). To determine whether cellular proteins are involved in this process, we performed UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG (promoter) sequence between genes 6 and 7 (IG7) and the 5' untranslational region of mouse hepatitis virus genomic RNA. We demonstrated that three different cellular proteins (p70, p48, and p35/38) bound to the promoter sequence of the template RNA. Deletion analyses of the template RNA mapped the binding site of p35/38 at the consensus transcription initiation signal. In contrast, the binding of p70 and p48 was less specific. p35/38 is the same protein as the one previously identified to bind to the complementary strand of the leader RNA; its binding affinity to the leader was approximately 15 times stronger than that to IG7. Site-directed mutagenesis of the IG sequence revealed that mutations in the consensus sequence of IG7 (UCUAAUCUAAAC to UCGAAAC and GCUAAAG), which resulted in reduced subgenomic mRNA transcription, also caused correspondingly reduced levels of p35/38 binding. These results demonstrated that the extent of protein binding to the IG sequences correlated with the amounts of subgenomic mRNAs transcribed from the IG site. These studies suggest that these RNA-binding proteins are involved in coronavirus RNA transcription and may represent transcription factors.

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Selected References

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  1. Armstrong J., Smeekens S., Rottier P. Sequence of the nucleocapsid gene from murine coronavirus MHV-A59. Nucleic Acids Res. 1983 Feb 11;11(3):883–891. doi: 10.1093/nar/11.3.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brayton P. R., Lai M. M., Patton C. D., Stohlman S. A. Characterization of two RNA polymerase activities induced by mouse hepatitis virus. J Virol. 1982 Jun;42(3):847–853. doi: 10.1128/jvi.42.3.847-853.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Budzilowicz C. J., Wilczynski S. P., Weiss S. R. Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3' end of the viral mRNA leader sequence. J Virol. 1985 Mar;53(3):834–840. doi: 10.1128/jvi.53.3.834-840.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Compton S. R., Rogers D. B., Holmes K. V., Fertsch D., Remenick J., McGowan J. J. In vitro replication of mouse hepatitis virus strain A59. J Virol. 1987 Jun;61(6):1814–1820. doi: 10.1128/jvi.61.6.1814-1820.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Jeong Y. S., Makino S. Evidence for coronavirus discontinuous transcription. J Virol. 1994 Apr;68(4):2615–2623. doi: 10.1128/jvi.68.4.2615-2623.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Lai M. M. Coronavirus leader-RNA-primed transcription: an alternative mechanism to RNA splicing. Bioessays. 1986 Dec;5(6):257–260. doi: 10.1002/bies.950050606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Stohlman S. A., Baric R. S., Nelson G. N., Soe L. H., Welter L. M., Deans R. J. Specific interaction between coronavirus leader RNA and nucleocapsid protein. J Virol. 1988 Nov;62(11):4288–4295. doi: 10.1128/jvi.62.11.4288-4295.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zhang X., Lai M. M. Unusual heterogeneity of leader-mRNA fusion in a murine coronavirus: implications for the mechanism of RNA transcription and recombination. J Virol. 1994 Oct;68(10):6626–6633. doi: 10.1128/jvi.68.10.6626-6633.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang X., Liao C. L., Lai M. M. Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. J Virol. 1994 Aug;68(8):4738–4746. doi: 10.1128/jvi.68.8.4738-4746.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van der Most R. G., de Groot R. J., Spaan W. J. Subgenomic RNA synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation. J Virol. 1994 Jun;68(6):3656–3666. doi: 10.1128/jvi.68.6.3656-3666.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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