Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1991 Nov;65(11):6031–6041. doi: 10.1128/jvi.65.11.6031-6041.1991

A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion.

S Makino 1, M Joo 1, J K Makino 1
PMCID: PMC250269  PMID: 1656085

Abstract

A system that exploits defective interfering (DI) RNAs of mouse hepatitis virus (MHV) for deciphering the mechanisms of coronavirus mRNA transcription was developed. A complete cDNA clone of MHV DI RNA containing an inserted intergenic region, derived from the area of genomic RNA between genes 6 and 7, was constructed. After transfection of the in vitro-synthesized DI RNA into MHV-infected cells, replication of genomic DI RNA as well as transcription of the subgenomic DI RNA was observed. S1 nuclease protection experiments, sequence analysis, and Northern (RNA) blotting analysis revealed that the subgenomic DI RNA contained the leader sequence at its 5' end and that the body of the subgenomic DI RNA started from the inserted intergenic sequence. Two subgenomic DI RNAs were synthesized after inserting two intergenic sites into the MHV DI RNA. Metabolic labeling of virus-specific protein in DI RNA replicating cells demonstrated that a protein was translated from the subgenomic DI RNA, which can therefore be considered a functional mRNA. Transfection study of gel-purified genomic DI RNA and subgenomic DI RNA revealed that the introduction of the genomic DI RNA, but not subgenomic DI RNA, into MHV-infected cells was required for synthesis of the subgenomic DI RNA. A series of deletion mutations in the intergenic site demonstrated that the sequence flanking the consensus sequence of UCUAAAC affected the efficiency of subgenomic DI RNA transcription and that the consensus sequence was necessary but not sufficient for the synthesis of the subgenomic DI RNA.

Full text

PDF
6031

Images in this article

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. Baric R. S., Stohlman S. A., Razavi M. K., Lai M. M. Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription. Virus Res. 1985 Jul;3(1):19–33. doi: 10.1016/0168-1702(85)90038-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Lai M. M., Patton C. D., Stohlman S. A. Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length. J Virol. 1982 Nov;44(2):487–492. doi: 10.1128/jvi.44.2.487-492.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lai M. M., Stohlman S. A. RNA of mouse hepatitis virus. J Virol. 1978 May;26(2):236–242. doi: 10.1128/jvi.26.2.236-242.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Makino S., Shieh C. K., Keck J. G., Lai M. M. Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs. Virology. 1988 Mar;163(1):104–111. doi: 10.1016/0042-6822(88)90237-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Makino S., Shieh C. K., Soe L. H., Baker S. C., Lai M. M. Primary structure and translation of a defective interfering RNA of murine coronavirus. Virology. 1988 Oct;166(2):550–560. doi: 10.1016/0042-6822(88)90526-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Makino S., Taguchi F., Fujiwara K. Defective interfering particles of mouse hepatitis virus. Virology. 1984 Feb;133(1):9–17. doi: 10.1016/0042-6822(84)90420-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Makino S., Taguchi F., Hirano N., Fujiwara K. Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain. Virology. 1984 Nov;139(1):138–151. doi: 10.1016/0042-6822(84)90335-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Makino S., Yokomori K., Lai M. M. Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal. J Virol. 1990 Dec;64(12):6045–6053. doi: 10.1128/jvi.64.12.6045-6053.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [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. Sethna P. B., Hofmann M. A., Brian D. A. Minus-strand copies of replicating coronavirus mRNAs contain antileaders. J Virol. 1991 Jan;65(1):320–325. doi: 10.1128/jvi.65.1.320-325.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sethna P. B., Hung S. L., Brian D. A. Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5626–5630. doi: 10.1073/pnas.86.14.5626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Skinner M. A., Siddell S. G. Coronavirus JHM: nucleotide sequence of the mRNA that encodes nucleocapsid protein. Nucleic Acids Res. 1983 Aug 11;11(15):5045–5054. doi: 10.1093/nar/11.15.5045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Soe L. H., Shieh C. K., Baker S. C., Chang M. F., Lai M. M. Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J Virol. 1987 Dec;61(12):3968–3976. doi: 10.1128/jvi.61.12.3968-3976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stohlman S. A., Lai M. M. Phosphoproteins of murine hepatitis viruses. J Virol. 1979 Nov;32(2):672–675. doi: 10.1128/jvi.32.2.672-675.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sturman L. S., Holmes K. V., Behnke J. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J Virol. 1980 Jan;33(1):449–462. doi: 10.1128/jvi.33.1.449-462.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weiss B., Nitschko H., Ghattas I., Wright R., Schlesinger S. Evidence for specificity in the encapsidation of Sindbis virus RNAs. J Virol. 1989 Dec;63(12):5310–5318. doi: 10.1128/jvi.63.12.5310-5318.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yokomori K., La Monica N., Makino S., Shieh C. K., Lai M. M. Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology. 1989 Dec;173(2):683–691. doi: 10.1016/0042-6822(89)90581-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zimmern D., Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. doi: 10.1073/pnas.75.9.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van der Most R. G., Bredenbeek P. J., Spaan W. J. A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol. 1991 Jun;65(6):3219–3226. doi: 10.1128/jvi.65.6.3219-3226.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES