Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Feb 11;114(1):39–51. doi: 10.1016/0042-6822(81)90250-6

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM☆

Julian L Leibowitz 1,2, Kirk C Wilhelmsen 1,3, Clifford W Bond 1,4
PMCID: PMC7131044  PMID: 7281517

Abstract

Seven virus-specific, polyadenylated RNA species have been identified in mouse cells infected with the murine coronaviruses MHV-A59 (A59V) or MHV-JHM (JHMV). MHV-infected 17CL·1 cells were labeled with [32P]orthophosphate in the presence of actinomycin D and the cytoplasmic RNA was extracted and analyzed by agarose gel electrophoresis. These RNA species range in size from 6.3 × 105 to 6.1 × 106 daltons. The A59V and JHMV-specific RNAs have identical molecular weights and comigrate in agarose gels. The largest intracellular RNA species is identical to RNA isolated from purified virions, as determined by agarose gel electrophoresis and oligonucleotide fingerprint studies of ribonuclease T1 digests. Oligonucleotide fingerprints of the six subgenomic RNAS show that the sequences they contain are present in virion RNA, confirming their virus-specific nature. The fingerprinting studies also demonstrate that the six subgenomic RNA species make up a nested set. The sequences present in each RNA species are also present in all larger RNA species. These larger RNAs also contain additional sequences consistent with their greater size. The subgenomic RNAs fulfull many of the criteria for mRNAs. Possible mechanisms for generating these RNAs are discussed.

Footnotes

☆

This work was supported in part by NIH Grants NS 07078, NS 13898, and NS 15211 from the National Institute of Neurological and Communicative Disorders and Stroke and a grant from the National Multiple Sclerosis Society. Portions of it were presented at the 79th Annual Meeting of the American Society for Microbiology in Los Angeles in May of 1979 and at the Symposium on the Biochemistry and Biology of Coronaviruses in Würzburg, West Germany, in October 1980.

References

  1. Bailey J.M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal. Biochem. 1976;70:75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  2. Bailey O.T., Pappenheimer A.M., Cheever F.S., Daniels J.B. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. II. Pathology. J. Exp. Med. 1949;90:195–221. doi: 10.1084/jem.90.3.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bond C.W., Leibowitz J.L., Robb J.A. Pathogenic murine coronaviruses. II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V. Virology. 1979;94:371–384. doi: 10.1016/0042-6822(79)90468-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borun T.W., Scharff M.D., Robbins E. Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochem. Biophys. Acta. 1967;149:302–304. doi: 10.1016/0005-2787(67)90715-0. [DOI] [PubMed] [Google Scholar]
  5. Brezeski H., Kennedy S.I.T. Synthesis of alphavirus-specified RNA. J. Virol. 1978;25:630–640. doi: 10.1128/jvi.25.2.630-640.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guy J.S., Brian D.A. Bovine coronavirus genome. J. Virol. 1979;29:293–300. doi: 10.1128/jvi.29.1.293-300.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Horwitz M.S. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 1971;8:675–683. doi: 10.1128/jvi.8.5.675-683.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lai M.M.C., Stohlman S.A. RNA of mouse hepatitis virus. J. Virol. 1978;26:236–242. doi: 10.1128/jvi.26.2.236-242.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laskey R.A., Mills A.D. Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Lett. 1977;82:314–316. doi: 10.1016/0014-5793(77)80609-1. [DOI] [PubMed] [Google Scholar]
  10. Lee Y.F., Kitamura N., Nomoto A., Wimmer E. Sequence studies of poliovirus RNA. IV. Nucleotide sequence complexities of poliovirus type 1, type 2, and two type 1 defective interfering particles RNAs1 and fingerprint of the poliovirus type 3 genome. J. Gen. Virol. 1979;44:311–322. doi: 10.1099/0022-1317-44-2-311. [DOI] [PubMed] [Google Scholar]
  11. Leibowitz, J. L., and Weiss, S. R. (in press). Murine coronavirus RNA. In “Proceedings of the Symposium on the Biology and Biochemistry of Coronaviruses” (V. terMeulen and S. G. Siddell, eds.). Plenum, New York.
  12. Lomniczi B. Biological properties of avian coronavirus RNA. J. Gen. Virol. 1977;36:531–533. doi: 10.1099/0022-1317-36-3-531. [DOI] [PubMed] [Google Scholar]
  13. Lomniczi B., Kennedy I. Genome of infectious bronchitis virus. J. Virol. 1977;24:99–107. doi: 10.1128/jvi.24.1.99-107.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Macnaughton M.R., Madge M.H. The genome of human coronavirus strain 229E. J. Gen Virol. 1978;39:497–504. doi: 10.1099/0022-1317-39-3-497. [DOI] [PubMed] [Google Scholar]
  15. McMaster G.K., Carmichael G.C. Vol. 74. 1977. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange; pp. 4835–4838. (Proc. Nat. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mishra N.K., Ryan W.L. Ribonucleic acid synthesis in porcine cell cultures infected with transmissible gastroenteritis virus. Amer. J. Vet. Res. 1973;34:185–188. [PubMed] [Google Scholar]
  17. Pettersson U., Sambrook J. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J. Mol. Biol. 1973;73:125–130. doi: 10.1016/0022-2836(73)90164-2. [DOI] [PubMed] [Google Scholar]
  18. Plagemann P.G.W., Swim H.E. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 1966;91:2317–2326. doi: 10.1128/jb.91.6.2317-2326.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robb J.A., Bond C.W. Coronaviridae. In: Fraenkel-Conrat H., Wagner R.R., editors. Vol. 14. Plenum; New York: 1979. pp. 193–247. (Comprehensive Virology). [Google Scholar]
  20. Robb J.A., Bond C.W. Pathogenic murine coronaviruses. I. Characterization of biological behavior in vitro and virus specific intracellular RNA of strongly neurotropic JHMV and weakly neurotropic A59V viruses. Virology. 1979;94:352–370. doi: 10.1016/0042-6822(79)90467-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Robb J.A., Bond C.W., Leibowitz J.L. Pathogenic murine coronaviruses. III. Biological and biochemical characterization of temperature sensitive mutants of JHMV. Virology. 1979;94:385–399. doi: 10.1016/0042-6822(79)90469-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schochetman G., Stevens R.H., Simpson R.W. Presence of infectious polyadenylated RNA in the coronavirus avian bronchitis virus. Virology. 1977;77:772–782. doi: 10.1016/0042-6822(77)90498-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Siddell S.G., Wege H., Barthel A., terMeulen V. Coronavirus JHM: Cell-free synthesis of structural protein p60. J. Virol. 1980;33:10–17. doi: 10.1128/jvi.33.1.10-17.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stern D.F., Kennedy S.I.T. Coronavirus multiplication strategy. I. Identification and characterization of virus-specified RNA. J. Virol. 1980;34:665–674. doi: 10.1128/jvi.34.3.665-674.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stern D.F., Kennedy S.I.T. Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome. J. Virol. 1980;36:440–449. doi: 10.1128/jvi.36.2.440-449.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sturman L.S. Characterization of a coronavirus. I. Structural proteins: effects of preparative conditions on the migration of protein in polyacrylamide gels. Virology. 1977;77:637–649. doi: 10.1016/0042-6822(77)90488-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sturman L.S., Takemoto K.K. Enhanced growth of a murine coronavirus in transformed mouse cells. Infect. Immun. 1972;6:501–507. doi: 10.1128/iai.6.4.501-507.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tannock G.A., Hierholzer J.C. Presence of genomic polyadenylate and absence of detectable virion transcriptase in human coronavirus OC-43. J. Gen. Virol. 1978;39:29–39. doi: 10.1099/0022-1317-39-1-29. [DOI] [PubMed] [Google Scholar]
  29. Tyrell D.A.J., Alexander D.J., Almeida J.D., Cunningham C.H., Easterday B.C., Garwes D.J., Hierholzer J.C., Kapikian A., Macnaughton M.R., McIntonsh K. Coronaviridae: Second Report. Intevirology. 1978;10:321–328. doi: 10.1159/000148996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. vanderZeist, B. (in press). Isolation and characterization of the virus RNA and the subgenomic mRNAs of mouse hepatitis virus MHV A59. In “Proceedings of the Symposium on the Biology and Biochemistry of Coronaviruses” (V. terMeulen and S. G. Siddell, eds.). Plenum, New York.
  31. Wege H., Müller, terMeulen V. Genomic RNA of the murine coronavirus JHM. J. Gen. Virol. 1978;41:217–227. doi: 10.1099/0022-1317-41-2-217. [DOI] [PubMed] [Google Scholar]
  32. Wege H., Wege H., Nagashima K., terMeulen V. Structural polypeptides of the murine coronavirus JHM. J. Gen. Virol. 1979;42:37–47. doi: 10.1099/0022-1317-42-1-37. [DOI] [PubMed] [Google Scholar]
  33. Weiss S. R., and Leibowitz, J. L. (in press). Comparison of the RNAs of murine and human coronaviruses. In “Proceedings of the Symposium on the Biology and Biochemistry of Coronaviruses” (V. terMeulen and S. G. Siddell, eds.). Plenum, New York.
  34. Wilt F.H. The dynamics of maternal poly (A)-containing mRNA in fertilized sea urchin eggs. Cell. 1977;11:673–681. doi: 10.1016/0092-8674(77)90084-8. [DOI] [PubMed] [Google Scholar]
  35. Yogo Y., Hirano N., Hino S., Shibuta H., Matumoto M. Polyadenylate in the virion RNA of mouse hepatitis virus. J. Biochem. 1977;82:1103–1108. doi: 10.1093/oxfordjournals.jbchem.a131782. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES