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. 2004 Jun 9;108(2):424–434. doi: 10.1016/0042-6822(81)90449-9

Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59)

Willy JM Spaan 1, Peter JM Rottier 1, Marian C Horzinek 1, Bernard AM Van Der Zeijst 1,1
PMCID: PMC7130792  PMID: 6258295

Abstract

We have determined the kinetics of virus production and virus-specific RNA synthesis in Sac(−) cells infected with mouse hepatitis virus strain A59 (MHV-A59). Immunofluorescence showed that all cells became infected at a multiplicity of 10 PFU/cell. The virus was concentrated and purified to obtain the high titered stocks needed for these one-step growth experiments. Release of virus into the culture medium started 4 hr after infection (pi) and was complete at 10 hr pi. Synthesis of virus-specific RNA, measured by the incorporation of [3H]uridine in the presence of 1 μg/ml actinomycin D, also started at 4 hr pi and its maximum rate occurred between 6 and 8 hr pi. RNA labeled during this period was isolated from infected cells. About 50% of this RNA bound to oligo(dT)-cellulose; this material was denatured with glyoxal-dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. Seven RNA species with the following molecular weights were present: 5.6 × 106 (RNA1), 4.0 × 106 (RNA2), 3.0 × 106 (RNA3), 1.4 × 106 (RNA4), 1.2 × 106 (RNA5), 0.9 × 106 (RNA6), and 0.6 × 106 (RNA7). RNA1 comigrated with the viral genome. Artifacts caused by defective interfering particles or breakdown of RNA were excluded. To determine whether these RNA species were functional as messengers in infected cells, virus-specific RNAs present in polyribosomes were analyzed. EDTA treatment was used to discriminate between RNA present in polyribosomes and in EDTA-resistant, presumably ribonucleoprotein, particles. Most (91%) of RNA1 was present in EDTA-resistant particles; the remainder and all other RNAs synthesized between 6 and 8 hr pi were present in polyribosomes. We conclude that MHV-A59 has six subgenomic mRNAs. Since the total molecular mass (11.1 × 106 daltons) of these messengers is about twice that of the viral genome, sequence homologies must exist between the mRNAs. The position of these homologous regions and the translation products of each of the mRNAs remain to be determined.

References

  1. Baltimore D. Expression of animal virus genomes. Bacteriol. Rev. 1971;35:235–241. doi: 10.1128/br.35.3.235-241.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cheever F.S., Daniels J.B., Pappenheimer A.M., Bailey O.T. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myeline. I. Isolation and biological properties of the virus. J. Exp. Med. 1949;90:181–194. doi: 10.1084/jem.90.3.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deininger P., Esty A., Laporte P., Friedmann T. Nucleotide sequence and genetic organization of the polyoma late region: Features common to the polyoma early region and SV40. Cell. 1979;18:771–779. doi: 10.1016/0092-8674(79)90130-2. [DOI] [PubMed] [Google Scholar]
  4. Dick G.W.A., Niven J.S.F., Gledhill A.W. A virus related to that causing hepatitis in mice (MHV) Brit. J. Exp. Pathol. 1956;37:90–98. [PMC free article] [PubMed] [Google Scholar]
  5. Fiers W., Contreras R., Haegeman G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature (London) 1978;273:113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  6. Gledhill A.W., Andrewes C.H. A hepatitis virus of mice. Brit. J. Exp. Pathol. 1951;22:559–568. [PMC free article] [PubMed] [Google Scholar]
  7. Godson G.N., Sinsheimer R.L. The replication of bacteriophage MS2. VI. Interaction between bacteriophage RNA and cellular components in MS2-infected Escherichia coli. J. Mot. Biol. 1967;23:495–521. doi: 10.1016/s0022-2836(67)80121-9. [DOI] [PubMed] [Google Scholar]
  8. Haspel M.V., Lampert P.W., Oldstone M.B.A. Vol. 75. 1978. Temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination; pp. 4033–4036. (Proc. Nat. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herndon R.M., Griffon D.E., McCormick U., Weiner L.P. Mouse hepatitis virus-induced recurrent demyelination. Arch. Neurol. 1975;32:32–35. doi: 10.1001/archneur.1975.00490430054008. [DOI] [PubMed] [Google Scholar]
  10. Herndon R.M., Price P.L., Weiner L.P. Regeneration of oligodendroglia during recovery from demyelinating disease. Science. 1977;195:693–694. doi: 10.1126/science.190678. [DOI] [PubMed] [Google Scholar]
  11. Hierholzer J.C., Broderson J.R., Murphy F.A. New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice. Infect. Immun. 1979;24:508–522. doi: 10.1128/iai.24.2.508-522.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hunter T.R., Hunt T., Knowland J., Zimmern D. Messenger RNA for the coat protein of tobacco mosaic virus. Nature (London) 1976;260:759–764. doi: 10.1038/260759a0. [DOI] [PubMed] [Google Scholar]
  13. Kääriäinen L., Söderlund H. Structure and replication of alpha-viruses. Curr. Top. Microbial. Immunol. 1978;81:15–69. doi: 10.1007/978-3-642-46388-4_2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lampert P.W., Sims J.K., Kniazeff A.J. Mechanism of demyelination in JHM virus encephalomyelitis. Electron microscopic studies. Acta Neuropathol. 1973;24:76–85. doi: 10.1007/BF00691421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laskey R.A., Mills A.D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 1975;56:335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  17. McIntosh K. Coronaviruses: a comparative review. Curr. Top. Microbial. Immunol. 1974;63:85–129. [Google Scholar]
  18. McMaster G.K., Carmichael G.G. 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]
  19. Manaker R.A., Piczak C.V., Miller A.A., Stanton M.F. A hepatitis virus complicating studies with mouse leukemias. J. Nat. Cancer Inst. 1961;27:29–45. [PubMed] [Google Scholar]
  20. Nagashima K., Wege H., Ter Meulen V. Early and late CNS-effects of coronavirus infection in rats. Adv. Exp. Med. Biol. 1978;100:395–409. doi: 10.1007/978-1-4684-2514-7_28. [DOI] [PubMed] [Google Scholar]
  21. Nagashima K., Wege H., Meyermann R., Ter Meulen V. Demyelinating encephalomyelitis induced by a long-term coronavirus infection in rats. Acta Neuropathol. 1979;45:205–213. doi: 10.1007/BF00702672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nelson J.B. Acute hepatitis associated with mouse leukemia. I. Pathological features and transmission of the disease. J. Exp. Med. 1952;96:293–302. doi: 10.1084/jem.96.4.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Rothenberg E., Donoghue D.J., Baltimore D. Analysis of a 5′ leader sequence on murine leukemia virus 21S RNA: Heteroduplex mapping with long reverse transcriptase products. Cell. 1978;13:435–451. doi: 10.1016/0092-8674(78)90318-5. [DOI] [PubMed] [Google Scholar]
  26. Rowe W.P., Hartley J.W., Capps W.I. Vol. 112. 1963. Mouse hepatitis virus infection as a highly contagious prevalent, enteric infection of mice; pp. 161–165. (Proc. Soc. Exp. Biol. Med.). [DOI] [PubMed] [Google Scholar]
  27. Sabesin S.M. Isolation of a latent murine hepatitis virus from cultured mouse liver cells. Amer. J. Gastroenterol. 1972;58:259–274. [PubMed] [Google Scholar]
  28. Sangar D.V. The replication of picornaviruses. J. Gen. Viral. 1979;45:1–13. doi: 10.1099/0022-1317-45-1-1. [DOI] [PubMed] [Google Scholar]
  29. Siddell S.G., Wege H., Barthel A., Ter Meulen 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]
  30. 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]
  31. Tyrrell D.A.J., Almeida J.D., Cunningham C.H., Dowdle W.R., Hofstad M.S., McIntosh K., Fajima M., Zakstelskaya L.Ya., Easterday B.C., Kapikian A., Bingham R.W. Coronaviridae. Intervirology. 1975;5:76–82. doi: 10.1159/000149883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tyrrell 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., McIntosh K. Coronaviridae: Second report. Intervirology. 1978;10:321–328. doi: 10.1159/000148996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Virelizier J.L., Dayan A.D., Allison A.C. Neuropathological effects of persistent infection of mice by mouse hepatitis virus. Infect. Immun. 1975;12:1127–1140. doi: 10.1128/iai.12.5.1127-1140.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wege H., Müller A., Ter Meulen 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]
  35. Wege H., Wege H., Nagashima K., Ter Meulen V. Structural polypeptides of the murine coronavirus JHM. J. Gen. Viral. 1979;42:37–47. doi: 10.1099/0022-1317-42-1-37. [DOI] [PubMed] [Google Scholar]
  36. Weiland E., Mussgay M., Weiland F. Non-producer malignant tumor cells with rescuable sarcoma virus genome isolated from a recurrent Moloney sarcoma. J. Exp. Med. 1978;148:408–423. doi: 10.1084/jem.148.2.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weiner L.P. Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus) Arch. Neurol. 1973;28:298–303. doi: 10.1001/archneur.1973.00490230034003. [DOI] [PubMed] [Google Scholar]
  38. Zeijst van der B.A.M., Bloemers H.P.J. 4698 Isokinetic glycerol and sucrose gradients for density gradient centrifugation. In: Fasman G.D., editor. 3rd ed. Vol. 1. CRC Press; Cleveland: 1976. pp. 426–519. (Handbook of Biochemistry and Molecular Biology, Physical and Chemical Data). [Google Scholar]

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