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. 2004 Feb 6;166(1):66–75. doi: 10.1016/0042-6822(88)90147-X

Synthesis of virus-specific RNA in permeabilized murine coronavirus-infected cells

Julian L Leibowitz 1,1, James R Devries 1
PMCID: PMC7131142  PMID: 2842958

Abstract

We have developed a permeabilized cell system for assaying mouse hepatitis virus-specific RNA polymerase activity. This activity was characterized as to its requirements for mono- and divalent cations, requirements for an exogenous energy source, and pH optimum. This system faithfully reflects MHV-specific RNA synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. The system is efficient and the RNA products were identical to those observed in intact MHV-infected cells as judged by agarose gel electrophoresis and hybridization. Permeabilized cells appear to be an ideal system for studying coronavirus RNA synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems.

References

  1. Baric R.S., Stohlman S.A., Razavi M.K., Lai M.M.C. Characterization of leader-related small RNAs in coronavirus-infected cells: Further evidence for leader-primed mechanism of transcription. Virus Res. 1985;3:19–33. doi: 10.1016/0168-1702(85)90038-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boursnell M.E.G., Brown T.D., Foulds I.J., Green P.F., Tomley F.M., Binns M.M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J. Gen. Virol. 1987;68:57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  3. Brayton P.R., Lai M.M.C., Patton C.D., Stohlman S.A. Characterization of two RNA polymerase activities induced by mouse hepatitis virus. J. Virol. 1982;42:847–853. doi: 10.1128/jvi.42.3.847-853.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brayton P.R., Stohlman S.A., Lai M.M.C. Further characterization of mouse hepatitis virus RNA-dependent RNA polymerases. Virology. 1984;133:197–201. doi: 10.1016/0042-6822(84)90439-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Budzilowicz C.J., Wilczynski S.P., Weiss S.R. Three intragenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the Tend of the viral mRNA leader sequence. J. Virol. 1985;53:834–840. doi: 10.1128/jvi.53.3.834-840.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabirac G.F., Mulloy J.J., Strayer D.S., Sell S., Leibowitz J.L. Transcriptional mapping of early RNA from regions of the genome of the Shope fibroma and malignant rabbit fibroma virus genomes. Virology. 1986;153:53–69. doi: 10.1016/0042-6822(86)90007-3. [DOI] [PubMed] [Google Scholar]
  7. Cheley S., Anderson R., Cuples M.J., Lee Chan E.C.M., Morris V.L. Intracellular murine hepatitis virus virus-specific RNAs contain common sequences. Virology. 1981;112:596–604. doi: 10.1016/0042-6822(81)90305-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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;61:1814–1820. doi: 10.1128/jvi.61.6.1814-1820.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Condra J.H., Lazzarini R.A. Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells. J. Virol. 1980;36:796–804. doi: 10.1128/jvi.36.3.796-804.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denison M.R., Perlman S. Translation and processing of mouse hepatitis virus virion RNA in a cell-free system. J. Virol. 1986;60:12–18. doi: 10.1128/jvi.60.1.12-18.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denison M., Perlman S. Identification of putative polymerase gene product in cells infected with murine coronavirus A59. Virology. 1987;157:565–568. doi: 10.1016/0042-6822(87)90303-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dennis D.E., Brian D.A. RNA-dependent RNA polymerase activity in coronavirus-infected cells. J. Virol. 1982;42:153–164. doi: 10.1128/jvi.42.1.153-164.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fahrney D.E., Gold A.M. Sulfonylfluorides. I. Rates of reaction with acetylcholinesterases, α-chymotrypsin and trypsin. J. Amer. Chem. Soc. 1963;85:997–1003. [Google Scholar]
  14. Grun J.B., Brinton M.A. Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. J. Virol. 1986;60:1113–1124. doi: 10.1128/jvi.60.3.1113-1124.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayashi T.T., McFarlane K. Comparison of endogenous and exogenous RNA primers of poly(U) polymerase in rat hepatic ribosomes. Biochem. J. 1979;177:895–902. doi: 10.1042/bj1770895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lai M.M.C., Baric R.S., Brayton P.R., Stohlman S.A. Vol. 81. 1984. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus; pp. 3626–3630. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lai M.M.C., 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;39:823–834. doi: 10.1128/jvi.39.3.823-834.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lai M.M.C., Patton C.D., Baric R.S., Stohlman S.A. Presence of leader sequences in the mRNA of mouse hepatitis virus. J. Virol. 1983;46:1027–1033. doi: 10.1128/jvi.46.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lai M.M.C., Patton C.D., Stohlman S.A. Further characterization of mRNAs of mouse hepatitis virus: Presence of common 5′-end nucleotides. J. Virol. 1982;41:557–565. doi: 10.1128/jvi.41.2.557-565.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lai M.M.C., 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;44:487–492. doi: 10.1128/jvi.44.2.487-492.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laskowski M., Sealock R.W. Protein protease inhibitors-Molecular aspects. In: Boyer P.D., editor. The Enzymes. 3rd ed. Academic Press; New York: 1972. pp. 375–473. [Google Scholar]
  22. Leibowitz J.L., Weiss S.R., Paavola E., Bond C.W. Cell-free translation of murine coronavirus RNA. J. Virol. 1982;43:905–913. doi: 10.1128/jvi.43.3.905-913.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leibowitz J.L., Wilhelmsen K.C., Bond C.W. Thevirus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM. Virology. 1981;114:39–51. doi: 10.1016/0042-6822(81)90250-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lerach H., Diamond D., Wozney J.M., Boedtker H. RNA molecular weight determinations of gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977;96:4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Mahey B.W.J., Siddell S., Wege H., termellen V. RNA-dependent RNA polymerase activity in murine coronavirusinfected cells. J. Gen. Virol. 1983;64:103–111. doi: 10.1099/0022-1317-64-1-103. [DOI] [PubMed] [Google Scholar]
  27. Robb J.A., Bond C.W. Pathogenic murine coronaviruses. I. Characterization of biologic behavior in vitro and virus specific intracellular RNA of strongly neurotropic JHMV and weakly neurotropic A59V viruses. Virology. 19790;94:352–370. doi: 10.1016/0042-6822(79)90467-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ruoho A., Kyte J. Vol. 71. 1974. Photoaffinity labeling of the ouabainbinding site on (Na+ + K+) adenosine triphosphatase; pp. 2352–2356. (Proc. Nail. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sawicki S.G., Sawicki D.L. Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis. J. Virol. 1986;57:328–334. doi: 10.1128/jvi.57.1.328-334.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schmidt I., Skinner M., Siddell S. Nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus MHV-1HM. J. Gen. Virol. 1987;68:47–56. doi: 10.1099/0022-1317-68-1-47. [DOI] [PubMed] [Google Scholar]
  31. Southern E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 1975;98:503–518. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. Spaan W.J., Delius H., Skinner M., Armstrong J., Rottier P., Smeekens S., van der Zeiist B.A.M., Siddell S.G. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBOJ. 1983;2:1839–1844. doi: 10.1002/j.1460-2075.1983.tb01667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spaan W.J., Rottier P.J., Horzinek M.C., van der Zeiist B.A.M. Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59) Virology. 1981;108:424–434. doi: 10.1016/0042-6822(81)90449-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spaan W.J., Rottier P.J., Horzinek M.C., van der Zeiist B.A. Sequence relationships between the genome and the intracellular RNA species 1, 3, 6, and 7 of mouse hepatitis virus strain A59. J. Virol. 1982;42:432–439. doi: 10.1128/jvi.42.2.432-439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Strayer D.S., Cabirac G.F., Sell S., Leibowitz J.L. Malignant rabbit fibroma virus: Observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor. J. Natl. Cancer Inst. 1983;71:91–104. [PubMed] [Google Scholar]
  38. Strayer D.S., Skaletsky E., Cabirac G.F., Sharp P.A., Corbeil L.B., Sell S., Leibowitz J.L. Malignant rabbit fibroma virus causes secondary immunosupression in rabbits. J. Immunol. 1983;130:399–404. [PubMed] [Google Scholar]
  39. 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]
  40. Weiss S.R., Leibowitz J.L. Characterization of murine coronavirus RNA by hybridization with virus-specific cDNA probes. J. Gen. Virol. 1983;64:127–133. doi: 10.1099/0022-1317-64-1-127. [DOI] [PubMed] [Google Scholar]
  41. Wittek R., Hanggi M., Hiller G. Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J. Virol. 1984;49:371–378. doi: 10.1128/jvi.49.2.371-378.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yogo Y., Hirano N., Hind 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]

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