Skip to main content
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Feb 6;157(2):509–515. doi: 10.1016/0042-6822(87)90293-5

In Vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain A59

Carol J Budzilowicz 1, Susan R Weiss 1,1
PMCID: PMC7131581  PMID: 3029985

Abstract

The complete nucleotide sequence of nonstructural gene 5 of coronavirus mouse hepatitis virus (MHV) strain A59 has been determined. This sequence contains two potential open reading frames which overlap by five nucleotides. The putative protein products predicted from the sequence are a basic 13,000-Da polypeptide and a 9600-Da polypeptide containing an unusually long hydrophobic amino terminus. RNAs transcribed in vitro from DNAs containing each of the open reading frames in pGEM vectors direct the synthesis in vitro of polypeptides of the sizes predicted by the sequence. An RNA transcript containing both of the open reading frames directs the synthesis primarily of the polypeptide corresponding to the downstream open reading frame. These data suggest that MHV-A59 messenger RNA 5 potentially encodes two proteins and may be preferentially translated from an internal AUG initiation codon.

References

  1. Armstong J., Niemann H., Smeekens S., Rottier P., Warren G. Sequence and topology of a model intracellular membrane protein, El glycoprotein, from a coronavirus. Nature (London) 1984;308:751–762. doi: 10.1038/308751a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Vol. 69. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose; pp. 1408–1412. (Proc. Natl, Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biggins M.D., Gibson T.J., Hong G.F. Vol. 80. 1983. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination; pp. 3963–3965. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boursnell M.E.G., Brown T.D.K. Sequencing of the coronavirus IBV genomic RNA: A 95-base open reading frame encoded by mRNA B. Gene. 1984;29:87–92. doi: 10.1016/0378-1119(84)90169-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Budzilowicz C.J., Wilczynski S.P., Weiss S.R. Three intergenic regions of coronavirus MHV-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]
  7. Cheley S., Anderson R., Cupples M.J., Lee Chan E.C.M., Morris V.L. Intracellular murine hepatitis 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. Curran J.A., Richardson C., Kolakofsky D. Ribosome initiation at alternative AUGs on the Sendai virus P/C mRNA. J. Virol. 1986;57:684–687. doi: 10.1128/jvi.57.2.684-687.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ernst H., Shatkin A. Vol. 82. 1985. Reovirus hemagglutinin mRNA codes for two polypeptides in overlapping reading frames; pp. 48–52. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fritensky B., Lis J., Wu R. Portable microcomputer software for nucleotide sequence analysis. Nucleic Acids Res. 1982;10:6451–6463. doi: 10.1093/nar/10.20.6451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gough N.M., Webb E.A., Corey S., Adams J.M. Molecular cloning of seven immunoglobulin K chain messenger ribonucleic acids. Biochemistry. 1980;19:2702–2710. doi: 10.1021/bi00553a026. [DOI] [PubMed] [Google Scholar]
  12. Hiebert S.W., Patterson R.G., Lamb R.A. Identification and predicted sequence of a previously unrecognized small hydrophobic protein SH, of the paramyxovirus simian virus 5. J. Virol. 1985;55:744–751. doi: 10.1128/jvi.55.3.744-751.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs L., Spaan W.J.M., Horzinek M.C., van Der Zeiist B.A.M. Synthesis of subgenomic mRNAs of mouse hepatitis virus is initiated independently: Evidence from UV transcriptional mapping. J. Virol. 1981;39:823–834. doi: 10.1128/jvi.39.2.401-406.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984;12:857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krieg P.A., Melton D.A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984;12:7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kyte J., Doolittle R.F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982;157:105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  17. 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; p. 3626. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lai M.M.C., Brayton P.R., Armen P.C., Patton C.D., Pugh C., Stohlman S.A. Mouse hepatitis virus A59: Messenger RNA structure and genetic localization of the sequence divergence from a 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]
  19. Lai M.M.C., Patton C.D., 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]
  20. 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]
  21. 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]
  22. Leibowitz J.L., Wilhelmson K.C., Bond C.W. The virus-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]
  23. Maizel J.V., Jr . Polyacrylamide gel electrophoresis of viral proteins. In: Maramorosh K., Koprowski H., editors. Vol. 5. Academic Press; New York: 1971. pp. 179–246. (Methods in Virology). [Google Scholar]
  24. Manaker R.A., Piczak C.V., Miller A.A., Stanton M.F. A hepatitis virus complicating studies with murine leukemia. J. Natl. Cancer Inst. 1961;27:29–51. [PubMed] [Google Scholar]
  25. Messing J., Crea R., Seeburg P. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981;9:309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rottier P.J.M., Spaan W.J.M., Horzinek M.C., van Derzeijst B.A.M. Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevisoocytes. J. Virol. 1981;38:20–26. doi: 10.1128/jvi.38.1.20-26.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Siddell S., Wege H., ter Meulen V. The structure and function of coronaviruses. Curr. Top. Microbiol. Immunol. 1982;99:131–163. doi: 10.1007/978-3-642-68528-6_4. [DOI] [PubMed] [Google Scholar]
  28. Skinner M.A., Ebner D., Siddell S.G. Corona virus MHV-JHM mRNA 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame. J. Gen. Virol. 1985;66:581–592. doi: 10.1099/0022-1317-66-3-581. [DOI] [PubMed] [Google Scholar]
  29. Skinner M.A., Siddell S.G. Coding sequence of coronavirus MHV-JHM mRNA 4. J. Gen. Virol. 1985;66:593–596. doi: 10.1099/0022-1317-66-3-593. [DOI] [PubMed] [Google Scholar]
  30. Spaan W.J.M., Delius H., Skinner M.A., Armstrong J., Rottier P.J.W., Smeekens S., Siddell S.G., van Der Zeiist B.A.M. Transcription strategy of coronaviruses: Fusion of noncontiguous sequences during mRNA synthesis. In: Rottier P.J.M., van der Zeijst B.A.M., Spaan W.J.M., Horzinek M.C., editors. Molecular Biology and Pathogenesis of Coronaviruses. Plenum; New York: 1984. pp. 173–186. [DOI] [PubMed] [Google Scholar]
  31. Spaan W.J.M., Rottier P.J.M., Horzinek M.C., van Der Zeiist B.A.M. Sequence relationships among the genome and 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]
  32. 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]
  33. Zebedee S.L., Richardson C.D., Lamb R.A. Characterization of the influenza virus M2 integral membrane protein and expression at the infected cell surface from cloned cDNA. J. Virol. 1985;56:502–511. doi: 10.1128/jvi.56.2.502-511.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES