Characterization of leader-related small RNAs in coronavirus-infected cells: Further evidence for leader-primed mechanism of transcription (original) (raw)

Abstract

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70–82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Keywords: Mouse hepatitis virus, transcription, small leader related RNAs, coronavirus, temperature-sensitive mutant

References

  1. Armstrong J., Niemann H., Smeckens S., Rottier P., Warren G. Sequence and topology of a model intracellular membrane protein e1 glycoprotein from a coronavirus. Nature (London) 1984;308:751–752. doi: 10.1038/308751a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baric R.S., Stohlman S.A., Lai M.M.C. Characterization of replicative intermediate RNA of mouse hepatitis virus: Presence of leader RNA sequences on nascent chains. J. Virol. 1983;48:633–640. doi: 10.1128/jvi.48.3.633-640.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bittner M., Kupferer P., Morris C.F. Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal. Biochem. 1980;102:459–471. doi: 10.1016/0003-2697(80)90182-7. [DOI] [PubMed] [Google Scholar]
  4. Boothroyd J.C., Cross G.A.M. Transcripts coding for the different variant surface glycoproteins of Trypanosoma brucei have a short identical exon at their 5'-end. Gene. 1982;20:281–289. doi: 10.1016/0378-1119(82)90046-4. [DOI] [PubMed] [Google Scholar]
  5. Bouloy M., Plotch S.J., Krug R.M. Vol. 75. 1978. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro; pp. 4886–4890. (Proc. Natl. Acad. Sci. U.S.A.). [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brayton P.R., Ganges R.G., Stohlman S.A. Host cell nuclear function and murine hepatitis virus replication. J. Gen. Virol. 1981;56:457–460. doi: 10.1099/0022-1317-56-2-457. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Campbell D.A., Thornton D.A., Boothroyd J.C. Apparent discontinuous transcription of Trypanosoma brucei variant surface antigen genes. Nature (London) 1984;311:350. doi: 10.1038/311350a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colonno R.J., Banerjee A.K. A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro. Cell. 1976;8:197–204. doi: 10.1016/0092-8674(76)90003-9. [DOI] [PubMed] [Google Scholar]
  11. Grinnell B.W., Wagner R.R. Nucleotide sequence and secondary structure of VSV leader RNA and homologous DNA involved in inhibition of DNA-dependent transcription. Cell. 1984;36:533–543. doi: 10.1016/0092-8674(84)90246-0. [DOI] [PubMed] [Google Scholar]
  12. Jacobs L., Spaan W.J.M., Horzinek M.C., Van der Zeijst B.A.M. Synthesis of subgenomic mRNAs of mouse hepatitis virus is initiated independently: evidence from U.V. transcriptional mapping. J. Virol. 1981;34:401–406. doi: 10.1128/jvi.39.2.401-406.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kurilla M.G., Piwnica-Worms H., Keene J.D. Vol. 79. 1982. Rapid and transient localization of the leader RNA of vesicular stomatitis virus in the nuclei of infected cells; pp. 5240–5244. (Proc. Natl. Acad. Sci. U.S.A.). [DOI] [PMC free article] [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. Lai M.M.C., Brayton P.R., Armen A.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]
  16. 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]
  17. Lai M.M.C., Patton C.D., Stohlman S.A. Replication of mouse hepatitis virus: Negative-stranded RNA and replicative form RNA are of genomic length. J. Virol. 1982;44:487–492. doi: 10.1128/jvi.44.2.487-492.1982. [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., 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. U.S.A.). [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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;114:39–51. doi: 10.1016/0042-6822(81)90250-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Longacre S., Hibner V., Raibaud A., Eisen H., Baitz T., Giroud C., Baltz D. DNA rearrangements and antigenic variation in Trypanosoma equiperdium: Multiple expression linked sites in independent isolates of trypanosomes expressing the same antigen. Mol. Cell. Biochem. 1983;3:399–409. doi: 10.1128/mcb.3.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McGowan J.J., Emerson S.U., Wagner R.R. The plus-strand leader RNA of VSV inhibits DNA dependent transcription of adenovirus and SV40 genes in a soluble whole cell extract. Cell. 1982;28:325–333. doi: 10.1016/0092-8674(82)90350-6. [DOI] [PubMed] [Google Scholar]
  23. Milhausen M., Nelson R.G., Sather S., Selkirk M., Agabian N. Identification of a small RNA containing the trypanosoma spliced leader: A donor of shared 5' sequences of trypanosomated mRNAs? Cell. 1984;38:721–729. doi: 10.1016/0092-8674(84)90267-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patterson J.L., Cadradilla C., Holloway B.P., Obijeski J.F., Kolakofsky D. Multiple leader RNAs and messenger RNAs are transcribed from the La Crosse virus small genome segment. Cell. 1983;33:791–799. doi: 10.1016/0092-8674(83)90021-1. [DOI] [PubMed] [Google Scholar]
  25. Plotch S.J., Bouloy M., Ulmanen I., Krug R.M. A unique cap (M7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate primers that initiate viral RNA transcription. Cell. 1981;23:847–858. doi: 10.1016/0092-8674(81)90449-9. [DOI] [PubMed] [Google Scholar]
  26. Sawicki P.L., Kaariainen L., Lambek C., Gomatos P.J. Mechanism for control of synthesis of Semliki Forest virus 26S and 42S RNA. J. Virol. 1978;25:19–27. doi: 10.1128/jvi.25.1.19-27.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Siddell S., Wege H., Barthel A., ter Meulen V. Coronavirus JHM: intracellular protein synthesis. J. Gen. Virol. 1981;53:145–155. doi: 10.1099/0022-1317-53-1-145. [DOI] [PubMed] [Google Scholar]
  28. Skinner M.A., Siddell S.G. Coronavirus JHM: nucleotide sequence of the mRNA that encodes nucleocapsid protein. Nucleic Acids Res. 1983;11:5045–5054. doi: 10.1093/nar/11.15.5045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spaan W.M., Rottier P.J.M., Horzinek M.C., van der Zeijst 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]
  30. Spaan W., Delius H., Skinner M., Armstrong J., Rottier P., Smeekens S., van der Zeijst B.A.M., Siddell S.G. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J. 1983;2:1839–1844. doi: 10.1002/j.1460-2075.1983.tb01667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stohlman S.A., Fleming J.O., Patton C.D., Lai M.M.C. Synthesis and subcellular location of the murine Coronavirus nucleocapsid protein. Virology. 1983;130:527–532. doi: 10.1016/0042-6822(83)90106-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sturman L.S. Characterization of a coronavirus. I. Structural proteins: effect 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]
  33. Thomas P.S. Vol. 77. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose; pp. 5201–5205. (Proc. Natl. Acad. Sci. U.S.A.). [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wege H., Wege K., Nagashima K., ter Meulen V. Structural polypeptides of the murine coronavirus JHM. J. Gen. Virol. 1979;42:37–42. doi: 10.1099/0022-1317-42-1-37. [DOI] [PubMed] [Google Scholar]
  35. Wege H., Siddell S., ter Meulen V. The biology and pathogenesis of coronaviruses. Curr. Top. Microbiol. Immunol. 1982;99:165–200. doi: 10.1007/978-3-642-68528-6_5. [DOI] [PubMed] [Google Scholar]
  36. Wilhelmsen K.C., Leibowitz J.L., Bond C.W., Robb J.A. The replication of murine coronaviruses in enucleated cells. Virology. 1981;110:252–1230. doi: 10.1016/0042-6822(81)90027-1. [DOI] [PMC free article] [PubMed] [Google Scholar]