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. 2004 Feb 23;156(2):321–330. doi: 10.1016/0042-6822(87)90412-0

The 5′-end sequence of the murine coronavirus genome: Implications for multiple fusion sites in leader-primed transcription

Chien-Kou Shieh 1, Lisa H Soe 1, Shinji Making 1, Ming-Fu Chang 1, Stephen A Stohlman 1, Michael MC Lai 1,1
PMCID: PMC7130777  PMID: 3027981

Abstract

The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5′-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5′-end of the mouse hepatitis virus genomic RNA. The 5′-terminal sequences are identical to the leader sequences present at the 5′-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3′-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5′-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7–18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

References

  1. Armstrong J., Niemann H., Smeekens 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., Shieh C.K., Stohlman S.A., Lai M.M.C. Analysis of intracellular small RNAs of mouse hepatitis virus: Evidence for discontinuous transcription. Virology. 1987;156:342–354. doi: 10.1016/0042-6822(87)90414-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Brown T.D.K., Boursnell M.E.G., Binns M.M., Tomely F.M. Cloning and sequencing of 5′ terminal sequences from avian infectious bronchitis virus genomic RNA. J. Gen. Virol. 1986;67:221–228. doi: 10.1099/0022-1317-67-2-221. [DOI] [PubMed] [Google Scholar]
  8. Budzilowicz C.J., Wilczynski S.P., Weiss S.R. Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3′ end 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]
  9. Dagert M., Ehrlich S.D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979;6:23–29. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  10. D'Alessio J.M. RNA sequencing. In: Rickwood D., Hames B.D., editors. Gel Electrophoresis of Nucleic Acids. IRL Press; Eynsham: 1982. pp. 173–197. [Google Scholar]
  11. Grunstein M., Hogness D.S. Vol. 72. 1975. Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene; pp. 3961–3965. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gubler U., Hoffman B.J. A simple and very efficient method for generating cDNA libraries. Gene. 1983;25:263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  13. Henikoff S., Kelly J.D., Cohen E.H. Transcription terminates in yeast distal to a control sequence. Cell. 1983;33:607–614. doi: 10.1016/0092-8674(83)90441-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kapke P.A., Brian D.A. Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology. 1986;151:41–49. doi: 10.1016/0042-6822(86)90102-9. [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., 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]
  19. 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]
  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. Makino S., Fujioka N., Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J. Virol. 1985;54:329–336. doi: 10.1128/jvi.54.2.329-336.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Makino S., Stohlman S.A., Lai M.M.C. Vol. 83. 1986. Leader sequences of murine coronavirus mRNAs can be freely reassorted: Evidence for the role of free leader RNA in transcription; pp. 4204–4208. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Makino S., Taguchi F., Fuj1wara K. Defective interfering particles of mouse hepatitis virus. Virology. 1984;133:9–17. doi: 10.1016/0042-6822(84)90420-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Makino S., Taguchi F., Hirano N., Fujiwara K. Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain. Virology. 1984;139:138–151. doi: 10.1016/0042-6822(84)90335-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maniatis T., Fritsch E.F., Sambrook J. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY: 1982. (Molecular Cloning—A Laboratory Manual). [Google Scholar]
  26. Martin F.H., Tinoco I., Jr. DNA-RNA hybrid duplexes containing oligo (dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. Nucleic Acids Res. 1980;8:2295–2299. doi: 10.1093/nar/8.10.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maxam A.M., Gilbert W. Sequencing end-labelled DNA with base-specific chemical cleavages. In: Grossman L., Moldave K., editors. Vol. 65. Academic Press; Orlando, FL: 1980. pp. 499–560. (Methods in Enzymology). [DOI] [PubMed] [Google Scholar]
  28. 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. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mills D.R., Dabkin C., Kramer F.R. Template-determined, variable rate of RNA chain elongation. Cell. 1978;15:541–550. doi: 10.1016/0092-8674(78)90022-3. [DOI] [PubMed] [Google Scholar]
  30. Nomoto A., Imura N. A convenient sequencing method for 5′ protein-linked RNAs. Nucleic Acids Res. 1979;7:1233–1246. doi: 10.1093/nar/7.5.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pedersen F.S., Haseltine W.A. A micromethod for detailed characterization of high molecular weight RNA. In: Grossman L., Moldave K., editors. Vol. 65. Academic Press; Orlando, FL: 1980. pp. 680–687. (Methods in Enzymology). [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A.R. Vol. 74. 1977. DNA sequencing with chain-terminating inhibitors; pp. 5463–5467. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Skinner M.A., Ebner D., Siddell S.G. Coronavirus 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]
  34. Skinner M.A., Siddell S.G. Coronavirus JH M: Nucleotide sequence of the mRNA that encodes nucleocapsid protein. Nucleic Acids Res. 1983;15:5045–5054. doi: 10.1093/nar/11.15.5045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Thomas P.S. Vol. 77. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose; pp. 5201–5205. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wilhelmsen K.C., Leibowitz J.L., Bond C.W., Robb J.A. The replication of murine coronaviruses in enucleated cells. Virology. 1981;110:225–230. doi: 10.1016/0042-6822(81)90027-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zaret K.S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982;28:563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]

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