Abstract
The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.
References
- Abraham S., Kienzle T.E., Lapps W.E., Brian D.A. Sequence and expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7, and 9.5 kDa encoded between the spike and membrane protein genes of the bovine coronavirus. Virology. 1990;177:488–495. doi: 10.1016/0042-6822(90)90513-Q. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boursnell M.E.G., Binns M., Brown T.D.K. Sequencing of coronavirus IBV genomic RNA: Three open reading frames in the 5′ “unique” region of mRNA D. J. Gen. Virol. 1985;66:2253–2258. doi: 10.1099/0022-1317-66-10-2253. [DOI] [PubMed] [Google Scholar]
- Boursnell M.E.G., Brown T.D.K., 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]
- Brierley I., Boursnell M.E.G., Binns M.M., Bilimoria B., Blok V.C., Brown T.D.K., Inglis S.C. An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV. EMBO. J. 1987;6:3779–3785. doi: 10.1002/j.1460-2075.1987.tb02713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brierley I., Digard P., Inglis S.C. Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot. Cell. 1989;57:537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown T.D.K., Boursnell M.E.G., Binns M.M. A leader sequence is present on mRNA A of avian infectious bronchitis virus. J. Gen. Virol. 1984;54:1437–1442. doi: 10.1099/0022-1317-65-8-1437. [DOI] [PubMed] [Google Scholar]
- Brown T.D.K., Boursnell M.E.G., Binns M.M., Tomley 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]
- Cavanagh D., Davis P.J. Evolution of avian coronavirus IBV: Sequence of the matrix glycoprotein gene and intergenic region of several serotypes. J. Gen. Virol. 1988;69:621–629. doi: 10.1099/0022-1317-69-3-621. [DOI] [PubMed] [Google Scholar]
- Cavanagh D., Brian D.A., Enjuanes L., Holmes K.V., Lai M.M.C., Laude H., Siddell S.G., Spaan W., Taguchi F., Talbot P.J. Recommendations of the coronavirus study group for the nomenclature of the structural proteins, mRNAs, and genes of coronaviruses. Virology. 1990;176:306–307. doi: 10.1016/0042-6822(90)90259-T. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Contreras R., Cheroutre H., Degrave W., Fiers W. Simple efficient in vitro synthesis of capped RNA useful for direct expression of cloned DNA. Nucleic Acids Res. 1982;10:6353–6362. doi: 10.1093/nar/10.20.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dawson P.S., Gough R.E. Antigenic variation in strains of avian infectious bronchitis virus. Archive fur die gesamte Virusforschung. 1971;34:32–39. doi: 10.1007/BF01250243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dotto G.P., Enea V., Zinder N.D. Functional analysis of bacteriophage f1 intergenic region. Virology. 1981;114:463–473. doi: 10.1016/0042-6822(81)90226-9. [DOI] [PubMed] [Google Scholar]
- Fuerst T.R., Niles E.G., Studier F.W., Moss B. Vol. 83. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase; pp. 8122–8127. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inglis S.C., Carroll A.R., Lamb R.A., Mary B.W.J. Polypeptides specified by the influenza virus genome. 1. Evidence for eight distinct gene products specified by fowl plague virus. Virology. 1976;74:489–503. doi: 10.1016/0042-6822(76)90355-x. [DOI] [PubMed] [Google Scholar]
- Inglis S.C., McGeoch D.J., Mahy B.W.J. Polypeptides specified by the influenza virus genome. 2. Assignment of protein coding functions to individual genome segments by in vitro translation. Virology. 1977;78:522–536. doi: 10.1016/0042-6822(77)90128-3. [DOI] [PubMed] [Google Scholar]
- Kozak M. Analysis of 5′ noncoding sequences from 699 verbrate messenger RNAs. Nucleic Acids Res. 1987;15:8125–8149. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozak M. The scanning model for translation: An update. J. Mol. Biol. 1989;108:229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krieg P.A., Melton D.A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNA. Nucleic Acids Res. 1984;12:7057–7071. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U.K. Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lai M.M.C. Coronavirus leader-RNA-primed transcription: An alternative mechanism to RNA splicing. BioEssays. 1986;5:257–260. doi: 10.1002/bies.950050606. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leibowitz J.L., Perlman S., Weinstock G., Devries J.R., Budzilowcz C., Weissemann J.M., Weiss S.R. Detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame. Virology. 1988;164:156–164. doi: 10.1016/0042-6822(88)90631-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Russel M., Kidd S., Kelley M.R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45:333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
- Schochetman G., Stevens R.H., Simpson R.W. Presence of infectious polyadenylated RNA in the coronavirus avian bronchitis virus. Virology. 1977;77:772–782. doi: 10.1016/0042-6822(77)90498-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Smith A.R., Boursnell M.E.G., Binns M.M., Brown T.D.K., Inglis S.C. Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus. J. Gen. Virol. 1990;71:3–11. doi: 10.1099/0022-1317-71-1-3. [DOI] [PubMed] [Google Scholar]
- Stanley K.K., Luzlo J.P. Construction of a new family of high efficiency bacterial expression vectors: Identification of cDNA clones coding for human liver proteins. EMBO J. 1984;3:1429–1434. doi: 10.1002/j.1460-2075.1984.tb01988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stern D.F., Kennedy S.I.T. Coronavirus multiplication strategy. I. Identification and characterisation of virus specified RNA species to the genome. J. Virol. 1980;34:665–674. doi: 10.1128/jvi.34.3.665-674.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Stern D.F., Sefton B.M. Coronavirus multiplication: Location of genes for virion proteins on the avian infectious bronchitis virus genome. J. Virol. 1984;50:22–29. doi: 10.1128/jvi.50.1.22-29.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wesley R.D., Cheung A.K., Michael D.D., Woods R.D. Nucleotide sequence of coronavirus TGEV genomic RNA: Evidence for 3 mRNA species between the peplomer and matrix protein genes. Virus Res. 1989;13:87–100. doi: 10.1016/0168-1702(89)90008-7. [DOI] [PMC free article] [PubMed] [Google Scholar]