Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Virology. 1992 Jun; 188(2): 666–675.
Published online 2004 Feb 11. doi: 10.1016/0042-6822(92)90521-P
PMCID: PMC7131960
PMID: 1316677

TGEV corona virus ORF4 encodes a membrane protein that is incorporated into virions

Murielle Godet, Rene L'Haridon, Jean-Francois Vautherot, and Hubert Laude1
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

The coding potential of the open reading frame ORF4 (82 amino acids) of transmissible gastroenteritis virus (TGEV) has been confirmed by expression using a baculovirus vector. Five monoclonal antibodies (MAbs) raised against the 10K recombinant product immunoprecipitated a polypeptide of a similar size in TGEV-infected cells. Immunofluorescence assays performed both on insect and mammalian cells revealed that ORF4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in ORF4 sequence. Two epitopes were localized within the last 21 C-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated ORF4 recombinant protein. Since the relevant MAbs were found to induce a cell surface fluorescence, these data suggest that ORF4 may be an integral membrane protein having a Cexo-Nendo orientation. Anti-ORF4 MAbs were also used to show that ORF4 polypeptide may be detected in TGEV virion preparations, with an estimated number of 20 molecules incorporated per particle. Comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to TGEV ORF4. Our results led us to propose that ORF4 represents a novel minor structural polypeptide, tentatively designated SM (small membrane protein).

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. [PMC free article] [PubMed] [Google Scholar]
  • Boursnell M.E.G., Binns M.M., Brown 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. [PubMed] [Google Scholar]
  • Britton P., Carmenes R.S., Page K.W., Garwes D.J., Parka F. Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae. Mol. Microbiol. 1988;2(1):89–99. [PubMed] [Google Scholar]
  • Britton P., Otin C.L., Alonso J.M.M., Parka F. Sequence of the coding regions from the 3.0 kb and 3.9 kb mRNA subgenomic species from a virulent isolate of transmissible gastroenteritis virus. Arch. Virol. 1989;105:165–178. [PMC free article] [PubMed] [Google Scholar]
  • Brown M., Faulkner P. A plaque assay for nuclear polyhedrosis viruses using a solid overlay. J. Gen. Virol. 1977;36:361–364. [Google Scholar]
  • Budzilowicz C.J., Weiss S.R. In vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain A59. Virology. 1987;157:509–515. [PMC free article] [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. [PMC free article] [PubMed] [Google Scholar]
  • Corpet F. Multiple sequence alignment with hierarchical clustering. Nucl. Acids Res. 1988;16:10,881–10,891. [PMC free article] [PubMed] [Google Scholar]
  • Delmas B., Laude H. Assembly of coronavirus spike protein into trimers and its role in epitope expression. J. Virol. 1990;64:5367–5375. [PMC free article] [PubMed] [Google Scholar]
  • Delmas B., Rasschaert D., Godet M., Gelfi J., Laude H. Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S. J. Gen. Virol. 1990;71:1313–1323. [PubMed] [Google Scholar]
  • Delmas B., Laude H. Carbohydrate-induced conformational changes strongly modulate the antigenicity of coronavirus TGEV glycoproteins S and M. Virus Res. 1991;20:107–120. [PMC free article] [PubMed] [Google Scholar]
  • Gaedigk-Nitschko K., Schlesinger M.J. The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids. Virology. 1990;175:274–281. [PubMed] [Google Scholar]
  • Garoff H., Frischauf A-M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cDNA coding for Semliki Forest virus membrane glycoproteins. Nature. 1980;288:236–241. [PubMed] [Google Scholar]
  • Garwes D.J., Stewart F., Britton P. The polypeptide of Mr 14000 of porcine transmissible gastroenteritis virus: Gene assignment and intracellular location. J. Gen. Virol. 1989;70:2495–2499. [PubMed] [Google Scholar]
  • Geysen H.M., Meloen R.H., Barteling S.J. Vol. 81. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid; pp. 3998–4002. (Proc. Natl. Acad. Sci. USA). [PMC free article] [PubMed] [Google Scholar]
  • Godet M., Rasschaert D., Laude H. Processing and antigenicity of entire and anchor-free spike glycoprotein S of coronavirus TGEV expressed by recombinant baculovirus. Virology. 1991;185:732–740. [PMC free article] [PubMed] [Google Scholar]
  • Hartmann E., Rapoport T.A., Lodish H.F. Vol. 86. 1989. Predicting the orientation of eukaryotic membrane-spanning proteins; pp. 5786–5790. (Proc. Natl. Acad. Sci. USA). [PMC free article] [PubMed] [Google Scholar]
  • Hay A.J., Wolstenholme A.J., Skehel J.J., Smith M.H. The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 1985;4:3021–3024. [PMC free article] [PubMed] [Google Scholar]
  • Hiebert S.W., Paterson 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. [PMC free article] [PubMed] [Google Scholar]
  • Hiebert S.W., Richardson C.D., Lamb R.A. Cell surface expression and orientation in membranes of the 44-amino-acid SH protein of simian virus. 5. J. Gen. Virol. 1988;62:2347–2357. [PMC free article] [PubMed] [Google Scholar]
  • Kapke P.A., Brian D.A. Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology. 1986;151:41–49. [PMC free article] [PubMed] [Google Scholar]
  • Kapke P.A., Tung F.Y.T., Brian D.A. Nucleotide sequence between the peplomer and matrix protein genes of the porcine transmissible gastroenteritis coronavirus identifies three large open reading frames. Virus Genes. 1988;2(3):293–294. [PMC free article] [PubMed] [Google Scholar]
  • Kiefhaber T., Rudolph R., Kohler H.H., Buchner J. Protein aggregation in vitro and in vivo: A quantitative model of the kinetic competition between folding and aggregation. Bio/Technology. 1991;9:825–829. [PubMed] [Google Scholar]
  • Kitts P.A., Ayres M.D., Possee R.D. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucl. Acids Res. 1990;18:5667–5672. [PMC free article] [PubMed] [Google Scholar]
  • Lai M.M.C. Coronavirus: Organization, replication and expression of genome. Annu. Rev. Microbiol. 1990;44:303–333. [PubMed] [Google Scholar]
  • Lamb R.A., Zebedee S.L., Richardson C.D. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell. 1985;40:627–633. [PubMed] [Google Scholar]
  • Laude H., Chapsal J.M., Gelfi J., Labiau S., Grosclaude J. Antigenic structure of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against virion proteins. J. Gen. Virol. 1986;67:119–130. [PubMed] [Google Scholar]
  • L'Haridon R.M., Bourget P., Lefevre F., La Bonnardiere C. Production of an hybridoma library to recombinant porcine alpha I interferon: A very sensitive assay (ISBA) allows the detection of a large number of clones. Hybridoma. 1991;10(1):35–47. [PubMed] [Google Scholar]
  • Leibowitz J.L., Perlman S., Weinstock G., Devries J.R., Budzilowicz 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. [PMC free article] [PubMed] [Google Scholar]
  • Liljeström P., Lusa S., Huylebroeck D., Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: The small 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 1991;65:4107–4113. [PMC free article] [PubMed] [Google Scholar]
  • Liu D.X., Cavanagh D., Green P., Inglis S.C. A polycistronic m RNA specified by the coronavirus infectious bronchitis virus. Virology. 1991;184:531–544. [PMC free article] [PubMed] [Google Scholar]
  • Luckow V.A., Summers M.D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989;170:31–39. [PubMed] [Google Scholar]
  • Olmsted R.A., Collins P.L. The 1 A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species. J. Virol. 1989;63:2019–2029. [PMC free article] [PubMed] [Google Scholar]
  • Raabe T., Siddell S. Nucleotide sequence of the human coronavirus HCV 229E mRNA 4 and mRNA 5 unique regions. Nucl. Acids Res. 1989;17(15):6387. [PMC free article] [PubMed] [Google Scholar]
  • Rasschaert D., Duarte M., Laude H. Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions. J. Gen. Virol. 1990;71:2599–2607. [PubMed] [Google Scholar]
  • Rasschaert D., Gelfi J., Laude H. Enteric coronavirus TGEV: Partial sequence of the genomic RNA, its organization and expression. Biochimie. 1987;69:591–600. [PMC free article] [PubMed] [Google Scholar]
  • Roseto A., Bobulesco P., Laporte J., Escaig J., Gaches D., Peries J. Bovine enteric coronavirus structure as studied by a freeze-drying technique. J. Gen. Virol. 1982;63:241–245. [PubMed] [Google Scholar]
  • Sambrook J., Fritsch E.F., Maniatis T. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY: 1989. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  • Scharf S.J., Horn G.T., Erlich H.A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986;233:1076–1078. [PubMed] [Google Scholar]
  • Schmidt M.F.G. Acylation of viral spike glycoproteins: A feature of enveloped RNA viruses. Virology. 1982;116:327–338. [PMC free article] [PubMed] [Google Scholar]
  • Skinner M.A., Ebner D., Siddell S.G. Coronavlrus MHV-1HM mRNA 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame. J. Gen. Virol. 1985;66:581–592. [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. [PubMed] [Google Scholar]
  • Spaan W., Cavanagh D., Horzinek M.C. Coronavlruses: Structure and genome expression. J. Gen. Virol. 1988;69:2939–2952. [PubMed] [Google Scholar]
  • Sugrue R.J., Hay A.J. Structural characteristics of the M2 protein of influenza A viruses: Evidence that it forms a tetrameric channel. Virology. 1991;180:617–624. [PMC free article] [PubMed] [Google Scholar]
  • Summers M.D., Smith G.E. Texas Agricultural Experiment Station Bulletin No. 1555 Publishers College Station; Texas: 1987. (A manual of methods for baculovirus vectors and insect cell culture procedures). [Google Scholar]
  • Welch W.J., Sefton B.M. Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki forest virus. J. Virol. 1980;33:230–237. [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. [PMC free article] [PubMed] [Google Scholar]
  • Wesley R.D., Woods R.D., Cheung A.K. Genetic basis for the pathogenesis of transmissible gastroenteritis virus. J. Virol. 1990;64:4761–4766. [PMC free article] [PubMed] [Google Scholar]
  • Woloszyn N., Boireau P., Laporte J. Nucleotide sequence of the bovine enteric coronavirus BECV F15 mRNA 5 and mRNA 6 unique regions. Nucl. Acids Res. 1990;11:1303. 18(5) [PMC free article] [PubMed] [Google Scholar]
  • Zebedee S.L., Lamb R.A. Influenza A virus M2 protein: Monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 1988;62:2762–2772. [PMC free article] [PubMed] [Google Scholar]
Articles from Virology are provided here courtesy of Elsevier
-