Skip to main content
NCBI home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2006 Oct 27;2(4):497–505. doi: 10.1111/j.1365-2958.1988.tb00056.x

The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli

P Britton 1,✉, R S Cármenes 1,†, K W Page 1, D J Garwes 1
PMCID: PMC7168440  PMID: 2845226

Summary

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29459 polypeptide and β‐galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

References

  1. Armstrong, J. , Niemann, H. , Smeekens, S. , Rottier, P. , and Warren, G. (1984) Sequence and topology of a model intracellular membrane protein. E1 glycoprotein, from a coronavirus. Nature 308, 751–752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boursnell, M.E.G. , Brown, T.D.K. , and Binns, M.M. (1984) Sequence of the membrane protein gene from avian coronavirus IBV. Virus Res 1, 303–313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Britton, P. (1981) Ph.D. Thesis, University of Edinburgh.
  4. Britton, P. , Murfitt, D. , Parra, F. , Jones‐Mortimer, M.G. , and Kornberg, H.L. (1982) Phosphotransferase‐mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages λlex(crr) and λgsr(tgs). EMBO J 1, 907–911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Britton, P. , Lee, L.G. , Murfitt, D. , Boronat, A. , Jones‐Mortimer, M.C. , and Komberg, H.L. (1984) Location and direction of transcription of the ptsH and ptsl genes on the Escherichia coli K12 genome. J Gen Microbiol 130, 861–868. [DOI] [PubMed] [Google Scholar]
  6. Britton, P. , Garwes, D.J. , Millson, G.C. , Page, K. , Bountiff, L. , Stewart, F. , and Walmsiey, J. (1986) Towards a genetically‐engineered vaccine against porcine transmissible gastroenteritis virus In Biomolecular Engineering in the European Community. Final Report. Magnien E. (Ed.). The Netherlands : Martinus Nijhoff, pp. 301–313. [Google Scholar]
  7. Britton, P. , Garwes, D.J. , Page, K. , and Walmsiey, J. (1987) Expression of porcine transmissible gastroenteritis virus genes in E. coli as β‐galactosidase chimaeric proteins In Coronaviruses. Lai M.M.C., and Stohlman S.A. (Eds) New York ; Plenum Press; Adv Exp Med Biol 218, 55–64. [DOI] [PubMed] [Google Scholar]
  8. Britton, P. , Cármenes, R.S. , Page, K.W. , Garwes, D.J. , and Parra, F. (1988) Sequence of the nucleoprotein from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae . Mol Microbiol 2, 89–99. [PubMed] [Google Scholar]
  9. Brown, T.D.K. , Boursnell, M.E.G. , and Binns, M.M. (1984)A leader sequence is present on mRNA A of avian infectious bronchitis virus . J Gen Virol 65, 1437–1442. [DOI] [PubMed] [Google Scholar]
  10. Budzilowicz, C.J. , Wilczynski, S.P. , and Weiss, S.R. (1985) 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 53, 834–840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dennis, D.E. , and Brian, DA . (1981) Coronavirus cell‐associated RNA‐dependent RNA polymerase In Biochemstry and Biology of Coronaviruses. ter Meulen V., Siddell S., and Wege H. (Eds) New York : Plenum Press; Adv Exp Med Biol 142, 155–170. [DOI] [PubMed] [Google Scholar]
  12. Devereux, J. , Haeberli, P. , and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. NucI Acids Res 12: 387–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garwes, D.J. , and Pocock, D.H. (1975) The polypeptide structure of transmissible gastroenteritis virus. J Gen Virol 29, 25–34. [DOI] [PubMed] [Google Scholar]
  14. Garwes, D.J. , Pocock, D.H. , and Wijaszka, T.M. (1975) Identification of heat‐dissociable RNA complexes in two porcine corona‐viruses. Nature 257, 508–510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garwes, D.J. , Bountiff, L. , Millson, G.C. , and Elleman, G.J. (1984). Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line In Molecular Biology and Pathogenesis of Coronaviruses. Rottier P.J.M., van der Zeijst B.A.M., Spaan W.J.M., and Horzinek M.C. (Eds) New York : Plenum Press; Adv Exp Med Biol 173, 79–93. [DOI] [PubMed] [Google Scholar]
  16. Garwes, D.J. , Stewart, F. , and Elleman, C.J. (1987) Identification of epitopes of immunological importance on the peplomer of porcine transmissible gastroenteritis virus In Coronaviruses. Lai M.M.C., and Stohlman S.A. (Eds) New York : Plenum Press; Adv Exp Med Biol 218, 509–515. [DOI] [PubMed] [Google Scholar]
  17. Hanahan, D. (1985) Techniques for transformation of Escherichia coli In DNA Cloning: A Practical Approach. Vol. II Glover D.M. (Ed.). Oxford : IRL Press, 109–135. [Google Scholar]
  18. Jacobs, L. , van der Zeijst, B.A.M. , and Horzinek, M.C. (1986) Characterization and translation of transmissible gastroenteritis virus mRNAs. J Virol 57, 1010–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jimenez, G. , Correa, I. , Melgosa, M.P. , Bullido, M.J. , and Enjuanes, L. (1986) Critical epitopes in transmissible gastroenteritis virus neutralization. J Virol 60, 131–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kapke, P.A. , and Brian, D.A. (1986) Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology 151:41–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kapke, P.A. , Tung, F.Y.C. , Brian, DA , Woods, R.D. , and Wesley, R. (1987) Nucleotide sequence of the porcine transmissible gastroenteritis coronavirus matrix protein In Coronaviruses. Lai M.M.C, and Stohlman S.A. (Eds) New York : Plenum Press; Adv Exp Med Biol 218, 117–122. [DOI] [PubMed] [Google Scholar]
  22. Klenk, H.D. , and Rott, R. (1980) Cotranslational and posttranslational processing of viral glycoproteins. Curr Top Microbiol Immunol 90, 19–48. [DOI] [PubMed] [Google Scholar]
  23. Kozak, M. (1983) Comparison of initiation of protein synthesis in prokaryotes, eukaryotes and organelles. Microbiol Rev 47, 1–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. [DOI] [PubMed] [Google Scholar]
  25. Kyte, J. , and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157; 105–132. [DOI] [PubMed] [Google Scholar]
  26. Lai, M.M.C. , Baric, R.S. , Brayton, P.R. , and Stohlman, S.A. (1984) Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc Natl Acad Sci USA 81, 3626–3630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lapps, W. , Hogue, B.G. , and Brian, D.A. (1987) Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes. Virology 157, 47–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laude, H. , Rasschaert, D. , and Huet, J.C. (1987) Sequence and N‐terminal processing of the transmembrane protein E1 of the coronavirus transmissible gastroenteritis virus. J Gen Virol 68, 1687–1693. [DOI] [PubMed] [Google Scholar]
  29. Maniatis, T. , Fritsch, E.F. , and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor , New York : Cold Spring Harbor Laboratory Press. [Google Scholar]
  30. McGeoch, D.J. (1985) On the predictive recognition of signal peptide sequences. Virus Res 3, 271–286. [DOI] [PubMed] [Google Scholar]
  31. Niemann, H. , and Klenk, H.D. (1981) Coronavirus glycoprotein E1, a new type of viral glycoprotein. J Mol Biol 53, 993–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Niemann, H. , Geyer, R. , Klenk, H.D. , Linder, D. , Stirm, S. , and Wirth, M. (1984) The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the 0‐glycosidically linked oligosaccharides of glycoprotein E1. EMBO J 3, 665–670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pfleiderer, M. , Skinner, M.A. , and Siddell, S.G. (1986) Coronavirus MHV‐JHM: nucleotide sequence of the mRNA that encodes the membrane protein. Nucl Acids Res 14, 6338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rasschaert, D. , and Laude, H. (1987) The predicted structure of the peplomer protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J Gen Virol 68, 1883–1890. [DOI] [PubMed] [Google Scholar]
  35. Rasschaert, D. , Delmas, B. , Charley, B. , Grossclaude, J. , Gelfi, J. , and Laude, H. (1987a) Surface glycoproteins of transmissible gastroenteritis virus: functions and gene sequence In Corona‐viruses. Lai M.M.C, and Stohlman S.A. (Eds) New York : Plenum Press; Adv Exp Med Biol 218, 109–116. [DOI] [PubMed] [Google Scholar]
  36. Rasschaert, D. , Geifi, J. , and Laude, H. (1987b) Enteric coronavirus TGEV: partial sequence of the genomic RNA, its organisation and expression. Biochimie 69, 591–600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rottier, P. , Welling, G.W. , Welling‐Wester, S. , Niesters, H.G. , Lenstra, J.A. , and van der Zeijst, B.A.M. (1986) Predicted membrane topology of the coronavirus protein El. Biochemistry 25, 1335–1339. [DOI] [PubMed] [Google Scholar]
  38. Ruther, U. , and Muller‐Hill, B. (1983) Easy identification of cDNA clones. EMBO J 2, 1791–1794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shieh, C‐K. , Soe, L.H. , Makino, S. , Chang, M‐F. , Stohlman, S.A. , and Lai, M.M.C (1987) The 5′‐end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader‐primed transcription. Virology 156, 321–330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Silhavy, T.J. , Berman, M.L. , and Enquist, L.W. (1984) Experiments With Gene Fusions. Cold Spring Harbor , New York : Cold Spring Harbor Laboratory Press. [Google Scholar]
  41. Spaan, W.J.M. , Delius, H. , Skinner, M. , Armstrong, J. , Rottier, P. , Smeekens, S. , van der Zeijst, B.A.M. , and Siddell, S.G. (1983) Coronavirus mRNA synthesis involves fusion of non‐contiguous sequences. EMBO J 2, 1839–1844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Staden, R. (1982) An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucl Acids Res 10, 2951–2961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stern, D.F. , and Sefton, B.M. (1982a) Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins. J Virol 44, 804–812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stern, D.F. , and Sefton, B.M. (1982b) Coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins. J Virol 44, 794–803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14, 4683–4690. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular Microbiology are provided here courtesy of Wiley

RESOURCES