Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Jan 30;173(2):683–691. doi: 10.1016/0042-6822(89)90581-3

Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus

Kyoko Yokomori 1, Nicola La Monica 1, Shinji Makino 1, Chien-Kou Shieh 1, Michael MC Lai 1,1
PMCID: PMC7118923  PMID: 2556847

Abstract

We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected.

References

  1. Baric R.S., Nelson G.W., Fleming J.O., Deans R.J., Keck J.G., Casteel N., Stohlman S.A. Interactions between coronavirus nucleocapsid protein and viral RNAs: Implication for viral transcription. J. Virol. 1988;62:4280–4287. doi: 10.1128/jvi.62.11.4280-4287.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cavanagh D. Structural polypeptides of Coronavirus IBV. J. Gen. Virol. 1981;53:93–103. doi: 10.1099/0022-1317-53-1-93. [DOI] [PubMed] [Google Scholar]
  3. Dalziel R.G., Lampert P.W., Talbot P.J., Buchmeier M.J. Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence. J. Virol. 1986;59:463–471. doi: 10.1128/jvi.59.2.463-471.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dea S., Tussen P. Identification of the structural proteins of turkey enteric coronavirus. Arch. Virol. 1988;99:173–186. doi: 10.1007/BF01311068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fleming J.O., Shubin R.A., Sussman M.A., Casteel N., Stohlman S.A. Monoclonal antibodies to the matrix (E1) glycoprotein of mouse hepatitis virus protect mice from encephalitis. Virology. 1989;168:162–167. doi: 10.1016/0042-6822(89)90415-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fleming J.O., Trousdale M.D., El-Zaatari F.A.K., Stohlman S.A., Weiner L.P. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol. 1986;58:869–875. doi: 10.1128/jvi.58.3.869-875.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirano N., Fujiwara K., Hino S., Matsumoto M. Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture. Arch. Gesamte Virusforsch. 1974;44:298–302. doi: 10.1007/BF01240618. [DOI] [PubMed] [Google Scholar]
  8. Hogue B.G., Brian D.A. Structural proteins of human respiratory coronavirus OC43. Virus Res. 1986;5:131–144. doi: 10.1016/0168-1702(86)90013-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holmes K.V., Doller E.W., Sturman L.S. Tunicamycin-resistant glycosylation of coronavirus glycoprotein: Demonstration of a novel type of viral glycoprotein. Virology. 1981;115:334–344. doi: 10.1016/0042-6822(81)90115-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson D.C., Spear P.G. O-linked oligosaccharides are acquired by Herpes simplex virus glycoproteins in the Golgi apparatus. Cell. 1983;32:987–997. doi: 10.1016/0092-8674(83)90083-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kakach L.T., Suzich J.A., Collett M.S. Rift valley fever virus M segment: Phlebovirus expression strategy and protein glycosylation. Virology. 1989;170:505–510. doi: 10.1016/0042-6822(89)90442-x. [DOI] [PubMed] [Google Scholar]
  12. King B., Brian D.A. Bovine coronavirus structural proteins. J. Virol. 1982;42:700–707. doi: 10.1128/jvi.42.2.700-707.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. King B., Potts B.J., Brian D.A. Bovine coronavirus hemagglutinin protein. Virus Res. 1985;2:53–59. doi: 10.1016/0168-1702(85)90059-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lai M.M.C., Stohlman S.A. Comparative analysis of RNA genome of mouse hepatitis virus. J. Virol. 1981;38:661–670. doi: 10.1128/jvi.38.2.661-670.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leibowitz J.L., Weiss S.R., Paavola E., Bond C.W. Cell-free translation of murine coronavirus RNA. J. Virol. 1982;43:905–913. doi: 10.1128/jvi.43.3.905-913.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Luytjes W., Bredenbeek P.J., Noten A.F.H., Horzinek M., Spaan W.J.M. Sequence of mouse hepatitis virus A59 mRNA 2: Indications for RNA recombination between coronaviruses and influenza C virus. Virology. 1988;166:415–422. doi: 10.1016/0042-6822(88)90512-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Makino S., Lai M.M.C. Evolution of the 5′-end of genomic RNA of murine coronavirus during passages in vitro. Virology. 1989;169:227–232. doi: 10.1016/0042-6822(89)90060-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Makino S., Taguchi F., Fujiwara 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]
  23. Makino S., Taguchi F., Hayami M., Fujiwara K. Characterization of small plaque mutants of mouse hepatitis virus, JHM strain. Microbiol. Immunol. 1983;27:445–454. doi: 10.1111/j.1348-0421.1983.tb00603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Niemann H., Boschek B., Evans D., Rosing M., Tamura T., Klenk H.-D. Post-translational glycosylation of coronavirus glycoprotein E1: Inhibition by monensin. EMBO J. 1982;1:1499–1504. doi: 10.1002/j.1460-2075.1982.tb01346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Niemann H., Heistergerg-Moutsis G., Geyer R., Klenk H.-D., Wirth M. Glycoprotein E1 of MHV-A59: Structure of the O-linked carbohydrates and construction of full length recombinant cDNA clones. Adv. Exp. Med. Biol. 1984;173:201–213. doi: 10.1007/978-1-4615-9373-7_20. [DOI] [PubMed] [Google Scholar]
  26. Niemann H., Klenk H.-D. Coronavirus glycoprotein E1, a new type of viral glycoprotein. J. Mol. Biol. 1981;153:993–1010. doi: 10.1016/0022-2836(81)90463-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parker M.D., Cox G.J., Deregt D., Fitzpatrick D.R., Babiuk L.A. Cloning and in vitro expression of the gene for the E3 haemagglutinin glycoprotein of bovine coronavirus. J. Gen. Virol. 1989;70:155–164. doi: 10.1099/0022-1317-70-1-155. [DOI] [PubMed] [Google Scholar]
  28. Rottier P., Brandenberg D., Armstrong J., van der Zeijst B., Warren G. Vol. 81. 1984. Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: The E1 glycoprotein of Coronavirus mouse hepatitis virus A59; pp. 1421–1425. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rottier P.J., Horzinek M.C., van der Zeijst B.A.M. Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: Effect of tunicamycin. J. Virol. 1981;40:350–357. doi: 10.1128/jvi.40.2.350-357.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rottier P.J., Rose J.K. Coronavirus E1 glycoprotein expressed from cloned cDNA localizes in the Golgi region. J. Virol. 1987;61:2042–2045. doi: 10.1128/jvi.61.6.2042-2045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rottier P.J., Welling G.W., Welling-Wester S., Niesters H.G., Lenstra J.A., van der Zeijst B.A.M. Predicted membrane topology of the coronavirus protein E1. Biochemistry. 1986;25:1335–1339. doi: 10.1021/bi00354a022. [DOI] [PubMed] [Google Scholar]
  32. Shieh C.-K., Lee H.-J., Yokomori K., La Monica N., Makino S., Lai M.M.C. Identification of a new transcriptional initiation site and the corresponding functional gene 2b in murine coronavirus RNA genome. J. Virol. 1989;63:3729–3736. doi: 10.1128/jvi.63.9.3729-3736.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siddell S.G. Coronavirus JHM: Tryptic peptide fingerprinting of virion proteins and intracellular polypeptides. J. Gen. Virol. 1982;62:259–269. doi: 10.1099/0022-1317-62-2-259. [DOI] [PubMed] [Google Scholar]
  34. Siddell S. Coronavirus JHM: Coding assignments of subgenomic mRNAs. J. Gen. Virol. 1983;64:113–125. doi: 10.1099/0022-1317-64-1-113. [DOI] [PubMed] [Google Scholar]
  35. Siddell S.G., 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]
  36. Soe L.H., Shieh C.K., Baker S.C., Chang M.F., Lai M.M.C. Sequence and translation of the murine coronavirus 5′-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J. Virol. 1987;61:3968–3976. doi: 10.1128/jvi.61.12.3968-3976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stohlman S.A., Lai M.M.C. Phosphoprotein of mouse hepatitis viruses. J. Virol. 1979;32:672–675. doi: 10.1128/jvi.32.2.672-675.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Storz J., Rott R., Kaluza G. Enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment. Infect Immun. 1981;31:1214–1222. doi: 10.1128/iai.31.3.1214-1222.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sturman L.S., Holmes K.V. The molecular biology of coronaviruses. Adv. Virus Res. 1983;28:35–112. doi: 10.1016/S0065-3527(08)60721-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sturman L.S., Holmes K.V., Behnke J. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J. Virol. 1980;33:449–462. doi: 10.1128/jvi.33.1.449-462.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sturman L.S., Ricard C.S., Holmes K.V. Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: Activation of cell fusing activity of virions by trypsin and separation of two different 90K cleavage fragments. J. Virol. 1985;56:904–911. doi: 10.1128/jvi.56.3.904-911.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tabor S., Richardson C.C. Vol. 82. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes; pp. 1074–1078. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Takatsuki A., Kohno K., Tamura G. Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Agric. Biol. Chem. 1975;39:2089–2091. [Google Scholar]
  44. Talbot P.J. Hemagglutination by murine hepatitis virus. Absence of detectable activity in strains 3, A59, and S grown in DBT cells. Intervirology. 1989;30:117–120. doi: 10.1159/000150083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thomas S.M., Lamb R.A., Paterson R.G. Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5. Cell. 1988;54:891–902. doi: 10.1016/S0092-8674(88)91285-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vlasak R., Luytjes W., Leider J., Spaan W., Palese P. The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity. J. Virol. 1988;62:4686–4690. doi: 10.1128/jvi.62.12.4686-4690.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vlasak R., Luytjes W., Spaan W., Palese P. Vol. 85. 1988. Human and Bovine coronaviruses recognize sialic acid-containing receptor similar to those of influenza C viruses; pp. 4526–4529. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wege H., Muller A., ter Meulen V. Genomic RNA of the murine coronavirus JHM. J. Gen. Virol. 1978;41:217–227. doi: 10.1099/0022-1317-41-2-217. [DOI] [PubMed] [Google Scholar]
  49. Yoshikura H., Tajima S. Role of protease in mouse hepatitis virus-induced cell fusion. Virology. 1981;113:503–511. doi: 10.1016/0042-6822(81)90178-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES