Detection of Mouse Hepatitis Virus Nonstructural Proteins Using Antisera Directed Against Bacterial Viral Fusion Proteins

  • Philip W. Zoltick
  • Julian L. Leibowitz
  • James DeVries
  • Catherine J. Pachuk
  • Susan R. Weiss
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 276)

Abstract

Mouse hepatitis virus, strain A59 cDNAs were inserted into the procaryotic fusion vector pGE374. RecA/viral/LacZ tripartite fusion proteins were synthesized from these plasmids and purified from E. coli. Antisera were raised in rabbits against these fusion proteins. Viral nonstructural proteins were detected in infected murine fibroblasts and glial cells. The anti-gene B, 0RF1 sera detect a 3OK cytoplasmic protein while the anti-gene E, ORF2 sera detect a 9.6K protein. Sera raised against proteins encoded in cDNAs from 5′ portions of gene A immunoprecipitate the 200–250K polypeptides synthesized in vitro from genome RNA. Antisera raised against proteins encoded in both 5′ and 3′ portions of gene A immunoprecipitate membrane associated polypeptides of 150K and >600K from MHV infected cells.

Keywords

Hepatitis Codon Recombination Influenza Fractionation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Spaan, W.J.M., Cavanagh, D., Horzinek, M.C. 1988. Coronaviruses. Structure and genome expression. J. Gen. Virol. 69:2939–2952.PubMedCrossRefGoogle Scholar
  2. 2.
    Pachuk, C.J., Bredenbeek, P.J., Zoltick, P.W., Spaan, W.J.M. and Weiss, S.R. (1988). Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis Coronavirus, strain A59. Virology 171:141–148.CrossRefGoogle Scholar
  3. 3.
    Luytjes, W., Bredenbeek, P.J., Noten, A.F.S., Horzinek, M.C., Spaan, W.J.M. (1988).Sequence of mouse hepatitis virus mRNA 2: Indications for RNA recombination between coroanviruses and influenza C virus. Virology 166:415–422.PubMedCrossRefGoogle Scholar
  4. 4.
    Skinner, M.A., Siddell, S.G. (1985).Coding sequence of Coronavirus MHV-JHM mRNA4. J. Gen. Virol. 66:593–596.PubMedCrossRefGoogle Scholar
  5. 5.
    Skinner, M.A., Ebner, D., Siddell, S.G. (1985). Coronavirus MHV-JHM mRNA 5 has a sequence arrangement which potentially allows translation of a second downstream open reading frame. J. Gen. Virol. 66:581–592.PubMedCrossRefGoogle Scholar
  6. 6.
    Budzilowicz, C.J., Weiss, S.R. (1987). In vitro sythesis of two polypeptides from a nonstructural gene of coronvirus mouse hepatitis virus, strain A59. Virology 157:509–515.PubMedCrossRefGoogle Scholar
  7. 7.
    Leibowitz, J.L., DeVries, J.R., Haspel, M.V. (1982). Genetic analysis of murine hepatitis virus. J. Virol. 42:1080–1087.PubMedGoogle Scholar
  8. 8.
    Denison, M.R., Perlman, S. (1986). Translation and processing of mouse hepatitis virus virion RNA in a cell free system. J. Virol. 60:12–18.PubMedGoogle Scholar
  9. 9.
    Soe, L., Shieh,C.K., Baker,S., Chang, M.F., Lai, M.M.C. (1987) . Sequence and translation of murine Coronavirus 5’end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J. Virol. 61:3968–3976.PubMedGoogle Scholar
  10. 10.
    Leibowitz, J.L., Perlman, S., Weinstock, G., DeVries, J.R., Budzilowicz, C.J., Weissmann, J.M., Weiss, S.R. (1988). Detection of a murine Coronavirus nonstructural protein encoded in a downstream open reading frame. Virology 164:156– 164.PubMedCrossRefGoogle Scholar
  11. 11.
    Ebner, D., Siddell, S. (1986). Identification of the MHV-JHM mRNA 4 gene product using fusion protein antisera. In Advances in Experimental Medicine and Biology, Lai and Stohlman, eds. Plenum Press, N.Y. pp. 39–45.Google Scholar
  12. 12.
    Leibowitz, J.L., Weiss, S.R., Paavola, E., Bond, C.W. (1982). Cell-free translation of murine Coronavirus RNA. J. Virol. 43:905–913.PubMedGoogle Scholar
  13. 13.
    Leibowitz, J.L., Wilhelmson, D.C., Bond, C.W. (1981). The virus specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM. Virol. 114:39–51.CrossRefGoogle Scholar
  14. 14.
    Sturman, L.S., Takemoto, K.K. (1972).Enhanced growth of a murine Coronavirus in transformed mouse cells. Infect. Immunity 6:501–507.Google Scholar
  15. 15.
    Lavi, E., Suzumura, A., Hirayama, M., Silberburg, D.H., Weiss, S.R. (1987). Coroanvirus MHV-A59 causes a persistent productive infection in glial cell cultures. Microbiol Pathogenesis 3:79–86.CrossRefGoogle Scholar
  16. 16.
    Zoltick, P.W., Leibowitz, J.L., DeVries, J.R., Weinstock, G., Weiss, S.R. A general method for the induction and screening of antisera directed against polypeptides encoded by cDNAs:Generation of antiserum specific for Coronavirus MHV-A59 putative polyerase gene. Gene in press.Google Scholar
  17. 17.
    Brayton, P.R., Lai, M.M.C., Patton, C.D., Stohlman, S.A.(1982).Characterization of two RNA polymerase activities induced by mouse hepatitis virus. J. Virol. 42:847–853.PubMedGoogle Scholar
  18. 18.
    Gubler, U., Hoffman, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25:263–269, 1983.PubMedCrossRefGoogle Scholar
  19. 19.
    Budzilowicz, C.B., Wilczynski, S.P., Weiss, S.R. (1985).Three intergenic regions of 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.PubMedGoogle Scholar
  20. 20.
    Zoltick, P.W., Leibowitz, J.L., Oleszak, E., Weiss, S.R. Mouse hepatitis virus gene B 0RF1 is expressed in the cytoplasm of mouse fibroblasts and glial cells. Submitted to Virology.Google Scholar
  21. 21.
    Krieg, P.A., Melton,D.A. (1984) . Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Research,12:7075–7070.CrossRefGoogle Scholar
  22. 22.
    Deng, G., and Wu, R., (1981). An improved procedure for utilizing terminal transferase to add homopolymers to the 3’ terminus of DNA. Nuc. Acids. Res. 9:4173–4188.CrossRefGoogle Scholar
  23. 23.
    Boursnell, M.E.G., Brown, T.D.K., Foulds, I.J., Green, P.H., Tomley, F.,. Binns, M.M. (1987). The complete sequence of the genome of avian infectious bronchitis virus. J. Gen. Virol. 68:57–77.PubMedCrossRefGoogle Scholar
  24. 24.
    Leibowitz, J.L. DeVries, J.R.(1988). Synthesis of virus-specific RNA in permeabilized murine Coronavirus infected cells. Virology 166:66–75.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Philip W. Zoltick
    • 1
  • Julian L. Leibowitz
    • 2
  • James DeVries
    • 2
  • Catherine J. Pachuk
    • 1
  • Susan R. Weiss
    • 1
  1. 1.Department of MicrobiologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA
  2. 2.Department of Pathology and Laboratory MedicineUniversity of Texas Health Sciences CenterHoustonUSA

Personalised recommendations