Advertisement

Using a Defective-Interfering RNA System to Express the HE Protein of Mouse Hepatitis Virus for Studying Viral Pathogenesis

  • Xuming Zhang
  • David Hinton
  • Sungmin Park
  • Ching-Len Liao
  • Michael M. C. Lai
  • Stephen Stohlman
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 440)

Abstract

We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin’esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.

Keywords

Virus Titer Viral Antigen Plaque Form Unit Helper Virus Viral Pathogenesis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Deregt, D., and Babiuk, L. A., 1987, Monoclonal antibodies to bovine Coronavirus: Characteristics and topographical mapping of neutralizing epitopes on the E2 and E3 glycoproteins, Virology 161: 410–420.PubMedCrossRefGoogle Scholar
  2. Deregt, D., Gifford, G.A., Khalid Ijaz, M., Watts, T.C., Gilchrist, J.E., Haines, D.M., and Babiuk, L.A., 1989, Monoclonal antibodies to bovine Coronavirus glycoproteins E2 and E3: demonstation of in vivo neutralizing activity, J. Gen. Virol. 70:993–998.PubMedCrossRefGoogle Scholar
  3. Fleming, J.O., Stohlman, S.A., Harmon, R.C., Lai, M.M.C., Frelinger, J.A., and Weiner, L.P., 1983, Antigenic relationship of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus, Virology 131:296–307.PubMedCrossRefGoogle Scholar
  4. Gagnetan, S., Gout, O., Dubois-Dalcq, M., Rottier, P., Rossen, J., and Holmes, K.V., 1995, Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein, J. Virol. 69: 889–895.Google Scholar
  5. Herrler, G., Rott, R., Klenk, H.D., Müller, H.P., Shukda, A.K., and Schauer, R., 1985, The receptor-destroying enzyme of influenza C is virus neuraminidate-O-acetyl-esterase, EMBO J. 4:1503–1506.PubMedGoogle Scholar
  6. Herrler, G., Rott, R., and Klenk, H.D., 1985, Neuraminic acid is involved in the binding of influenza C virus to erythrocytes, Virology 141: 144–147.PubMedCrossRefGoogle Scholar
  7. Hirano, N., Fujiwara, K., Hino, S., and Matsumoto, M., 1974, Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture, Arch. Gesamte Virusforsch. 44, 298–302.PubMedCrossRefGoogle Scholar
  8. Lai, M.M.C., 1992, RNA recombination in animal and plant viruses, Microbiol. Rev. 56: 61–79.PubMedGoogle Scholar
  9. Lavi, E., Gilden, D.H., Highkin, M.K., Weiss, S.R., 1986, The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation, Lab. Ani. Sci. 36:130–135.Google Scholar
  10. Lee, H.-J., Shieh, C.-K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J., Bagdzyahdzhyan, A., and Lai, M. M. C, 1991, The complete sequence (22 kilobases) of murine Coronavirus gene 1 encoding the putative proteases and RNA polymerase, Virology 180: 567–582.PubMedCrossRefGoogle Scholar
  11. Liao, C.-L., and Lai, M. M. C, 1994, Requirement of the 5’-end genomic sequence as an upstream cis-acting element for Coronavirus subgenomic mRNA transcription, J. Virol. 68: 4727–4737.PubMedGoogle Scholar
  12. Liao, C.-L., Zhang, X. M., and Lai, M. M. C, 1995, Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase, Virology 208: 319–327.PubMedCrossRefGoogle Scholar
  13. Lin, Y.-J., and Lai, M. M. C, 1993, Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication, J. Virol. 67: 6110–6118.PubMedGoogle Scholar
  14. Makino, S., Shieh, C.-K., Soe, L. H., Baker, S. C, and Lai, M. M. C, 1988, Primary structure and translation of a defective-interfering RNA of murine Coronavirus, Virology 166: 550–560.PubMedCrossRefGoogle Scholar
  15. Makino, S., Joo, M., and Makino, J. K., 1991, A system for study of Coronavirus mRNA synthesis: A regulated, expressed subgenomic defective-interfering RNA results from intergenic site insertion, J. Virol. 65: 6031–6041.PubMedGoogle Scholar
  16. Yokomori, K., La Monica, N., Makino, S., Shieh, C.-K., and Lai, M.M.C., 1989, Biosynthesis, structure, and biological activities of envelope protein gp65 of murine Coronavirus, Virology 173: 683–691.PubMedCrossRefGoogle Scholar
  17. Yokomori, K., Baker, S.C., Stohlman, S.A., and Lai, M.M.C., 1992, Hemagglutinin-esterase (HE)-specific monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus, J. Virol. 66:2865–2874.PubMedGoogle Scholar
  18. Yokomori, K., Asanaka, M., Stohlman, S.A., Makino, S., Shubin, R.A., Gilmore, W., Weiner, L.R, Wang, F.I., and Lai, M.M.C., 1995, Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression, J. Neurovirol. 1:330–339.PubMedCrossRefGoogle Scholar
  19. Zhang, X. M., Liao, C.-L., and Lai, M. M. C, 1994, Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis, J. Virol. 68: 4738–4746.PubMedGoogle Scholar
  20. Zhang, X.M., Hinton, D.R., Cue, D., Stohlman, S., and Lai, M.M.C., 1997: Expression of gamma interferon by a Coronavirus defective-interfering RNA vector and its effect on viral replication, spread and pathogenicity, Virology. In press.Google Scholar

Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Xuming Zhang
    • 1
    • 5
  • David Hinton
    • 1
    • 2
  • Sungmin Park
    • 3
  • Ching-Len Liao
    • 3
  • Michael M. C. Lai
    • 1
    • 3
    • 4
  • Stephen Stohlman
    • 1
    • 4
  1. 1.Department of NeurologyUniversity of Southern California School of MedicineLos AngelesUSA
  2. 2.Department of PathologyUniversity of Southern California School of MedicineLos AngelesUSA
  3. 3.Department of Molecular Microbiology and ImmunologyUniversity of Southern California School of MedicineLos AngelesUSA
  4. 4.Howard Hughes Medical InstituteUniversity of Southern California School of MedicineLos AngelesUSA
  5. 5.Department of Microbiology and ImmunologyUniversity of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations