Expression of interferon-gamma by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity.
A defective-interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV) was developed as a vector for expressing interferon-gamma (IFN-gamma). The murine IFN-gamma gene was cloned into the DI vector under the control of an MHV transcriptional promoter and transfected into MHV-infected cells. IFN-gamma was secreted into culture medium as early as 6 hr posttransfection and reached a peak level (up to 180 U/ml) at 12 hr posttransfection. The DI-expressed IFN-gamma (DE-IFN-gamma) exhibited an antiviral activity comparable to that of recombinant IFN-gamma and was blocked by a neutralizing monoclonal antibody against IFN-gamma. Treatment of macrophages with DE-IFN-gamma selectively induced the expression of the cellular inducible nitric oxide synthase and the IFN-gamma-inducing factor (IGIF) but did not affect the amounts of the MHV receptor mRNA. Antiviral activity was detected only when cells were pretreated with IFN-gamma for 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with IFN-gamma during or after infection. Furthermore, addition of IFN-gamma together with MHV did not prevent infection, but appeared to prevent subsequent viral spread. MHV variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to IFN-gamma treatment in vitro, with the most virulent strain being most resistant to IFN-gamma treatment. Infection of susceptible mice with DE-IFN-gamma-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the CNS and less virus replication, than that caused by virus containing a control DI vector. This study thus demonstrates the feasibility and usefulness of this MHV DI vector for expressing cytokines and may provide a model for studying the role of cytokines in MHV pathogenesis.
Zhang
X
X
Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033-1054, USA.
Hinton
D R
DR
Cua
D J
DJ
Stohlman
S A
SA
Lai
M M
MM
D000818:Animals; D003673:Defective Viruses; D015870:Gene Expression; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D006517:Murine hepatitis virus / Q000276:immunology* / Q000472:pathogenicity / Q000502:physiology; D012367:RNA, Viral; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D012367:RNA, Viral; D007371:Interferon-gamma