VIROLOGY233, 327338 (1997) ARTICLE NO.VY978598 Expression of Interferon-gby a Coronavirus Defective-Interfering RNA Vector and Its Effect on Viral Replication, Spread, and Pathogenicity Xuming Zhang,*,1David R. Hinton,*, Daniel J. Cua,*, Stephen A. Stohlman,*, and Michael M. C. Lai*, *Department of Neurology, Department of Pathology, Department of Molecular Microbiology and Immunology, and Howard Hughes Medical Institute, University of Southern California School of Medicine, Los Angeles, California 90033-1054 Received November 13, 1996; returned to author for revision March 12, 1997; accepted May 5, 1997 A defective-interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV) was developed as a vector for expressing interferon-g(IFN-g). The murine IFN-ggene was cloned into the DI vector under the control of an MHV transcriptional promoter and transfected into MHV-infected cells. IFN-gwas 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-g(DE-IFN-g) exhibited an antiviral activity comparable to that of recombinant IFN-gand was blocked by a neutralizing monoclonal antibody against IFN-g. Treatment of macrophages with DE-IFN-gselectively induced the expression of the cellular inducible nitric oxide synthase and the IFN-g-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-gfor 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with IFN-gduring or after infection. Furthermore, addition of IFN-gtogether 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-gtreatment in vitro, with the most virulent strain being most resistant to IFN-gtreatment. Infection of susceptible mice with DE-IFN-g-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.q 1997 Academic Press INTRODUCTION1992). Resistance to IFN-gmay lead to incomplete viral clearance and contribute to the establishment of persis- Interferon-g(IFN-g) is a pleiotropic cytokine produced tent infection (Moskophidis et al., 1994). By contrast, IFN- by activated CD4/and CD8/T cells and natural killer gis also involved in inflammatory processes. IFN-gin- cells (Trinchieri and Perussia, 1985; Pestka and Langer, duces the expression of many other inflammatory cyto- 1987; Ijzermans and Marquet, 1989), which exerts both kines, such as interleukin-1 (IL-1) and tumor necrosis antiviral and immunomodulatory effects. These include factor(TNF), andacts synergisticallywith thesecytokines the activation of mononuclear phagocytes, enhancement (Wong and Goeddel, 1986). The multitude of immuno- of the generation of oxygen-free radicals, modulation of modulatory effects of IFN-gmakes it a particularly inter- class I and II major histocompatability complex (MHC) esting cytokine for studying viral pathogenesis. In the antigen expression, and promotion of differentiation of central nervous system (CNS), no cells constitutively ex- both T and B cells (for reviews, see references by Pestka press IFN-g. During encephalomyelitis, for example as and Langer, 1987; Benveniste, 1992). It plays an im- a result of mouse hepatitis virus (MHV) infection, acti- portant role in the early phase of many viral infections vated NK cells and T cells which pass through the blood (Wheelock, 1965; Wong and Goeddel, 1986; Leist et al., brain barrier into the CNS express IFN-g(Bukowski et 1989; Klavinskis et al., 1989; Feducchi and Carrasco, al., 1983; Pearce et al., 1994). In addition to its effects on 1991; Ramsey et al., 1993; Heise and Virgin IV, 1995; mononuclear cells, IFN-gacts upon cells of the CNS, Rodriguez et al., 1995), inhibiting the replication of a vari- such as astrocytes, microglia, and macrophages (Ben- ety of viruses prior to activation of antiviral effector cyto- veniste, 1992). toxic T lymphocyte (CTL) or antibodies. Because of its MHV, a murine coronavirus, causes a variety of dis- antiviral activity, IFN-ghas been implicated in virus clear- eases in rodents, such as hepatitis, enteritis, and neuro- ance and resolution of viral infection (Ramshaw et al., logical diseases, depending on the viral strain (Cheever et al., 1949; Gledhill and Niven, 1955; Ishida et al., 1978). 1To whom correspondence and reprint requests should be ad- Even within the well-studied neuropathogenic JHM dressed at Department of Neurology, University of Souther