Expression of Hemagglutinin/Esterase by a Mouse Hepatitis Virus Coronavirus DefectiveInterfering RNA Alters Viral Pathogenesis Xuming Zhang,*,1David R. Hinton,*, Sungmin Park, Beatriz Parra, Ching-Len Liao,2 Michael M. C. Lai*, and Stephen A. Stohlman*, *Department of Neurology, Department of Pathology, and Department of Molecular Microbiology and Immunology, and Howard Hughes Medical Institute, University of Southern California School of Medicine, Los Angeles, California 90033 Received September 9, 1997; returned to author for revision October 2, 1997; accepted December 9, 1997 A defective-interfering (DI) RNA of mouse hepatitis virus (MHV) was developed as a vector for expressing MHV hemagglutinin/esterase (HE) protein. The virus containing an expressed HE protein (A59-DE-HE) 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. A similar virus (A59-DE-CAT) expressing the chloramphenicol acetyltransferase (CAT) was used as a control. These viruses were inoculated intracerebrally into mice, and the role of the HE protein in viral pathogenesis was evaluated. Results showed that all mice infected with parental A59 or A59-DE-CAT succumbed to infection by 9 days postinfection (p.i.), demonstrating that inclusion of the DI did not by itself alter pathogenesis. In contrast, 60% of mice infected with A59-DE-HE survived infection. HE- or CAT-specific subgenomic mRNAs were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the DI vector were expressed only in the early stage of viral infection. No significant difference in virus titer or viral antigen expression in brains was observed between A59-DE-HE- and A59-DE-CAT-infected mice, suggesting that virus replication in brain was not affected by the expression of HE. However, at day 3 p.i. there was a slight increase in the extent of inflammatory cell infiltration in the brains of the A59-DE-HE-infected mice. Surprisingly, virus titers in the livers of A59-DE-HE-infected mice were 3 log10lower than that of the A59-DE-CAT-infected mice at day 6 p.i. Also, substantially less necrosis and viral antigen were detected in the livers of the A59-DE-HE-infected mice. This may account for the reduced mortality of these mice. The possible contribution of the host immune system to this difference in pathogenesis was analyzed by comparing the expression of four cytokines. Results showed that both tumor necrosis factor-a and interleukin-6 mRNAs increased in the brains of the A59-DE-HE-infected mice at day 2 p.i., whereas interferon-g and interleukin-1a mRNAs were similar between A59-DE-HE- and A59-DE-CAT-infected mice. These data suggest that the transient expression of HE protein enhances an early innate immune response, possibly contributing to the eventual clearance of virus from the liver. This study indicates the feasibility of the DI expression system for studying roles of viral proteins during MHV infection. 1998 Academic Press INTRODUCTION Thedevelopmentofinfectious,full-lengthcDNA clones of viral RNA has provided a powerful tool for understanding the structure and function of individual viral genes and for studying their roles in viral pathogen- esis. Unfortunately, an infectious cDNA clone is currently not available for some RNA viruses with a large genome, such as coronaviruses. We have recently developed an alternative approach, namely, a defectiveinterfering (DI) RNA (Makino et al. 1985, 1988, 1991), which functions like a minigenome, as an expression vector (Liao and Lai, 1994). Such a vector can express viral or foreign proteins, including cytokines (Liao et al. 1995; Zhang et al. 1997), thus allowing more critical analysis of the functional role of these proteins in coronavirus pathogenesis. Mouse hepatitis virus (MHV), a murine coronavirus, is a member of the Coronaviridae. It contains a single- strand, positive-sense RNA genome of 31 kb (Lee et al. 1991). In addition to the genomic-length RNA, six or seven subgenomic mRNAs are synthesized in infected cells. These RNAs are conested at the 39 end and extend toward the 59 end to different lengths at intergenic (IG) regions (Lai et al. 1981). Each mRNA contains a leader RNA of approximately 70 nt at the 59 end, which is identical to the leader of the genomic RNA (Lai et al. 1983, 1984). The IG sequence serves as an initiation site and is required for the synthesis of a subgenomic mRNA. In general, each mRNA translates only one protein from the 59-most open reading frame (ORF) (Lai and Ca- vanagh, 1997). MHV contains four or five structural proteins. The spike (S) protein is the major surface glycoprotein of the 1To whom reprint requests should be addressed at present address: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 West Markham St., Mail Slot 511, Little Rock, Arkansas 72205-7199. 2Present address: Department of Microbiology and Immun