The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR ؊/؊ ) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-␤ in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-␤ production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-␤ production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-␤ mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-␤ by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.The type I interferon (IFN) response, consisting of IFN-␣/␤, represents one of the first lines of defense against viral infection. During viral infection of a cell, pathogen-associated molecular patterns, such as double-stranded RNA (dsRNA), are exposed and recognized by pattern recognition receptors (PRRs). These PRRs include Toll-like receptors (TLRs), such as TLR3, that reside on the cell membrane or in endosomal compartments and the cytoplasmic receptors RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiationassociated protein 5) (8). Recognition by PRRs leads to the activation of IRF-3 (IFN regulatory factor 3), which dimerizes and translocates to the nucleus. In the nucleus it associates with the promoter region of the IFN-␤ gene along with NF-B, AP-1, and CBP/p300, driving IFN-␤ transcription. Once IFN-␤ is produced, it is secreted from infected cells and is able to bind to the IFN-associated receptor (IFNAR) on neighboring cells, amplifying the production of IFN-␤ and inducing the expression of IFN-␣ and many IFN-stimulated genes (37, 41). This series of events generates an antiviral milieu that helps to limit viral spread. Therefore, it is not surprising that many viruses have developed mechanisms to subvert or alter the type I IFN response, making it more difficult for the host to combat viral infection (8,20).Mouse hepatitis virus (MHV), a group 2 coronavirus (CoV), is a positive-strand RNA virus. Depending on the strain, MHV can infect both the ...

Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Coronaviruses are divided into three groups, with group I and II viruses infecting mammals and group III viruses infecting avian species (76). Human coronaviruses (HCoV), such as the group I HCoV-229E and group II HCoV-OC43 viruses, cause approximately 5 to 30% of all human respiratory tract infections (76, 77). In late 2002, severe acute respiratory syndrome-associated coronavirus (SARSCoV) infected more than 8,000 people with approximately 750 deaths (43,44,80), demonstrating that HCoV could also cause more serious disease in humans. The recent discoveries of two new human coronaviruses, the group I HCoV-NL63 (69, 70) and the group II HCoV-HKU1 (18, 77), in patients suffering from respiratory illnesses have also added to the need to further our understanding of coronavirus pathogenesis.Mouse hepatitis virus (MHV), a group II coronavirus, has long been used as a tool for studying coronavirus biology and pathogenesis. MHV causes hepatic and central nervous system diseases of varying severity depending on the strain and is therefore used as a model for hepatitis, viral encephalitis, and demyelination (76). While there is much known about the general immune response to MHV (76), there is limited information about the alpha/beta interferon (IFN-␣/␤) response induced by MHV infection (21,63,75,82).

The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-aminoacid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.