i., since under these conditions viral titers in the livers and spleens of mutant virus-infected AZD-2281 mice were indistinguishable from those of wild-type virus-infected mice (Fig. (Fig.2a,2a, bottom panel). The assessment of IFN-�� revealed no significant differences in IFN-�� expression in the spleen, liver, and serum (Fig. (Fig.6a),6a), further suggesting that a potentially elevated level of IFN-�� production by MHV-N1348A-infected pDCs may not play a major role in the apparent lack of liver pathology in MHV-N1348A-infected mice. We next assessed TNF-�� expression in mice and observed comparable expression levels in wild-type MHV-A59 and MHV-N1348A infections (Fig. (Fig.6b).6b). In contrast, IL-6 expression was significantly reduced in the spleens and livers of MHV-N1348A-infected mice compared to that in the spleens and livers of wild-type virus-infected mice (Fig.

(Fig.6c),6c), indicating that, in vivo, MHV-N1348A infection rather selectively impacts the production of this inflammatory cytokine. FIG. 6. MHV-N1348A-induced IFN-�� and inflammatory cytokine expression in vivo. 129Sv mice (n = 5) were infected with 5 �� 104 PFU of wild-type MHV-A59 (MHV wt) or MHV-N1348A. The levels of IFN-�� (a), TNF-�� (b), and IL-6 … DISCUSSION Despite the long-recognized conservation of a viral X domain in coronaviruses and RNA viruses of the alpha-like supergroup, its biological role remained elusive. Here we provide conclusive evidence that the X domain of a murine CoV impacts viral pathogenicity. Specifically, a single-amino-acid substitution in the ADRP catalytic core of the hepatotropic MHV-A59 prevented induction of acute viral hepatitis in mice.

Mechanistically, our data support the idea that expression of the coronaviral X domain interferes with host inflammatory and possibly innate responses, since compared to those for wild-type virus infections, we observed increased IFN-�� expression in pDCs and diminished proinflammatory cytokine expression in DCs and macrophages following MHV ADRP mutant infection in vitro. Type I IFN and proinflammatory cytokine production is one of the earliest events in acute hepatitis. These mediators initiate the production of other cytokines and chemokines, resulting in the recruitment of inflammatory cells and the generation of an inflammatory microenvironment that can lead to extensive hepatocyte death and liver damage.

The phenotype of MHV-N1348A indicates that the MHV X domain is likely to be involved in inflammatory processes that exacerbate MHV-induced liver pathology. We AV-951 have thoroughly assessed MHV-N1348A replication in mice, using a variety of experimental settings. Most strikingly, the mutant virus did not induce acute viral hepatitis irrespective of whether infections were done with low, intermediate, or high viral doses.