Cell Host Neumann- Haefelin et al., 2007). Thus, the innate immune response is a particularly important defense against pathogen invasion of theliver.TypeIinterferon(IFN)isthefi rstlineofdefenseavailable to restrict viral infection including infections with human hepatitis viruses (Garc a-Sastre and Biron, 2006; Qu and Lemon, 2010). Kupffer cells (liver macrophages) found in the liver sinusoids are among the fi rst cells to encounter viruses and as such, along with endothelial cells present a barrier that must be breached in order to access hepatocytes in the liver parenchyma (Crispe, 2009). Indeed, it has been reported that IFN signaling in macro- phages is crucial for control of hepatitis induced by mouse hepatitisvirus(Cervantes-Barraga netal.,2009)andlymphocytic choriomeningitis virus (Lang et al., 2010). Type I IFN, primarily IFN-a/b, produced by virus-infected cells, induces expression of over four hundred interferon-stimu- lated genes (ISGs), whose products cooperate to induce an antiviral state. One potent antiviral IFN induced activity is the 20,50-oligoadenylate synthetase-ribonuclease L (OAS-RNase L) pathway (Sadler and Williams, 2008) (Figure 1). After infection, IFNs induce a group of OAS genes whose products are acti- vated by viral double-stranded RNA (dsRNA). OAS uses ATP to generate the 20,50-linked oligoadenylates with the structures px50A(20p50A)n; x = 13; n R 2 (2-5A). 2-5A binds to the ubiqui- tous cellular endoribonuclease RNase L, causing inactive RNase L monomers to form activated dimers (Dong and Silver- man, 1995). RNase L cleaves single-stranded regions of both viral and cellular messenger RNA (mRNA), leading to inhibition of viral replication and protein synthesis (Silverman, 2007). In addition, detection of the newly generated short RNAs by cellular pattern recognition receptors, MDA5 and RIG-I, further enhances IFN production and the ensuing antiviral activities (Malathi et al., 2007). The concentration of 2-5A is believed to be the primary factor controlling RNase L activation. The cellular enzyme 20-phosphodiesterase (20-PDE, also known as PDE12) has been proposed to reduce or prevent 2-5A-mediated activa- tion of RNase L by degrading 2-5A, presumably to prevent excessive tissue damage caused by the effects of RNase L (Kubota et al., 2004). The antiviral activity of the OAS-RNase L pathway has been demonstrated during infections of mice with numerous RNA and DNA viruses (Flodstro m-Tullberg et al., 2005; Li et al., 1998; Samuel et al., 2006; Scherbik et al., 2006; Washenberger et al., 2007; Xiang et al., 2002; Zhou et al., 1997). Cell Host Snijder et al., 2003). We show here that ns2 has in fact a 20,50-phosphodiesterase activity that can down- regulate intracellular levels of 2-5A, thereby preventing the acti- vation and antiviral activity of RNase L. Our results underscore the importance of the OAS-RNase L pathway in vivo for protec- tion of the host from developing viral hepatitis. RESULTS MHV A59 Resists RNase L Activity MHV differs from many other viruses in that it stimulates type I IFN production in only limited cell types, primarily in macro- phages (Roth-Cross et al., 2008) and plasmacytoid dendritic cells (Cervantes-Barragan et al., 2007). Furthermore MHV displays cell type-specifi c sensitivity to the antiviral effects of IFN treatment, with macrophage lineage cells being most successful in restricting MHV replication (Rose and Weiss, 2009; Zhao et al., 2011). We also observed that MHV was unable to prevent IFN induction by a coinfecting virus and was also unable to protect a coinfecting virus from the effects of IFN (Roth-Cross et al., 2007). Thus, we reasoned that MHV is unable tosubvert majorIFNsignaling pathways, butrathermay interfere with one or more IFN-induced antiviral activities. Indeed, a previous study showed that MHV-A59 infection does not promote RNase L-mediated degradation of 18S and 28S ribo- somal RNA (rRNA) in HeLa cells (Ye et al., 2007), suggesting that the OAS-RNase L pathway may be downregulated. To more directly evaluate the role of RNase L in MHV replication, we compared the replication of wild-type A59 in bone-marrow derived macrophages (BMMs) from wild-type C57BL/6 (B6) mice or from mice genetically ablated for RNase L expression RNase L?/?(B6). The kinetics of accumulation of A59 genome RNA were indistinguishable in the two cell types (Figure 2A), as was the extent of infectious virus replication (Figure 2D). In contrast, the genome RNA levels of Sendai virus (SeV) and encephalomyocarditis virus (EMCV) were approximately 16-fold and 64-fold higher at 12 hr after infection in BMMs derived from RNase L?/?mice, compared with B6 (Figures 2B and 2C, respectively). These results indicate that RNase L activity restricts the replication of both SeV and EMCV but not that of MHV A59. Figure 1. The Interferon-Induced OAS-RNase L Pathway After infection, viral RNA is detected by pattern recognition receptors RIG-I and MDA5, resulting in the inductio