O030 Evasion of the OAS-RNase L pathway by murine coronavirus ns2 protein is required for viral replication and hepatitis

INTRODUCTION: The 2′,5′-oligoadenylate synthetase-ribonuclease L (OAS-RNase L) system is a potent IFN induced antiviral pathway. Following infection, IFNs induce a group of OAS genes whose products are activated by viral double-stranded RNA. OAS uses ATP to generate 2′,5′-linked oligoadenylates (2-5A). 2-5A binds to and activates the ubiquitous cellular endoribonuclease RNase L causing cleavages of single stranded regions of both viral and cellular RNA thus inhibiting viral replication. 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. The intracellular concentration of 2-5A is believed to be the primary factor controlling RNase L activation. The liver contains abundant innate immune cells, which provide the first line of defense against pathogens. However, the factors that determine whether a virus can bypass this defense to access and infect the liver parenchyma are not well understood. The murine coronavirus, mouse hepatitis virus (MHV), strain A59, infection of mice provides a model for virus induced hepatitis. The MHV accessory protein, ns2, antagonizes the type I IFN response in macrophages and promotes the induction of hepatitis. Here we will describe how the ns2 protein facilitates the development of viral hepatitis by blocking OAS-RNase L pathway. METHODS: Bone marrow macrophages (BMM) from wild type (wt) and RNase L−/− mice were infected with A59 and ns2 mutant MHV. Viral titers were determined by plaque assays. RNase L activity was monitored by rRNA integrity in RNA chips. Intracellular levels of 2-5A were measured using RNase L activation assays. Effects of ns2 on 2-5A levels in cells were determined by transfecting ns2 or mutant ns2 cDNAs into HEK-293T cells. Recombinant ns2 and mutant ns2H126R proteins were purified and incubated with 2-5A in vitro and the 2-5A breakdown products were measured by HPLC. Hepatitis was determined by histology following inoculation A59 or ns2 mutant MHV into wt and RNase L−/− mice. RESULTS: We found evidence for a new molecular mechanism of subversion of the RNase L pathway in macrophages that regulates acute hepatitis during MHV infection. Coronavirus ns2 belongs to the LigT-like protein family, within the 2H phosphoesterase superfamily, some of which possess cyclophosphodiesterase activity (CPD). We have found that ns2 is not a CPD, but instead is a 2′,5′-phosphodiesterase (PDE) that cleaves, and thus eliminates 2-5A, the activator of RNase L. We observed that ns2 blocks the IFN inducible OAS-RNase L pathway to facilitate hepatitis development. Ns2 prevents activation of RNase L and consequently limits viral RNA degradation. An ns2 mutant virus was unable to replicate in the liver or induce hepatitis in wt mice, but was highly pathogenic in RNase L−/− mice. Thus, RNase L is a critical cellular factor for protection against viral infection of the liver and the resulting hepatitis. CONCLUSION: MHV accessory protein ns2 is a 2′,5′-PDE which degrades 2-5A and limits RNase L activation thus facilitating virus-induced hepatitis in mice.