Human norovirus (HuNoV) are positive sense RNA viruses belonging to the Caliciviridae family and are a major cause of acute gastroenteritis in developing and developed countries. However, the study of HuNoV has proven to be challenging due to the lack of effective tissue culture systems and small animal models. Despite its significant health burden, there are currently no effective treatments for HuNoV infections. Recently, the discovery of a closely related norovirus, Murine Norovirus (MNV) has advanced our understanding of norovirus biology and pathogenesis.
Here we investigated the association between MNV infection, stress granules and protein translation. We observed that stress granules (SG), which are cytoplasmic RNA granules that consist of translationally-silenced RNA and RNA binding-proteins, are not induced during MNV infection. Further, infected cells treated with arsenite, a known oxidative stressor to induce SG formation, were restricted in their ability to form SGs, suggesting a potential viral control for delayed SG formation. We also demonstrated that during infection, there was a progressive increase in phosphorylated eukaryotic initiation factor 2 alpha (eIF2a), which is the regulatory subunit of eIF2 complex involved in translation initiation. Phosphorylation of eIF2a leads to the general shutoff of host protein translation. Interestingly, MNV translation still occurs under these condition implying MNV may employ an alternative protein translation mechanism to facilitate intracellular replication. To confirm the increasing host translation shutoff, we treated infected cells with puromycin and showed that there is increasing stalling of translation via western blotting with anti-puromycin antibodies, which correlates to the increasing phosphorylation of eIF2a. Our subsequent analyses suggest that the translational repression is mediated via Protein kinase-R (PKR) and that some of the individual viral proteins regulate both the phosphorylation of eIF2a and inhibition of SG formation.
These results suggest MNV regulates the PKR-mediated innate immune response by promoting eIF2a phosphorylation and thereby regulating cellular protein translation. This observations may provide a link between MNV infection, translational control, and a mechanism of immune evasion.