Uncoupling PKR activation and translational arrest during norovirus infection

Fritzlar S, Chao YW, Aktepe TE and Mackenzie JM

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, at the University of Melbourne.

Human norovirus (HuNoV) are positive sense RNA viruses belonging to the Caliciviridae family and are a major cause of acute gastroenteritis. However, the study of HuNoV is 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) are not induced during MNV infection. Further, infected cells treated with sodium 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 (eIF2α), yet increased MNV translation still occurred under these conditions implying MNV may employ an alternative protein translation mechanism. 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 correlated to the increasing phosphorylation of eIF2α. Our subsequent analyses suggested that the translational repression is mediated via Protein kinase-R (PKR), but further investigation revealed that PKR activation and translational arrest were uncoupled during infection. These results suggest MNV may regulate the PKR-mediated response by promoting eIF2α phosphorylation but inhibits cellular protein translation by a currently unknown mechanism. These observations may provide a link between MNV infection, stress granules, PKR and translational control.