Processing of the hepatitis E virus polyprotein can be mediated by a cellular protease

The genomes of positive-sense RNA viruses encode polyproteins that are essential for controlling viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV), a leading cause of acute viral hepatitis, translates its positive-sense RNA genome to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using a sub-genomic replicon system, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases. Overall, our data supports a model where HEV uses host proteases to support its replication and could have uniquely evolved not to rely on a virally-encoded protease for replication. ### Competing Interest Statement The authors have declared no competing interest.