De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1's N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis.