Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV’s open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; further, the “pPCP” domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together our work provides a comprehensive model of the structure and function of HEV ORF1. Significance Statement The development of non-teratogenic and potent antiviral therapies against HEV have been hindered by an incomplete understanding of the viral replication cycle. Our work provides a mechanistic insight into the complex replicative cycle of this understudied human pathogen and identifies a novel domain-domain interaction that is vital for replicative fitness.