Abstract
Hepatitis E virus is a leading cause of acute viral hepatitis with high lethality to pregnant and immunocompromised people, and no reliable treatments. The genome contains a region predicted to be a cysteine protease (PCP) indispensable for viral replication. The N-terminal domain is unstructured and contains cysteines essential for viral survival that have been noted for metal ion binding, while the C-terminal domain is highly structured and is homologous to a fatty acid (FA)-binding fold. However, PCP's sequence limits are unclear, and molecular functions of each domain remain contested. It is unclear whether PCP is a protease involved in viral polyprotein cleavage, or a host-targeting protein with any catalytic activity. To explore PCP’s functional repertoire, we adopt a holistic approach utilizing multiple constructs of PCP that differ in their N- or C-terminal limits to mechanistically dissect the roles and interconnection of each domain. Our lab has established expression and purification methods for PCP, and discovered that the CTD of PCP can coordinate an Fe-S cluster, and that the structured CTD binds retinoids specifically. The chemical nature of the metallocofactor or fatty acid ligand have not been previously explored, and it is unknown how these contribute to the structure or function of PCP. Here, we propose that PCP has deubiquitinase activity as the MetT-Y polyprotein fragment, and that PCP binds retinoids, and interacts with host machinery to acquire and use Fe-S clusters and to inhibit antiviral response. These interconnected aims will provide a fundamental understanding of how PCP facilitates HEV replication through its activities and host-targeted interactions, and how they are modulated by metal and FA binding. Insights will guide treatment strategies for this virus that is of growing global biomedical significance.