Abstract
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide, but the understandings of the ORF1 remain incomplete. In this study, we use a biochemical framework to interrogate the HEV papain-like cysteine protease (PCP) region and find evidence for a cysteine-rich, intrinsically disordered N-terminal domain linked to a more structured C-terminal domain with structural similarity to retinol fatty acid–binding proteins. Biophysical characterization of PCP constructs shows the presence of an oxygen-sensitive metallocofactor consistent with [Fe–S] cluster. Bis-ANS hydrophobic-site reporter assays prove that PCP selectively binds retinal and retinoic acid, while retinol and functional group matched fatty acid controls do not induce comparable displacement. Perturbation of the metallocofactor shows that retinal and retinoic acid competition is highly dependent on the metal state, supporting a model in which the N-terminal cofactor modulates ligand selectivity at the C-terminal domain. Retinal and retinoic acid competitive binding persists in an ORF1-relevant MetT_Y_PCP construct, indicating biological relevance beyond isolated PCP processing. Collectively, these results identify PCP as a retinoid-binding metalloprotein and provide a framework for understanding metallobiology in viral proteins.