Hepatitis E Virus ORF1 Polyprotein Harbors a Pocket‐Like Cavity That Is Vital for Virus Replication and Represents a Novel Antiviral Target

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide, yet no FDA‐approved anti‐HEV medication available. Elucidating HEV replication machinery is therefore crucial for identifying novel antiviral targets, and consequently developing potent antivirals. The nonstructural ORF1 polyprotein is pivotal for HEV replication. Herein, it is revealed that the ORF1 X domain is a critical component of HEV replication machinery. Interestingly, the ADP‐ribose hydrolase activity of X domain per se is dispensable for HEV replication. Instead, the X domain supports HEV replication through its interdomain interaction with the RNA‐dependent RNA polymerase (RdRp). Structure‐based functional analysis reveals that X and RdRp jointly create a “pocket‐like” cavity (PC) at their interaction interface. Site‐directed mutagenesis disrupting the PC integrity completely abolishes HEV replication, demonstrating its crucial role in the viral life cycle. Through a PC‐targeted high‐throughput in silico screening pipeline, combined with molecular docking analysis, surface plasmon resonance assays, and advanced in vitro HEV models, saikosaponin D and liriopesides B are identified as potent HEV inhibitors targeting this critical interface. Collectively, this study identifies a novel structure within ORF1 polyprotein that is crucial for HEV replication, and demonstrates the feasibility of developing novel antivirals by targeting this PC structure.