Hepatitis E Virus RNA-Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and known as the viral replicase. The RNA-dependent RNA polymerase (RdRp) domain is responsible for the synthesis of viral RNA. Here, we identified a highly conserved a-helix located in the RdRp thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic a-helix extending from amino acid 1622 to 1647 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic a-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RdRp palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. In conclusion, structure-function analyses identified a conserved amphipathic a-helix in the thumb subdomain of the HEV RdRp with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RdRp thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.