The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication

Abstract

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis, with an estimated 20 million human infections annually. Its diverse host range presents a significant zoonotic threat, yet the molecular mechanisms underpinning HEV–host interactions remain poorly characterised. Here, we used transposon-based insertion screening of the HEV pORF1 polyprotein to identify a novel region within the viral PCP domain that permits HA-epitope insertion, enabling the first epitope tagging of this domain. Combined with V5 tagging of the hypervariable region (HVR), this approach enabled the generation of a replication-competent stable cell line expressing dual-tagged ORF1. Co-immunoprecipitation and TMT-based LC-MS proteomics revealed that pORF1 interacts with host proteins involved in endoplasmic reticulum-associated degradation (ERAD) and the 26S proteasome. Pharmacological inhibition demonstrated that both pathways are required for efficient HEV replication. Treatment with the FDA-approved proteasome inhibitor bortezomib altered pORF1 precursor abundance and led to loss of full-length pORF1 through enhanced proteolysis. Importantly, the kinetics of ORF1 degradation following proteasome inhibition correlated with immediate loss of HEV replication, demonstrating a functional link between pORF1 proteostasis and viral replication. Collectively, our findings identify the ERAD network as a previously unrecognised requirement for HEV replication and establish a role for the 26S proteasome in maintaining pORF1 proteostasis. Importance Hepatitis E virus (HEV) is classified in the Hepeviridae family, which currently comprises 10 species, and infects a wide range of animal hosts. Genetically divergent HEV strains have recently been identified within populations of rodents, bats and shrews. Thus, novel HEV zoonotic spillover events are of increasing concern. A detailed understanding of how HEV subverts host cellular machinery for replication is incomplete. Here, we demonstrate that the HEV pORF1 polyprotein interacts with components of the ERAD and proteasomal pathways. We further show that pharmacological inhibition of these networks significantly reduced HEV replication. Mechanistic analysis revealed that pORF1 homeostasis is dependent on 26S proteasomal function as bortezomib treatment resulted in aberrant HEV pORF1 proteolysis, corresponding with a reduction in genome replication. Collectively, our findings improve the understanding of HEV host-pathogen interactions and further characterise the roles of two critical pathways required during replication.