The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication
- 1University of Leeds
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.
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BibTeX
@misc{abualsaoud2026proteasome,
title = {{The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication}},
author = {K. M. Abualsaoud and F. J. Buchanan and O. M. Byford and E. M. Bartholomew and C. E. Williams and M. Kailani and R. Penrice-Randal and M. Harris and M. R. Herod},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.08.757567},
url = {https://www.biorxiv.org/content/10.64898/2026.10.08.757567v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication
AU - K. M. Abualsaoud
AU - F. J. Buchanan
AU - O. M. Byford
AU - E. M. Bartholomew
AU - C. E. Williams
AU - M. Kailani
AU - R. Penrice-Randal
AU - M. Harris
AU - M. R. Herod
PY - 2026
DA - 2026/10/08
PB - bioRxiv
DO - 10.64898/2026.10.08.757567
UR - https://www.biorxiv.org/content/10.64898/2026.10.08.757567v1
AB - 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.
ER -