2023 Fiscal Year Annual Research Report
Elucidation of host factors and the associated pathways responsible for cellular permissiveness to hepatitis E virus replication and identification of the potential inhibitors
| Project/Area Number |
22K15478
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| Research Institution | Jichi Medical University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2024-03-31
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| Keywords | Hepatitis E virus / Host cellular factor / Anti-HEV drug / Microarray / Cell culture / Replication efficiency / Reporter virus / Drug screening |
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| Outline of Annual Research Achievements |
Subclones of a single PLC/PRF/5 cell line demonstrated up to 10,000-folds difference in the permissiveness to hepatitis E virus (HEV) replication. Based on the results of RNA microarray analysis of highly permissive and poorly permissive subclones, several genes were silenced by using small interfering RNA (siRNA) followed by screening using eHEV-nanoKAZ and evaluation in cell culture. Among them, possible genes involved in cellular permissiveness to HEV were identified.
One hit gene was upregulated in a subclone with the highest susceptibility to HEV replication (PLC 9E8), and introduction of siRNA decreased HEV replication efficiency. It encodes ubiquitin ligase. Since ubiquitin was suggested to play a role in HEV life cycle, a screening on a ubiquitin-related compound library was performed by using three HEV reporter systems to search for candidate compound with activity against HEV: HEV-GLuc replicon (covering HEV RNA replication), HEV-nanoKAZ (covering HEV entry and RNA replication), and HEV-HiBiT (covering HEV particle formation and release). Among the hit compounds, one with strongest inhibition on luciferase activity was further evaluated in cultured cells. By 12 days post-inoculation, the highest dose (10 μM) already decreased the HEV RNA titer in culture supernatants of the infected cells to 1,000 lower than that of untreated control cells.
In summary, several genes potentially contributing to cellular susceptibility to HEV replication were identified. They can be the target for the development of novel and specific anti-HEV drug.
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