2022 Fiscal Year Research-status 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 / Cell culture / Host cellular factor / Cellular permissiveness / Replication efficiency / Microarray / Reporter virus |
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| Outline of Annual Research Achievements |
Hepatitis E virus (HEV) is increasingly recognized as the leading cause of acute hepatitis. In immunocompromised patients, HEV can cause chronic hepatitis. Currently, there is no specific anti-HEV drug available. Targeting cellular factors associated with HEV replication can be one of the methods to develop specific anti-HEV drug. In this context, cell culture is required. Subclones of PLC/PRF/5 cells have variable permissiveness to HEV replication even when inoculated with the same virus, suggesting that aside from viral factor itself, cellular factors might be involved in determining host susceptibility to HEV replication, and therefore, can be the target for development of specific anti-HEV drug.
Subclones of a single PLC/PRF/5 cell line demonstrated up to 10,000-folds difference in the permissiveness to HEV replication. Based on the results of RNA microarray analysis of highly permissive and poorly permissive PLC/PRF/5 subclones, 15 upregulated genes and 15 downregulated genes were selected. Small interfering RNA (siRNA)-mediated gene silencing was performed on the upregulated and downregulated genes, followed by screening using eHEV-nanoKAZ (Primadharsini et al., J Virol 2022) and evaluation in cell culture. Silencing of any of four of upregulated genes in highly permissive subclone resulted in decreased HEV replication efficiency, while silencing of any of four downregulated genes in the poorly permissive subclone resulted in slightly increased HEV replication efficiency.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The possible genes involved in cellular permissiveness to HEV replication were identified through siRNA-mediated gene silencing, followed by screening using HEV-nanoKAZ, and the results have been further validated through evaluation in cell culture. Experiments to overexpress the downregulated genes in the highly permissive subclone are currently ongoing to further confirm their effect on HEV replication efficiency. The identified genes were subjected to functional prediction program to determine possible pathways involved in the cell permissiveness to HEV replication.
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| Strategy for Future Research Activity |
Importance of the identified host genes at particular step of HEV life cycle will be investigated by using reporter HEVs established in our lab: HEV-nanoKAZ (entry), HEV-GLuc replicon (RNA replication), and HEV-HiBiT (particle formation or release). Furthermore, known inhibitors of the identified genes will be used to validate their effect on HEV replication efficiency in cultured cells.
Based on results of RNA microarray analysis, several pathways associated with cellular permissiveness to HEV replication have been predicted. Genes associated with the predicted pathways will be silenced and their importance in HEV life cycle will be examined by using the reporter HEVs. Moreover, screening on the pathway-specific compound library will be performed to search for the compounds with activity against HEV. Effectiveness and mechanism of action of the hit small compounds will be investigated.
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