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Super-Resolution Microscopy Analysis of Hepatitis B Viral cccDNA and Host Factors
Phuong Thi Bich Doan1, Kouki Nio1, Tetsuro Shimakami1
1Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takara-Machi, Kanazawa 920-8641, Japan.
Insights
The host gene DOCK11 is crucial for hepatitis B virus (HBV) persistence by regulating the transcription of viral cccDNA. DOCK11 facilitates the association of cccDNA with key proteins, promoting viral replication.
Area of Science:
- Virology
- Molecular Biology
- Hepatology
Background:
- Hepatitis B virus (HBV) infection persists due to stable viral covalently closed circular DNA (cccDNA).
- The host gene dedicator of cytokinesis 11 (DOCK11) was previously identified as essential for HBV persistence.
Purpose of the Study:
- To elucidate the mechanism by which DOCK11 regulates cccDNA transcription.
- To investigate the interactions between DOCK11 and host factors involved in HBV persistence.
Main Methods:
- Quantitative real-time PCR (qPCR) and fluorescence in situ hybridization (FISH) to measure cccDNA levels.
- Super-resolution microscopy, immunoblotting, and chromatin immunoprecipitation to identify protein interactions.
- FISH for subcellular localization of HBV nucleic acids and host factors.
Main Results:
- DOCK11 partially colocalized with histone modifications (H3K4me3, H3K27me3) and RNA Pol II but had limited direct roles in modification or transcription.
- DOCK11 regulated the subnuclear distribution of host factors and cccDNA.
- DOCK11 promoted the proximity of cccDNA to H3K4me3 and RNA Pol II, facilitating cccDNA transcription.
Conclusions:
- DOCK11 is a key regulator of HBV cccDNA transcription, not through direct histone modification but by controlling cccDNA localization.
- DOCK11 facilitates the association of cccDNA with H3K4me3 and RNA Pol II, essential for activating viral transcription.
- Targeting DOCK11 may offer a novel strategy to inhibit HBV persistence.
Abstract:
Infection with hepatitis B virus (HBV) cannot be cured completely because of the persistence of covalently closed circular DNA (cccDNA). We previously found that the host gene dedicator of cytokinesis 11 (DOCK11) was required for HBV persistence. In this study, we further investigated the mechanism that links DOCK11 to other host genes in the regulation of cccDNA transcription. cccDNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) in stable HBV-producing cell lines and HBV-infected PXB-cells®. Interactions between DOCK11 and other host genes were identified by super-resolution microscopy, immunoblotting, and chromatin immunoprecipitation. FISH facilitated the subcellular localization of key HBV nucleic acids. Interestingly, although DOCK11 partially colocalized with histone proteins, such as H3K4me3 and H3K27me3, and nonhistone proteins, such as RNA Pol II, it played limited roles in histone modification and RNA transcription. DOCK11 was functionally involved in regulating the subnuclear distribution of host factors and/or cccDNA, resulting in an increase in cccDNA closely located to H3K4me3 and RNA Pol II for activating cccDNA transcription. Thus, it was suggested that the association of cccDNA-bound Pol II and H3K4me3 required the assistance of DOCK11. DOCK11 facilitated the association of cccDNA with H3K4me3 and RNA Pol II.
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