Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan.
Cell Mol Gastroenterol Hepatol. 2023;15(3):533-558. doi: 10.1016/j.jcmgh.2022.10.008. Epub 2022 Oct 19.
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified.
The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay.
The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels.
HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.
乙型肝炎病毒（HBV）感染难以治愈，原因是共价闭合环状病毒 DNA（cccDNA）持续存在。我们对新建立的 HBV 阳性和 HBV 阴性肝癌细胞系进行了单细胞转录组分析，发现胞质分裂衔接蛋白 11（DOCK11）在 HBV 持续感染中起着至关重要的作用。然而，DOCK11 在 HBV 生命周期中的作用尚未阐明。
利用 HBV 感染模型，通过 Southern 印迹和实时检测聚合酶链反应测量各种肝细胞（包括 PXB 细胞）中的 cccDNA 水平。通过超分辨率显微镜、邻近连接分析和延时分析研究 HBV 衣壳的逆行运输途径。通过液相色谱-串联质谱、免疫印迹和酶联免疫吸附试验检测 DOCK11 的下游分子和潜在机制。
DOCK11 的过表达强烈增加了 cccDNA 水平，而 DOCK11 的抑制作用则降低了 cccDNA 水平。有趣的是，DOCK11 与高尔基体逆向运输蛋白在 TGN 中的功能相关，包括 Arf-GAP 与 GTP 酶结构域、锚蛋白重复和 Pleckstrin 同源结构域蛋白 2（AGAP2）和 ADP-核糖基化因子 1（ARF1），以及 HBV 衣壳，共同开启了 HBV 从早期内体（EE）到 TGN 再到内质网（ER）的替代逆行运输途径，从而避免了溶酶体降解。临床上，慢性乙型肝炎患者肝活检中 DOCK11 的水平在用恩替卡韦治疗后显著降低，这种降低与 HBV 表面抗原水平相关。
HBV 使用通过 EE-TGN-ER 的逆行运输途径进行感染，该途径由 DOCK11 促进，有助于维持 cccDNA。因此，DOCK11 是预防持续性 HBV 感染的潜在治疗靶点。
