乙型肝炎病毒通过跨高尔基网络利用逆行运输路线来避免溶

StephenW

乙型肝炎病毒通过跨高尔基网络利用逆行运输路线来避免溶酶体降解
李英义 1 , 黑木和之 1 , 岛上哲郎 1 , 村井和久 2 , 川口和典 1 , 白崎贵义 2 , 公树光辉 1 , 杉本西保 1 , 西川智树 1 , 冈田光 1 , 折田典明 1 , 高山秀夫 1 , Ying Wang 2 , Phuong Doan Thi Bich 1 , Astuya Ishida 1 , Satahiro Iwabuchi 3 , Shinichi Hashimoto 3 , Takeshi Shimaoka 4 , Noriko Tabata 5 , Miho Watanabe-Takahashi 6 , Kiyotaka Nishikawa 6 , Hiroshi Yanagawa 5 , Motoharu Seiki 1 , Kouji Matsushima 4、山下太郎 1、金子修一 1、本田雅男 7
隶属关系
隶属关系

    1
    日本金泽大学医学研究生院胃肠病学系。
    2
    日本金泽大学健康医学研究生院临床检验医学系。
    3
    日本和歌山医科大学高等医学研究所分子病理生理学系。
    4
    日本千叶东京理科大学生物医学科学研究所炎症和免疫疾病的分子调控部门。
    5
    Purotech Bio Inc，日本神奈川。
    6
    同志社大学分子生命科学系。
    7
    金泽大学医学研究生院胃肠病学系，日本金泽；日本金泽大学健康医学研究生院临床检验医学系。电子地址：[email protected]。

    PMID: 36270602 DOI: 10.1016/j.jcmgh.2022.10.008

抽象的

背景与目的：由于共价闭合环状病毒 DNA (cccDNA) 的持续存在，乙型肝炎病毒 (HBV) 感染难以治愈。我们对新建立的 HBV 阳性和 HBV 阴性肝细胞癌细胞系进行了单细胞转录组分析，发现胞质分裂的贡献者 11 (DOCK11) 在 HBV 持久性中起关键作用。然而，DOCK11 在 HBV 生命周期中的作用尚未明确。

方法：使用HBV感染模型，通过Southern印迹和实时检测PCR在包括PXB细胞在内的各种肝细胞中测量cccDNA水平。通过超分辨率显微镜、邻近连接测定和延时分析研究了 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了 DOCK11 的下游分子及其潜在机制。

结果： cccDNA 水平因 DOCK11 过表达而显着增加，并因 DOCK11 抑制而受到抑制。有趣的是，DOCK11 与反高尔基网络 (TGN) 中的逆行运输蛋白、具有 GTPase 结构域、锚蛋白重复和 pleckstrin 同源结构域的蛋白 2 和 ADP-核糖基化因子 1 以及 HBV 衣壳的 Arf-GAP 功能相关，以为 HBV 从早期内体 (EEs) 到 TGN 再到内质网 (ER) 开辟一条替代逆行运输途径，从而避免溶酶体降解。临床上，恩替卡韦治疗可显着降低慢性乙型肝炎患者肝活检组织中的 DOCK11 水平，这种降低与 HBV 表面抗原水平相关。

结论：HBV 利用通过 EEs-TGN-ER 的逆行运输途径进行感染，该途径由 DOCK11 促进并用于维持 cccDNA。因此，DOCK11 是预防持续性 HBV 感染的潜在治疗靶点。

关键词：AGAP2；码头11； cccDNA；逆行贩卖。

版权所有 © 2022 作者。由 Elsevier Inc. 出版。保留所有权利。

StephenW

Hepatitis B virus utilizes a retrograde trafficking route via the trans-Golgi network to avoid lysosomal degradation
Ying-Yi Li  1 , Kazuyuki Kuroki  1 , Tetsuro Shimakami  1 , Kazuhisa Murai  2 , Kazunori Kawaguchi  1 , Takayoshi Shirasaki  2 , Kouki Nio  1 , Saiho Sugimoto  1 , Tomoki Nishikawa  1 , Hikari Okada  1 , Noriaki Orita  1 , Hideo Takayama  1 , Ying Wang  2 , Phuong Doan Thi Bich  1 , Astuya Ishida  1 , Sadahiro Iwabuchi  3 , Shinichi Hashimoto  3 , Takeshi Shimaoka  4 , Noriko Tabata  5 , Miho Watanabe-Takahashi  6 , Kiyotaka Nishikawa  6 , Hiroshi Yanagawa  5 , Motoharu Seiki  1 , Kouji Matsushima  4 , Taro Yamashita  1 , Shuichi Kaneko  1 , Masao Honda  7
Affiliations
Affiliations

    1
    Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
    2
    Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan.
    3
    Department of Molecular Pathophysiology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan.
    4
    Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan.
    5
    Purotech Bio Inc, Kanagawa, Japan.
    6
    Department of Molecular Life Sciences, Doshisha University.
    7
    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. Electronic address: [email protected].

    PMID: 36270602 DOI: 10.1016/j.jcmgh.2022.10.008

Abstract

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.

Methods: cccDNA levels were measured by Southern blotting and real-time detection PCR 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.

Results: 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 and ADP-ribosylation factor 1, 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.

Conclusions: HBV utilizes 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.

Keywords: AGAP2; DOCK11; cccDNA; retrograde trafficking.

Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

StephenW

https://doi.org/10.1016/j.jcmgh.2022.10.008