HBV 转录物的治疗性关闭促进 SMC5/6 复合物的重新出现和体内

StephenW

HBV 转录物的治疗性关闭促进 SMC5/6 复合物的重新出现和体内病毒基因组的沉默

    Lena Allweiss1, Katja Giersch1, Andrea Pirosu1, http://orcid.org/0000-0003-3120-8958Tassilo Volz1,2, Robert C Muench3, Rudolf K Beran3, Stephan Urban2,4, Hassan Javanbakht3, Simon P Fletcher3, Marc Lütgehetmann2 ,5, http://orcid.org/0000-0002-0073-6689Maura Dandri1,2

    与德国汉堡 20251 汉堡-埃彭多夫大学医学中心内科 Maura Dandri 博士的通信； [email protected]

抽象的

目的 沉默和减少乙型肝炎病毒 (HBV) 宿主共价闭合环状 DNA (cccDNA) 的治疗策略具有治愈慢性 HBV 感染的潜力。我们旨在研究靶向所有 HBV 转录物的小干扰 RNA (siRNA) 或聚乙二醇化干扰素-α (peg-IFNα) 对病毒调节性 HBx 蛋白和染色体 5/6 复合物 (SMC5/6) 结构维持的影响。抑制cccDNA转录的宿主因子。特别是，我们评估了降低 HBV 转录物的干预措施是否可以实现和维持体内 cccDNA 转录的沉默。

用 siRNA 或 peg-IFNα 处理设计的 HBV 感染的人肝嵌合小鼠。通过定性 PCR、ELISA、免疫印迹和染色质免疫沉淀在治疗结束时和反弹阶段分析病毒学和宿主变化。 RNA原位杂交与免疫荧光相结合，在单细胞水平检测SMC6和HBV RNA。在反弹阶段使用进入抑制剂 myrcludex-B 以避免新的感染事件。

结果 siRNA 和 peg-IFNα 都强烈降低了所有 HBV 标志物，包括 HBx 水平，从而使 SMC5/6 在肝细胞中重新出现，实现了 HBV-RNA 的阴性化和 SMC5/6 与 cccDNA 的结合。只有 IFN 降低了 cccDNA 负载并增强了 IFN 刺激的基因。然而，抗病毒作用在治疗后并未持续，SMC5/6 再次降解。值得注意的是，在治疗结束时开始阻断病毒进入，阻碍了 SMC5/6 的重新降解。

结论 这些结果表明，消除包括 HBx 在内的所有 HBV 转录物的治疗促进了 HBV 微型染色体的表观遗传抑制，而需要保护人类肝细胞免受再感染的策略来维持 cccDNA 沉默。
数据可用性声明

所有与研究相关的数据都包含在文章中或作为补充信息上传。可向 [email protected] 索取其他数据（例如，详细的协议）。

StephenW

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo

    Lena Allweiss1, Katja Giersch1, Andrea Pirosu1, http://orcid.org/0000-0003-3120-8958Tassilo Volz1,2, Robert C Muench3, Rudolf K Beran3, Stephan Urban2,4, Hassan Javanbakht3, Simon P Fletcher3, Marc Lütgehetmann2,5, http://orcid.org/0000-0002-0073-6689Maura Dandri1,2

    Correspondence to Dr Maura Dandri, Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg 20251, Germany; [email protected]

Abstract

Objective Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo.

Design HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events.

Results Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6.

Conclusion These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.
Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information. Additional data (eg, detailed protocols) are available upon request to [email protected].

StephenW

https://gut.bmj.com/content/gutjnl/71/2/372.full.pdf