乙肝病毒DNA整合在病毒生命周期的早期通过NTCP依赖性包膜病

StephenW

J Virol. 2018 Feb 7. pii: JVI.02007-17. doi: 10.1128/JVI.02007-17. [Epub ahead of print]
Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.Tu T1, Budzinska MA2, Vondran FWR3,4, Shackel NA2,5,6, Urban S7,8.
Author information
1Department of Infectious Diseases, Molecular Virology, Heidelberg Hospital University, Germany [email protected].2Centenary Institute, The University of Sydney, Australia.3Regenerative Medicine and Experimental Surgery (ReMediES), Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.4German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany.5South Western Sydney Clinical School, University of New South Wales, Australia.6Liverpool Hospital, Gastroenterology, Sydney, Australia.7Department of Infectious Diseases, Molecular Virology, Heidelberg Hospital University, Germany.8German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.

AbstractChronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration.ImportanceHepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation.


PMID:29437961DOI:10.1128/JVI.02007-17

StephenW

J Virol。 2018年2月7日。pii：JVI.02007-17。 doi：10.1128 / JVI.02007-17。 [电子版提前打印]
乙肝病毒DNA整合在病毒生命周期的早期通过NTCP依赖性包膜病毒颗粒的摄取在体外感染模型中发生。
Tu T1，Budzinska MA2，Vondran FWR3,4，Shackel NA2,5,6，Urban S7,8。
作者信息

1
    德国海德尔堡医科大学传染病分子病毒学系[email protected]。
2
    澳大利亚悉尼大学百年研究所。
3
    再生医学与实验手术（ReMediES），德国汉诺威汉诺威医学院内脏和移植外科。
4
    德国汉诺威感染研究中心（DZIF），德国汉诺威Hannover-Braunschweig合作伙伴网站。
五
    澳大利亚新南威尔士大学西南悉尼临床学院。
6
    利物浦医院，消化，悉尼，澳大利亚。
7
    德国海德堡医院大学传染病分子病毒学系。
8
    德国传染病研究中心（DZIF），合作伙伴网站海德堡，海德堡，德国。

抽象

乙型肝炎病毒（HBV）慢性感染是全球肝病的主要原因。虽然HBV通过核附加型DNA（cccDNA）复制，但HBV DNA整合到宿主细胞基因组中通常在感染患者的肝脏中观察到。虽然有报道称为致癌性改变，但是HBV DNA整合的机制和时机尚未充分了解，主要是由于缺乏可检测到整合事件的体外感染模型。在这里，我们建立了一个体外系统，可以在HBV感染后可靠地检测到整合。在HBV感染后，我们使用反向巢式PCR在原代人肝细胞，HepaRG-NTCP，HepG2-NTCP和Huh7-NTCP细胞中测量HBV DNA整合。在所有细胞类型中以每10000个细胞> 1的比率检测整合，在Huh7-NTCP细胞中检测最一致。整合率在感染后3至9天内保持稳定。通过用200nM的HBV进入抑制剂Myrcludex B处理有效地阻断了HBV DNA整合，但用10μM替诺福韦，100U干扰素α或1μM衣壳组装抑制剂GLS4不能有效阻断HBV DNA整合。这表明在感染肝细胞后立即发生HBV DNA整合，并且可能独立于该模型中的从头HBV复制。现场分析显示，HBV DNA整合分布在整个人类基因组中。此外，整合的HBV DNA序列与作为主要前体的双链线性HBV DNA一致。因此，我们建立了体外系统来研究HBV DNA整合机制。重要性乙型肝炎病毒（HBV）是一种常见的血源性病原体，在慢性感染后可引起肝癌和肝硬化。 HBV DNA整合入宿主基因组发生在所有已知的嗜肝病毒科成员中，尽管这种形式对于病毒复制不是必需的。据报道，HBV DNA整合可促使肝癌形成和病毒感染的持续存在。然而，什么时候HBV DNA整合发生的机制尚不清楚。在这里，我们开发和表征了体外系统，以可靠地检测由真实的HBV感染事件导致的HBV DNA整合，并且与患者组织中发现的非常相似。使用这个模型，我们证明当感染第一次建立时已经发生了整合。重要的是，我们在这里提供了一个系统来分析涉及HBV整合的分子因素，可以用来制定策略来制止它的形成。

结论：
    29437961
DOI：
    10.1128 / JVI.02007-17