Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.
Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.
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DOI：
10.1111/cmi.13250
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发表时间：
2020-12
影响因子：
3.4
通讯作者：
McKeating JA
中科院分区：
文献类型：
--
作者：
Chakraborty A;Ko C;Henning C;Lucko A;Harris JM;Chen F;Zhuang X;Wettengel JM;Roessler S;Protzer U;McKeating JA
B型肝炎病毒（HBV）是一种有包膜的DNA病毒，含有部分双链松弛环状（rc）DNA。在感染后，rcDNA被递送到细胞核，在那里它被修复为共价闭合环状（ccc）DNA，其充当所有病毒RNA的转录模板。我们对HBV颗粒进入动力学和调节细胞内病毒运输和cccDNA形成的宿主途径的了解有限。牛磺胆酸钠共转运肽（NTCP）作为主要受体的发现允许对病毒生命周期中的这些早期步骤进行研究。我们采用同步感染方案来量化HBV进入动力学。HBV在4 ° C下附着于细胞不依赖于NTCP，然而，随后的颗粒摄取是NTCP依赖性的，并在感染后12小时达到饱和。HBV的摄取是网格蛋白和动力蛋白依赖的，肌动蛋白和微管蛋白在感染的前6小时起作用。细胞分级分离研究表明，HBV DNA在感染后6小时内在细胞核中，cccDNA在感染后24小时首次检测到。我们的研究显示，大多数（83%）细胞结合颗粒进入HepG2-NTCP细胞，然而，只有少数（<1%）细胞内rcDNA转化为cccDNA，突出表明这是体外建立感染的限速因素。这些知识突出了我们体外细胞培养系统的缺陷，并将为支持有效HBV复制的生理相关模型的设计和评估提供信息。
Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.
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影响因子：
6.7
作者：
Kitamura K;Que L;Shimadu M;Koura M;Ishihara Y;Wakae K;Nakamura T;Watashi K;Wakita T;Muramatsu M
通讯作者：
Muramatsu M
DOI：
10.3390/v9010021
发表时间：
2017-01-21
期刊：
Viruses
影响因子：
--
作者：
Gallucci L;Kann M
通讯作者：
Kann M
影响因子：
5.4
作者：
Gonçalves-Carneiro D;McKeating JA;Bailey D
通讯作者：
Bailey D
影响因子：
3.4
作者：
Herrscher, Charline;Pastor, Florentin;Blanchard, Emmanuelle
通讯作者：
Blanchard, Emmanuelle
影响因子：
5.4
作者：
Funk, A;Mhamdi, M;Sirma, H
通讯作者：
Sirma, H