乙肝病毒进入的多步调控

StephenW

                Multi-step regulation of hepatitis B virus entry                                                                        October 10, 2019                                                        
                                                Koichi Watashi, Senior Researcher from the National Institute of Infectious Diseases, Japan, explores the trick for cell invasion in reaching regulation of hepatitis B entryChronic hepatitis B virus (HBV) infection is estimated to affect approximately 257 million people worldwide. For achieving the World Health Organization (WHO) strategy to control hepatitis by 20301, understanding the HBV transmission is of particular importance. Viral transmission is determined by factors derived from both virus itself and host. One of such major factors in host is a cell surface protein that mediates the binding to virus, defined as a viral entry receptor. Sodium taurocholate cotransporting polypeptide (NTCP), a bile acid transporter, has been revealed to function as an HBV entry receptor in 20122. This protein, expressed exclusively in the liver, is an apparent factor determining the HBV infectivity to host.
However, it has been totally unclarified how NTCP mediates HBV entry and determines host susceptibility to HBV infection. In April 2019, Koichi Watashi’s group in National Institute of Infectious Diseases, Japan published a paper in Proc Natl Acad Sci USA that provides a new concept that HBV infection is determined by another molecule that regulates the function of virus-receptor complex, termed as an entry cofactor3.
Cell culture and animal model data so far demonstrate that NTCP is required for HBV infection. However, there is accumulating evidences suggesting that NTCP expression is not sufficient for reproducing HBV infection in cells: HBV infection is not necessarily observed in all the NTCP expressing cells, and the infection in susceptible cells depends on culture condition, irrespective of the NTCP expression level. This data raises the possibility that another factor(s) regulates HBV infection. Koichi Watashi’s group has been pursuing to identify a series of small molecules and bioactive ligands that suppress or facilitate hepatitis virus infection. Their strategy is to take advantage of these agents to probe the mechanisms underlying virus infection.
So far, they have identified a bunch of small molecules that inhibit HBV infection, including cyclosporins, tricyclic polyketides, synthetic retinoids, coumarines, macrolides, thiazolidinediones, and cyclic peptides, which were reported in the context of drug development4. In addition, the group has also found those rather facilitating HBV infection, one of which is epidermal growth factor (EGF). By focusing on this probe, they revealed that EGF receptor (EGFR) regulates HBV infection to NTCP-expressing cells. Even if expressing NTCP, HBV infection was no more observed by deficiency of expression or function of EGFR.
Mechanistically, while NTCP mediates HBV attachment to the cell surface, EGFR was shown to be involved in HBV translocation to the endosomes (Figure). Their data and previous evidences suggest that EGFR interacts with NTCP irrespective of HBV infection: EGFR-NTCP complex is likely to travel between the cell surface and the intracellular vesicles by following the cellular membrane trafficking pathway either in the presence or absence of infection. When HBV attaches to NTCP on the cell surface, it migrates with NTCP-EGFR inside the vesicles through the trafficking. Thus, NTCP functions for the attachment with HBV, and EGFR drives the internalization of HBV-NTCP complex.
Notably, if NTCP does not bind to EGFR, NTCP no longer functions as the entry receptor of HBV, which was shown by the blockade of NTCP-EGFR binding by introducing amino acid mutation and treatment with decoy peptide. This data clearly show that productive infection of HBV requires EGFR as an entry cofactor, in addition to NTCP.
Figure 1. HBV entry. After HBV binds to its receptor, NTCP, HBV-NTCP-EGFR complex is internalised into cells. Gefitinib blocks HBV internalisation through inactivation of EGFRHuman immunodeficiency virus (HIV) uses its receptor, CD4, for cell attachment and requires another involvement of coreceptor, CCR5 or CXCR4 for the following membrane fusion. Hepatitis C virus (HCV) attaches to host cells through CD81 and scavenger receptor class B type I (SR-BI), which then clusters with tight junction proteins, claudin-1 and occludin for internalization. The presenting multi-step regulation of HBV entry is analogous to such examples for the entry mechanism of these viruses. However, it is unique that HBV follows the cellular intrinsic pathway of the membrane protein trafficking that readily occurs in the absence of infection.
Moreover, as EGFR senses extracellular ligands to change its functionality, HBV susceptibility of cells may be affected by these extracellular ligands, not only by NTCP genetic condition. Next questions include whether only EGFR functions as an entry cofactor of HBV, given that cells express related receptor tyrosine kinases such as fibroblast growth factors, vascular endothelial growth factors, and platelet-derived growth factors. How the entry cofactor drives HBV internalization, and releases HBV afterwards for the productive viral infection? Good news is that this machinery is expected to serve as a target for anti-HBV development. Actually, a peptide that interrupted EGFR-NTCP binding blocked HBV internalization and infection.
Similarly, EGFR tyrosine kinase inhibitor, gefitinib, a clinically used anti-cancer drug, inhibited HBV infection. Further analysis of targeting this machinery for developing a new anti-HBV strategy is of great demand in the near future. Albeit still many blackbox, we have had a significant gain of understanding in this decade on the mechanism underlying HBV infection and determining the susceptibility to HBV. Further analysis on HBV infection is expected to develop new strategies to control the worldwide hepatitis.

StephenW

乙肝病毒进入的多步调控
十月10，2019
日本国立传染病研究所高级研究员渡口浩一（Koichi Watashi）探索了细胞侵袭达到调控乙型肝炎进入的窍门

据估计，慢性乙型肝炎病毒（HBV）感染影响全世界约2.57亿人。为了实现世界卫生组织（WHO）到20301年控制肝炎的战略，了解HBV传播尤为重要。病毒的传播取决于病毒本身和宿主的来源。宿主中的主要因素之一是介导与病毒结合的细胞表面蛋白，被定义为病毒进入受体。牛磺胆酸钠共转运多肽（NTCP）（一种胆汁酸转运蛋白）已在20122年被证明具有HBV进入受体的功能。该蛋白仅在肝脏中表达，是决定HBV对宿主感染性的明显因素。

但是，目前还不清楚NTCP如何介导HBV进入并确定宿主对HBV感染的易感性。 2019年4月，日本国立传染病研究所的Koichi Watashi的研究小组在美国国家自然科学研究院（Proc Natl Acad Sci USA）上发表了一篇论文，提出了一个新概念，即HBV感染由另一种调节病毒受体复合物功能的分子决定，该分子被称为进入辅助因子3。

迄今为止的细胞培养和动物模型数据表明，HBV感染需要NTCP。但是，越来越多的证据表明，NTCP表达不足以在细胞中繁殖HBV感染：不一定在所有表达NTCP的细胞中都观察到HBV感染，而且易感细胞中的感染取决于培养条件，而与NTCP表达水平无关。该数据增加了另一个因素调节HBV感染的可能性。晃一晃（Koichi Watashi）的研究小组一直在寻找能够抑制或促进肝炎病毒感染的一系列小分子和生物活性配体。他们的策略是利用这些试剂来探究病毒感染的潜在机制。

到目前为止，他们已经发现了一堆抑制HBV感染的小分子，包括环孢菌素，三环聚酮化合物，合成类维生素A，香豆素，大环内酯，噻唑烷二酮和环状肽，这些都是在药物开发过程中报道的4。此外，该小组还发现了那些相当促进HBV感染的疾病，其中之一是表皮生长因子（EGF）。通过关注该探针，他们揭示了EGF受体（EGFR）调节HBV对表达NTCP的细胞的感染。即使表达NTCP，也不再因EGFR的表达或功能不足而观察到HBV感染。

从机制上讲，尽管NTCP介导HBV附着在细胞表面，但EGFR被证明与HBV转运到内体有关（图）。他们的数据和先前的证据表明，无论HBV感染如何，EGFR都会与NTCP相互作用：在存在或不存在感染的情况下，通过遵循细胞膜运输途径，EGFR-NTCP复合物很可能在细胞表面和细胞内囊泡之间传播。当HBV附着在细胞表面的NTCP上时，它会与NTCP-EGFR在囊泡内通过运输迁移。因此，NTCP用于与HBV的结合，而EGFR驱动HBV-NTCP复合体的内在化。

值得注意的是，如果NTCP不与EGFR结合，则NTCP将不再充当HBV的进入受体，这可以通过引入氨基酸突变和诱饵肽处理来阻断NTCP-EGFR结合来证明。该数据清楚地表明，除了NTCP外，生产性感染HBV还需要EGFR作为进入辅助因子。
乙型肝炎的监管
图1. HBV条目。 HBV与其受体NTCP结合后，HBV-NTCP-EGFR复合物被内化到细胞中。吉非替尼通过灭活EGFR阻断HBV内在化

人类免疫缺陷病毒（HIV）使用其受体CD4进行细胞附着，并且需要另一种共感受器CCR5或CXCR4参与其后的膜融合。丙型肝炎病毒（HCV）通过CD81和I类清道夫受体B（SR-BI）附着在宿主细胞上，然后与紧密连接蛋白claudin-1和occludin聚集在一起进行内在化。当前对HBV进入的多步调节类似于这些病毒进入机制的示例。然而，独特的是HBV遵循膜蛋白运输的细胞内在途径，这种途径在没有感染的情况下容易发生。
此外，由于EGFR感觉到细胞外配体会改变其功能，因此细胞的HBV易感性可能不仅受NTCP遗传条件的影响，还受这些细胞外配体的影响。接下来的问题包括，鉴于细胞表达相关的受体酪氨酸激酶，例如成纤维细胞生长因子，血管内皮生长因子和血小板衍生的生长因子，因此是否只有EGFR可以作为HBV的进入辅助因子。进入辅助因子如何驱动HBV内在化并随后释放HBV以进行生产性病毒感染？好消息是，该机器有望成为抗HBV开发的目标。实际上，打断EGFR-NTCP结合的肽可阻断HBV内在化和感染。

同样，EGFR酪氨酸激酶抑制剂吉非替尼（gefitinib）是一种临床使用的抗癌药物，可抑制HBV感染。在不久的将来，对针对这种机器以开发新的抗乙肝病毒策略的进一步分析需求很大。尽管仍然有很多黑匣子，但在这十年中，我们对HBV感染的潜在机制以及确定HBV的易感性有了重要的了解。预期对HBV感染的进一步分析将开发出控制全球肝炎的新策略。