NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization
NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization
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乙型肝炎病毒内化过程中，NTCP寡聚化发生在NTCP- egfr相互作用的下游
DOI：
10.1128/jvi.00938-21
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发表时间：
2021-12-01
影响因子：
5.4
通讯作者：
Muramatsu, Masamichi
中科院分区：
文献类型：
--
作者：
Fukano, Kento;Oshima, Mizuki;Muramatsu, Masamichi
牛磺胆酸钠共转运多肽（NTCP）是乙型肝炎病毒（HBV）进入宿主细胞所必需的受体。迄今为止已经报道了许多针对NTCP的HBV进入抑制剂；这些抑制剂促进了对病毒进入过程的机制分析。然而，病毒与NTCP相互作用后HBV内化进入宿主细胞的机制仍不清楚。最近，我们报道了曲格列酮，一种噻唑烷二酮衍生物，特异性抑制HBV内化和NTCP寡聚化，从而抑制HBV感染。本研究使用曲格列酮作为化学探针来研究进入过程，评估NTCP寡聚化对HBV内化的贡献。利用表面等离子体共振和转运体动力学，我们发现曲格列酮直接与NTCP相互作用，并非竞争性地干扰NTCP介导的胆汁酸摄取，这表明曲格列酮与NTCP变构结合，而不是与胆汁酸结合袋结合。此外，丙氨酸扫描诱变显示，NTCP的苯丙氨酸274 （F274A）突变导致HBV易感性丧失，破坏了NTCP的寡聚化和HBV内化，而不影响病毒附着在细胞表面。NTCP与表皮生长因子受体（EGFR） （HBV内化的另一宿主辅助因子）相互作用的抑制剂阻碍了NTCP寡聚化。同时，共免疫沉淀分析显示，曲格列酮和NTCP中的F274A突变都不影响NTCP- egfr相互作用。这些发现表明，NTCP寡聚化始于NTCP- egfr相互作用的下游，然后引发HBV内化。这项研究为HBV进入机制提供了重要的见解。乙型肝炎病毒（HBV）感染是通过与牛磺胆酸钠共转运多肽（NTCP）的特异性相互作用介导的，NTCP是一种病毒进入受体。虽然病毒与受体的相互作用被认为可以触发病毒内化进入宿主细胞，但HBV内化的确切分子机制尚不清楚。在这项研究中，我们揭示了曲格列酮的作用模式，曲格列酮是一种HBV内化的特异性抑制剂，可以阻碍NTCP寡聚化，并鉴定出NTCP苯丙氨酸274是这种寡聚化所必需的残基。我们进一步分析了NTCP寡聚化与HBV内化之间的关系，这一过程是由表皮生长因子受体（EGFR）介导的，表皮生长因子受体是HBV内化的另一个重要宿主辅助因子。我们的研究提供了关于HBV进入机制的关键信息，并表明病毒受体的寡聚化可以作为药物发现的一个有吸引力的靶点。
Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms.IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.