Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release.
Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release.
复制标题
丙型肝炎病毒非结构 2 蛋白与宿主衔接蛋白 1 和 4 之间的相互作用协调病毒释放。
DOI：
10.1128/mbio.02233-17
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发表时间：
2018
期刊：
影响因子：
6.4
通讯作者：
Einav,Shirit
中科院分区：
文献类型：
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作者：
Xiao,Fei;Wang,Stanley;Barouch-Bentov,Rina;Neveu,Gregory;Pu,Szuyuan;Beer,Melanie;Schor,Stanford;Kumar,Sathish;Nicolaescu,Vlad;Lindenbach,BrettD;Randall,Glenn;Einav,Shirit
丙型肝炎病毒（HCV）通过分泌的无细胞颗粒或直接细胞间传播传播。然而，病毒宿主的决定因素管理的差异细胞内运输的细胞间和细胞间传播的病毒仍然未知。宿主衔接蛋白（AP）AP-1A、AP-1B和AP-4在高尔基体后区室中进行交通，后两者与基底外侧分选有关。我们报告说，AP-1A介导的HCV贩运过程中释放，而内吞适配器AP-2介导的进入和组装。我们证明了宿主激酶AAK 1和GAK通过控制这些网格蛋白相关AP来调节HCV感染。在这里，我们试图定义AP-4，网格蛋白非依赖性适配器，AP-1A和AP-1B在HCV感染中的作用。我们通过基于哺乳动物细胞的蛋白片段互补分析筛选HCV蛋白与AP-1A、AP-1B和AP-4的μ亚基之间的相互作用。在这次筛选中以及通过HCV感染细胞中的免疫共沉淀，非结构2（NS 2）蛋白成为这些衔接子的相互作用物。NS 2 C端两个以前未被识别的基于二亮氨酸的基序介导AP结合和HCV释放。亲和性和共培养试验表明，虽然所有三个衔接子介导HCV释放和无细胞传播，AP-1B和AP-4，但AP-1A，介导细胞间传播。活细胞成像显示HCV与AP-1A、AP-1B和AP-4共同运输，AP-4介导HCV在高尔基体后区室中的运输。最后，HCV细胞间传播受AAK 1和GAK调节，因此对AAK 1和GAK抑制剂治疗敏感。这些数据提供了一个机制的理解，HCV贩运在不同的释放途径，并揭示了在细胞间病毒spread.IMPORTANCEHCV传播通过无细胞感染或细胞间接触，屏蔽抗体中和，从而促进病毒持久性的AP的要求。然而，支配这种差异排序的因素仍然未知。通过整合蛋白质组学、RNA干扰、遗传学、活细胞成像和药理学方法，我们揭示了宿主衔接蛋白（AP）在病毒生命周期的不同晚期介导HCV交通的差异性选择。我们报道了AP-1A和AP-2分别在释放和组装过程中介导HCV的运输。在这里，我们证明了NS 2蛋白中的双亮氨酸基序介导AP-1A，AP-1B和AP-4结合和无细胞病毒释放。此外，我们发现，AP-4，以前没有涉及病毒感染的适配器，介导细胞到细胞的传播和HCV贩运。最后，我们展示了AAK 1和GAK，宿主激酶控制AP，以及对它们的抑制剂的敏感性的细胞到细胞的传播调节。这项研究提供了机制的见解，病毒宿主决定因素，促进丙型肝炎病毒的运输，具有潜在的影响，发病机制和抗病毒药的设计。
Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.IMPORTANCEHCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence. Yet, factors governing this differential sorting remain unknown. By integrating proteomic, RNA interference, genetic, live-cell imaging, and pharmacological approaches, we uncover differential coopting of host adaptor proteins (APs) to mediate HCV traffic at distinct late steps of the viral life cycle. We reported that AP-1A and AP-2 mediate HCV trafficking during release and assembly, respectively. Here, we demonstrate that dileucine motifs in the NS2 protein mediate AP-1A, AP-1B, and AP-4 binding and cell-free virus release. Moreover, we reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. Lastly, we demonstrate cell-to-cell spread regulation by AAK1 and GAK, host kinases controlling APs, and susceptibility to their inhibitors. This study provides mechanistic insights into virus-host determinants that facilitate HCV trafficking, with potential implications for pathogenesis and antiviral agent design.