Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly.
Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly.
复制标题
DOI：
10.1371/journal.ppat.1002845
复制
发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Einav S
中科院分区：
文献类型：
--
作者：
Neveu G;Barouch-Bentov R;Ziv-Av A;Gerber D;Jacob Y;Einav S
目前迫切需要新的治疗方法来对抗丙型肝炎病毒感染（HCV），这是一个重大的全球卫生问题。目前的传染性病毒生成模型表明，HCV病毒粒子在脂滴表面或其附近组装，在内质网获得包膜，并通过分泌途径排出。然而，HCV组装的机制，特别是病毒-宿主蛋白-蛋白相互作用在介导这一过程中的作用，人们知之甚少。我们在核心蛋白中发现了一个保守的迄今未被识别的YXXΦ基序（Φ是一个庞大的疏水残基）。该基序与宿主货物蛋白内的分类信号同源，已知其介导AP2M1、网格蛋白接头蛋白复合体2 （AP-2）的μ亚基结合和细胞内运输。通过微流体亲和分析、蛋白片段互补分析和感染细胞的共免疫沉淀，我们发现该基序介导了与AP2M1的核心结合。YXXΦ突变、沉默AP2M1表达或过表达AP2M1显性阴性突变对HCV RNA复制没有影响，然而，它们显著抑制细胞内和细胞外感染性，与病毒组装缺陷一致。定量共聚焦免疫荧光分析显示，core的YXXΦ基序介导AP2M1向脂滴募集，并且在core-AP2M1结合中断后观察到的HCV组装缺陷与core在脂滴上的积累、core与E2共定位减少以及core定位到反式高尔基网络（TGN）的减少有关，TGN是病毒颗粒成熟的假定位点。此外，已知的丝氨酸/苏氨酸激酶AAK1和GAK可刺激AP2M1与宿主货物蛋白的结合，调节核心-AP2M1的结合，对HCV组装至关重要。最后，已批准的抑制AAK1或GAK的抗癌药物不仅破坏核心- ap2m1结合，而且显著抑制HCV组装和感染性病毒的产生。这些结果验证了病毒-宿主相互作用对HCV组装和药物开发产生化合物至关重要。需要新的抗病毒策略来对抗HCV大流行。了解参与介导组装的病毒-宿主决定因素对于开发针对HCV生命周期这一阶段的药物至关重要。我们在核心中发现了一个保守的迄今未被识别的酪氨酸基序，它与宿主货物蛋白中被AP2M1（一种膜运输蛋白）识别的分选信号同源。通过将新的蛋白质组学技术与分子病毒学、RNAi、显性干扰和药理学方法相结合，我们发现该基序介导AP2M1和HCV组装的核心结合，并且AP2M1对HCV组装至关重要。Core的tryosine基序将AP2M1招募到病毒组装的脂滴上，而Core -AP2M1结合的破坏改变了Core的亚细胞定位和与包膜蛋白E2的共定位。AAK1和GAK是刺激AP2M1与货物蛋白相互作用的宿主激酶，调节核心-AP2M1结合，对HCV组装至关重要。最后，我们发现了结合AAK1或GAK并破坏核心- ap2m1结合和HCV组装的激酶抑制剂。我们的研究提供了对HCV组装机制的深入了解，并为药物开发提供了候选靶点和化合物。由于一些发现的化合物已被批准为抗癌药物，因此可能有机会将它们重新用作抗病毒药物。
Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development. Novel antiviral strategies are needed to combat the HCV pandemic. Understanding viral-host determinants involved in mediating assembly is critical for the development of drugs targeting this stage of the HCV life cycle. We identified a conserved heretofore unrecognized tyrosine motif within core, which is homologous to sorting signals within host cargo proteins that are recognized by AP2M1, a membrane trafficking protein. By combining novel proteomic techniques with molecular virology, RNAi, dominant-interfering, and pharmacological approaches, we show that this motif mediates core binding to AP2M1 and HCV assembly and that AP2M1 is essential for HCV assembly. Core's tryosine motif recruits AP2M1 to lipid droplets, the site of viral assembly, and disruption of core-AP2M1 binding alters core's sub-cellular localization and colocalization with the envelope protein E2. AAK1 and GAK, host kinases that stimulate AP2M1's interactions with cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, we discovered kinase inhibitors, which bind AAK1 or GAK and disrupt core-AP2M1 binding and HCV assembly. Our study provides insight into mechanisms of HCV assembly and yields candidate targets and compounds for pharmaceutical development. Since some of the discovered compounds are approved anti-cancer drugs, there may be an opportunity for repurposing them as antivirals.
登录
查看更多内容
影响因子：
3.7
作者：
Ariumi Y;Kuroki M;Maki M;Ikeda M;Dansako H;Wakita T;Kato N
通讯作者：
Kato N
影响因子：
6.7
作者：
Counihan NA;Rawlinson SM;Lindenbach BD
通讯作者：
Lindenbach BD
影响因子：
5.4
作者：
Cocquerel, L;Duvet, S;Dubuisson, J
通讯作者：
Dubuisson, J
影响因子：
5.4
作者：
Boulant, S;Vanbelle, C;Lavergne, JP
通讯作者：
Lavergne, JP
影响因子：
4.8
作者：
Boulant, Steeve;Montserret, Roland;McLauchlan, John
通讯作者：
McLauchlan, John