The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex.
The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex.
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DOI：
10.1371/journal.ppat.1000925
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发表时间：
2010-06-03
期刊：
影响因子：
6.7
通讯作者：
Sanderson MR
中科院分区：
文献类型：
--
作者：
Bergeron JR;Huthoff H;Veselkov DA;Beavil RL;Simpson PJ;Matthews SJ;Malim MH;Sanderson MR
HIV-1病毒感染因子（Vif）蛋白将E3泛素连接酶复合物（包括细胞蛋白延长蛋白B和C（EloBC）、cullin 5（Cul 5）和RING盒2（Rbx 2））募集至抗病毒蛋白APOBEC 3G（A3 G）和APOBEC 3F（A3 F），并诱导它们的多聚泛素化和蛋白酶体降解。在这项研究中，我们使用纯化的蛋白质和直接在体外结合试验，等温滴定量热法和NMR光谱来描述组装的Vif-EloBC三元复合物的分子机制。我们证明，Vif结合EloBC在两个位置，这两个相互作用诱导的SOCS框Vif以及EloBC的结构变化。特别是，除了以前建立的结合Vif的BC盒EloC，我们报告了一种新的相互作用之间的保守的Pro-Pro-Leu-Pro基序的Vif和C-末端结构域的EloB。使用基于细胞的测定，我们进一步表明，这种相互作用是必要的功能连接酶复合物的形成，从而建立了这个主题的作用。我们得出结论，HIV-1 Vif通过诱导折叠机制参与EloBC，不需要额外的辅助因子，并推测这些特征将Vif与其他EloBC特异性因子（如细胞SOCS蛋白）区分开来，并可能增强获得Vif功能治疗性抑制剂的前景。HIV-1是艾滋病的病原体。目前的疗法是基于抗病毒药物的鸡尾酒，抑制病毒复制所必需的病毒酶，但这种策略有几个缺点，包括耐药病毒株的发展。因此，干扰HIV-1复制的其他方面的药理学策略有可能增强HIV-1治疗。HIV-1 Vif蛋白是开发新的抗HIV-1疗法的有希望的靶标;它的功能是抵消细胞蛋白A3 G和A3 F，这是人类抗病毒防御机制的两种组分。Vif通过劫持细胞复合物（包括蛋白质EloB、EloC、Cul 5和Rbx 2）来实现这一点，然后通过降解从感染的细胞中消除A3 G和A3 F，从而逃避它们的抗病毒作用。在这里，我们使用纯化的蛋白质在体外重组的HIV-1 Vif的这种复合物的招聘。使用结构和生物化学方法，我们剖析了Vif与EloBC复合物相互作用的不同事件。我们的研究结果揭示了与已知招募这种复合物的细胞蛋白质的根本差异，表明Vif具有独特的功能，可以通过药理学干预进行靶向治疗，而不会干扰正常的细胞功能。这里报道的测定可用于发现这样的抑制剂。
The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function. HIV-1 is the etiologic agent of AIDS. Current therapies are based on cocktails of anti-viral drugs that inhibit viral enzymes essential for virus replication, but this strategy has several shortcomings, including the development of drug-resistant virus strains. Consequently, pharmacologic strategies that interfere with additional aspects of HIV-1 replication have the potential to enhance HIV-1 treatments. The HIV-1 Vif protein is a promising target for the development of new anti-HIV-1 therapeutics; it functions to counteract the cellular proteins A3G and A3F, two components of a human anti-viral defence mechanism. Vif accomplishes this by hijacking a cellular complex (comprising the proteins EloB, EloC, Cul5 and Rbx2), which then eliminates A3G and A3F from infected cells by degradation, therefore evading their anti-viral effect. Here, we used purified proteins to reconstitute in vitro the recruitment of this complex by HIV-1 Vif. Using structural and biochemical methods, we dissected the different events involved in Vif's interaction with the EloBC complex. Our results reveal fundamental differences with cellular proteins known to recruit this complex, suggesting that Vif possesses unique features that could be targeted by pharmacologic intervention, without disturbing normal cell functions. The assays reported here could be utilized for the discovery of such inhibitors.
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