Mechanism of Vpu action: the rest of the story

描述(由申请人提供)：HIV辅助蛋白VPU已知调节两种宿主蛋白，CD4和BST-2，但它被认为通过两种完全不同的机制做到这一点。CD4被认为是内质网中蛋白酶体降解的靶点，而BST-2被认为是高尔基体和内体循环中溶酶体降解的靶点。这两个靶点都被认为与细胞内的Skp-Cullin1-F-box(SCF)E3连接酶相互作用，以促进它们的作用，但哪种SCF连接酶是辅助因子仍存在争议。含有F-box蛋白ssTRCP-1或-2的SCF连接酶被认为是两个潜在的VPU辅助因子，但一些人认为还有其他形式的连接酶尚未被鉴定。由于通常使用不同的分析方法来研究这两个VPU目标，因此很难在它们之间进行直接比较。特别是，用于研究CD4的检测是在VPU表达水平远远高于正常感染的条件下进行的。长臂猿白血病病毒的糖蛋白(Galv Env)最近被确定为另一个VPU靶点。根据识别参数，Galv Env似乎类似于CD4，但根据其作用机制，它似乎更类似于BST-2。VPU需要SCF连接酶来靶向Galv Env，但ssTRCP-1和-2是必不可少的。此外，还发现在特定条件下，CD4的表达以BST-2和HIV Env非依赖的方式限制HIV-1的感染性，但这种限制可以被VPU缓解。基于这一观察结果，已经开发了一种新的方法来研究VPU在生理浓度下对CD4的靶向性。假设CD4、BST-2和Galv Env都受到单一机制的调节，并且存在一个先前未知的F-box蛋白在这种调节中发挥关键作用。基于传染性的试验将被用来鉴定VPU用于靶向Galv Env的SCF连接酶，并表征该连接酶在调节所有三个VPU靶点中的重要性。这项工作将分为以下具体目标：目的1.确定在没有HIV-1Env的情况下，CD4如何限制病毒的传染性，以及VPU如何克服这一限制。目的2.鉴定VPU用于调节Galv Env的F-box蛋白，并确定该F-box蛋白是否也具有调节CD4和BST-2的功能。这些研究将回答有关HIV-1 VPU活性机制的基本问题，并可能为治疗抗病毒药物的开发开辟新的途径。

DESCRIPTION (provided by applicant): The HIV accessory protein Vpu is known to modulate two host proteins, CD4 and BST-2, but it is thought to do so by two completely different mechanisms. CD4 is believed to be targeted in the endoplasmic reticulum for proteasomal degradation, while BST-2 is believed to be targeted in the Golgi apparatus and the recycling endosomes for lysosomal degradation. Both targets are believed to interact with cellular Skp-Cullin1-F-box (SCF) E3 ligases to facilitate their action, but it is disputed which kinds of SCF ligase act as co-factors. SCF ligases that contain the F- box proteins ssTRCP-1 or -2 are believed to be two potential Vpu cofactors, but some suggest that there are additional forms of the ligase that have not yet been identified. Because different assays are typically used to study these two Vpu targets, it has been difficult to draw direct parallels between them. In particular, the assays used to study CD4 are performed under conditions where Vpu is expressed at levels much higher than would be found in a normal infection. The glycoprotein from Gibbon ape leukemia virus (GaLV Env) has recently been identified as an additional Vpu target. Based on recognition parameters, GaLV Env appears to be analogous to CD4, but based on its mechanism of action, it appears more analogous to BST-2. Vpu requires an SCF ligase to target GaLV Env, but ssTRCP-1 and -2 are dispensable. In addition, CD4 expression was found to restrict HIV-1 infectivity under particular conditions in a BST-2 and HIV Env independent fashion, but this restriction could be alleviated by Vpu. A novel assay has been developed based on this observation for studying Vpu targeting of CD4 at physiological concentrations. It is hypothesized that CD4, BST-2, and GaLV Env are all modulated by a single mechanism and that there is a previously unidentified F-box protein that plays a critical role in this modulation An infectivity-based assay will be used to identify the SCF ligase used by Vpu to target GaLV Env, and characterize the importance of this ligase in the modulation of all three Vpu targets. The work will be divided into the following specific aims: Aim 1. To determine how CD4 restricts viral infectivity in the absence of HIV-1 Env and how Vpu overcomes this restriction. Aim 2. To identify the F-box protein used by Vpu to modulate GaLV Env and to determine if this F-box protein also functions in the modulation of CD4 and BST-2. These studies will answer fundamental questions about the mechanism of HIV-1 Vpu activity and could open up new avenues for the development of therapeutic antivirals.