Mechanism of Vpu action: the rest of the story

描述（由申请人提供）：已知HIV辅助蛋白Vpu调节两种宿主蛋白，CD4和BST-2，但它被认为是通过两种完全不同的机制来调节的。CD4被认为是内质网蛋白酶体降解的靶标，而BST-2被认为是高尔基体和循环内体溶酶体降解的靶标。这两种靶标都被认为与细胞Skp-Cullin1-F-box (SCF) E3连接酶相互作用以促进其作用，但哪种SCF连接酶作为辅助因子存在争议。含有F- box蛋白ssTRCP-1或-2的SCF连接酶被认为是两种潜在的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测定方法来研究这两种Vpu靶点，因此很难在它们之间得出直接的相似之处。特别是，用于研究CD4的检测是在Vpu表达水平远高于正常感染水平的条件下进行的。长臂猿白血病病毒（GaLV Env）的糖蛋白最近被确定为一个额外的Vpu靶标。从识别参数上看，GaLV Env与CD4类似，但从作用机制上看，它更类似于BST-2。Vpu需要SCF连接酶靶向GaLV Env，但ssTRCP-1和-2是可有可无的。此外，CD4表达在特定条件下以BST-2和HIV Env独立的方式限制HIV-1的感染，但Vpu可以缓解这种限制。基于这一观察结果，一种新的检测方法被开发出来，用于研究Vpu在生理浓度下靶向CD4。假设CD4， BST-2和GaLV Env都是由单一机制调节的，并且存在一种先前未被识别的F-box蛋白在这种调节中起关键作用。基于感染的检测将用于鉴定Vpu靶向GaLV Env的SCF连接酶，并表征该连接酶在所有三个Vpu靶点调节中的重要性。这项工作将分为以下具体目标：目标1。确定在HIV-1 Env缺失的情况下，CD4如何限制病毒的传染性，以及Vpu如何克服这种限制。目标2。目的鉴定Vpu调节GaLV Env的F-box蛋白，并确定该F-box蛋白是否也参与CD4和BST-2的调节。这些研究将回答有关HIV-1 Vpu活性机制的基本问题，并可能为开发治疗性抗病毒药物开辟新的途径。

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.