Understanding how HIV-1 Vpu and HIV-2 Env stimulate virus release

灵长类慢病毒编码一种保守的功能，该功能是有效生产病毒性慢病毒所必需的。 颗粒和体内致病性。对于HIV-1，这是由辅助蛋白Vpu进行的，而对于HIV-2， 该功能已定位于Env糖蛋白。此外，我们对简单逆转录病毒的研究， 鼠白血病病毒（MLV）的Env蛋白也具有类似的活性。我们发现，这三个 这些病毒释放增强剂（EVR）可以促进异源逆转录病毒的产生，这表明 它们作用于逆转录病毒出芽的一般细胞途径。EVR响应分析 无应答细胞系的发现提示人类细胞含有一种天然限制因子， 抑制逆转录病毒的萌芽，EVR已经进化到抵消。因此，这是我们的中心 假设EVR功能通常是逆转录病毒保守和重要的活性。 虽然Vpu的EVR活性在1989年首次被描述，但其增强病毒的机制尚不清楚。 释放仍然是未知的，代表了我们对艾滋病毒生物学知识的重大差距。更不是 了解HIV-2 Env的活性，它与Vpu既有相似之处，也有显著的差异。的 该提案中概述的研究计划将采取综合方法研究逆转录病毒EVR， 为了确定病毒蛋白的功能域，并了解宿主细胞的过程， 他们活动的目标。使用我们已经确定的三种不同的EVR，我们将精确地 确定这些蛋白质内的功能结构域，并评估它们是否具有相同的细胞靶点， 活性，或者是否通过不同途径获得共同的EVR表型。我们还将采取 我们已经确定的非Vpu反应性HeLa细胞系的优势，它保留了反应能力， 到HIV-2 Env EVR。对这种细胞系的初步研究已经表明了Vpu的机制 活动我们将直接研究EVR如何影响病毒组装和出芽以及它们对细胞增殖的影响。 运输途径，使用微阵列，生物化学和免疫荧光方法的组合。 最后，我们将尝试使用基于微阵列的方法克隆人细胞限制因子 分析复杂的cDNA和siRNA文库。 研究与公共卫生的相关性：艾滋病毒感染和艾滋病仍然是一个主要的公共卫生问题， 而抗艾滋病病毒药物只能针对有限的病毒靶点。HIV-1 Vpu蛋白是HIV病毒的必需蛋白。 致病性，但其作用机制尚不清楚。我们的目标是研究这背后的机制 重要HIV功能，从而确定药物开发的新靶点。

Primate lentiviruses code for a conserved function that is necessary for the efficient production of viral particles and in vivo pathogenicity. For HIV-1 this is carried out by the accessory protein Vpu, while for HIV-2, the function has been mapped to the Env glycoprotein. In addition, our studies with the simple retrovirus, murine leukemia virus (MLV), have identified a similar activity in its Env protein. We have found that all three of these enhancers of virus release (EVRs) can boost the production of heterologous retroviruses, suggesting that they act on the general cellular pathways involved in retrovirus budding. Analysis of EVR-responsive and non-responsive cell lines has led to the suggestion that human cells contain a natural restriction factor that suppresses retrovirus budding and which the EVRs have evolved to counteract. Therefore, it is our central hypothesis that EVR function is a conserved and important activity for retroviruses in general. Although the EVR activity of Vpu was first described in 1989, the mechanism whereby it enhances virus release is still unknown and represents a significant gap in our knowledge of HIV biology. Even less is understood about the HIV-2 Env activity, which shows both similarities and striking differences with Vpu. The research plan outlined in this proposal will take a comprehensive approach to studying retroviral EVRs, both in order to identify the functional domains of the viral proteins involved, and to understand the host cell processes that are the targets of their activity. Using the three different EVRs that we have identified, we will precisely define the functional domains within these proteins and assess whether they have the same cellular target and activity, or whether a common EVR phenotype is achieved through different pathways. We will also take advantage of a non-Vpu-responsive HeLa cell line that we have identified, which retains the ability to respond to the HIV-2 Env EVR. Preliminary studies of this cell lines are already suggesting a mechanism for Vpu `s activity. We will directly examine how EVRs affect virus assembly and budding and their influence on cellular trafficking pathways, using a combination of microarray, biochemical and immunofluorescence approaches. Finally, we will attempt to clone the human cell restriction factor using approaches based on the microarray analysis of complex cDNA and siRNA libraries. Relevance of research to public health: HIV infection and AIDS continue to be a major public health problem, with anti-HIV drugs only available against limited viral targets. The HIV-1 Vpu protein is essential for HIV pathogenicity but its mechanism of action is unclear. It is our goal to investigate the mechanism behind this important HIV function and thereby identify new targets for drug development.