Nef Function in HIV Infectivity

描述（由申请人提供）：Nef是HIV-1和其他灵长类慢病毒的毒力因子，对快速发展为艾滋病至关重要。在细胞培养中，Nef以一种未知的机制增加HIV-1子代病毒粒子的感染性。我们最近发现动力蛋白2 （Dyn2）是网格蛋白介导的运输的关键调节因子，作为一种新的Nef结合伙伴，它具有增强病毒感染性的能力。显性阴性的Dyn2或通过RNAi去除Dyn2可有效抑制Nef对HIV-1感染的作用。此外，在Dyn2缺失的细胞中，这种功能可以通过异位表达的Dyn2而不是Dyn1来恢复，Dyn1是一种不结合Nef的密切相关的异构体。Nef的传染性增强也依赖于网格蛋白，因为它在网格蛋白缺失的细胞中减少，在表达AP180的网格蛋白结合结构域的细胞中被严重抑制，该结构域阻断了网格蛋白包被的窝的形成。本应用主要关注Nef- dyn2相互作用在增强HIV-1传染性和复制中的作用，因为Nef的这一功能仍然知之甚少。一个目标是阐明是什么使Nef能够与Dyn2相互作用，而不是与密切相关的同型异构体相互作用。特别是，由于赋予Nef结合的两个Dyn2结构域介导了Dyn2组装，我们建议确定Nef结合和Dyn2组装是否相连。另一个目标是了解Dyn2及其与Nef的相互作用与HIV-1感染和复制的相关性。我们的初步结果还表明，Nef影响HIV-1病毒体的中和敏感性，这可能对HIV-1在体内的复制具有重要意义。因此，我们建议研究Nef对HIV-1感染和中和的影响之间的关系，Nef对中和影响的广度，以及Nef是否影响抗体与Env三聚体的结合。HIV-1的Nef蛋白对艾滋病感染者的发展至关重要。Nef通过一种未知的机制直接增强病毒颗粒的传染性，我们最近发现一种名为dynamin 2的人类蛋白质与Nef结合，并且是其增强病毒传染性的能力所必需的。由于Nef的这种活性可能影响HIV-1在人体内传播的能力，我们建议研究是什么使Nef结合动力蛋白2而不是密切相关的蛋白，并研究这种相互作用与HIV-1感染人类细胞和引起疾病的能力的相关性。

DESCRIPTION (provided by applicant): Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We have recently identified dynamin 2 (Dyn2), a key regulator of clathrin-mediated trafficking, as a novel Nef binding partner that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by RNAi potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, since it was diminished in clathrin- depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation. This application focuses on the role of the Nef-Dyn2 interaction in the enhancement of HIV-1 infectivity and replication, because this function of Nef remains poorly understood. One goal is to elucidate what enables Nef to interact with Dyn2 but not with closely related isoforms. In particular, since the two Dyn2 domains which confer Nef binding mediate Dyn2 assembly, we propose to determine whether Nef binding and Dyn2 assembly are linked. Another goal is to understand the relevance of Dyn2 and its interaction with Nef for HIV-1 infectivity and replication. Our preliminary results also indicate that Nef affects the neutralization sensitivity of HIV-1 virions, which could have important implications for HIV-1 replication in vivo. We therefore propose to examine the relationship between the effects of Nef on HIV-1 infectivity and neutralization, the breadth of the effect of Nef on neutralization, and whether Nef affects antibody binding to the Env trimer. PUBLIC HEALTH RELEVANCE The Nef protein of HIV-1 is crucial for the development of AIDS in infected individuals. Nef directly enhances the infectivity of viral particles by an unknown mechanism, and we have recently shown that a human protein called dynamin 2 binds to Nef and is required for its ability to enhance virus infectivity. Since this activity of Nef is likely to affect the ability of HIV-1 to spread in people, we propose to examine what enables Nef to bind dynamin 2 but not closely related proteins, and to investigate the relevance of this interaction for the ability of HIV-1 to infect human cells and to cause disease.