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，对快速进展为AIDS至关重要。在细胞培养中，Nef通过未知的机制增加HIV-1子代病毒粒子的感染性。我们最近确定了发动蛋白2（Dyn 2），网格蛋白介导的贩运的关键调节，作为一种新的Nef结合伴侣，这是其增加病毒感染性的能力所必需的。显性阴性Dyn 2或通过RNAi耗尽Dyn 2有效地抑制Nef对HIV-1感染性的作用。此外，在Dyn 2耗尽的细胞中，Nef的这种功能可以通过异位表达Dyn 2而不是Dyn 1来拯救，Dyn 1是一种不结合Nef的密切相关的同种型。Nef的感染性增强也依赖于网格蛋白，因为它在网格蛋白耗尽的细胞中减少，并且在表达AP 180的网格蛋白结合结构域的细胞中被深度抑制，所述AP 180的网格蛋白结合结构域阻断网格蛋白包被的小窝形成。本申请的重点是Nef-Dyn 2相互作用在增强HIV-1感染性和复制中的作用，因为Nef的这种功能仍然知之甚少。一个目标是阐明是什么使Nef与Dyn 2相互作用，但不与密切相关的亚型。特别是，由于两个Dyn 2域赋予Nef结合介导Dyn 2组装，我们建议确定Nef结合和Dyn 2组装是否连接。另一个目标是了解Dyn 2的相关性及其与Nef的相互作用对HIV-1感染和复制的影响。我们的初步结果还表明，Nef影响HIV-1病毒粒子的中和敏感性，这可能对HIV-1在体内的复制有重要意义。因此，我们建议研究Nef对HIV-1感染性和中和作用的影响之间的关系，Nef对中和作用的影响范围，以及Nef是否影响抗体与Env三聚体的结合。HIV-1的Nef蛋白对感染者发生艾滋病至关重要。Nef通过一种未知的机制直接增强病毒颗粒的感染性，我们最近发现一种名为发动蛋白2的人类蛋白质与Nef结合，并且是其增强病毒感染性的能力所必需的。由于Nef的这种活性可能会影响HIV-1在人群中传播的能力，因此我们建议检查是什么使Nef能够结合发动蛋白2而不是密切相关的蛋白质，并调查这种相互作用与HIV-1感染人类细胞并导致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.