The Role of Rab6A in HIV-1 Env-mediated Entry into Monocyte-Derived Macrophages

描述（由申请人提供）：HIV-1基因组仅由9个基因组成，因此高度依赖宿主细胞因子进行复制。最近的研究，通过全基因组siRNA筛选，已经确定了许多可能的“HIV-1依赖性因子”HDFs。然而，这些分析之间存在很大的不一致性，并且大多数潜在的撞击尚未在人类原代靶细胞中得到验证。因此，本研究的目的是验证最近发现的一种可能的HIV-1 env介导宿主因子Rab6A GTPase在原代人单核细胞源性巨噬细胞（MDMs）中的作用，并确定该蛋白促进HIV-1感染的机制。此外，由于Rab6A在两种密切相关的异构体Rab6A和Rab6A'中普遍表达，这两种异构体在细胞中具有相似但不同的作用，因此将分别研究这两种异构体。与所有gtpase类似，Rab6A（A/A’）功能依赖于特定的辅助蛋白，这些辅助蛋白允许在活性gtp结合和非活性gdp结合形式之间循环。我们假设激活的Rab6A（A/A’）是HIV-1 env介导的MDMs有效感染所必需的，并且Rab6A（A/A’）的激活依赖于Rab6A（A/A’）特异性鸟嘌呤核苷酸交换因子Ric1/Rgp1的活性。我们将利用伪病毒单轮感染实验来确定Rab6A和Rab6A'促进HIV-1在原代人巨噬细胞中复制的机制。我们的研究可能会揭示一种新的治疗途径来对抗HIV-1/AIDS，并有助于剖析Rab6A（a / a’）在原代人巨噬细胞中的基本生物学作用。

DESCRIPTION (provided by applicant): The HIV-1 genome consists of only 9 genes and thus, depends highly on host cellular factors to replicate. Recent studies, through genome-wide siRNA screens, have identified many possible "HIV-1 dependency factors" HDFs. However, there lies a large inconsistency between these analyses and the majority of potential hits have not been verified in primary human target cells. Therefore, the goal of this proposal is to validat a recently identified possible HIV-1 Env-mediated host factor, Rab6A GTPase, in primary human monocyte-derived macrophages (MDMs) and define the mechanism by which this protein facilitates HIV-1 infection. Additionally, because Rab6A is ubiquitously expressed in two closely related isoforms, Rab6A and Rab6A', that have similar but distinct roles in the cell, both isoforms will be individually investigated. Similar to all GTPases, Rab6A(A/A') function is dependent on specific accessory proteins that allow cycling between active-GTP bound and inactive-GDP bound forms. We hypothesize that activated Rab6A(A/A') is required for efficient HIV-1 Env-mediated infection of MDMs and activation of Rab6A(A/A') is dependent on Ric1/Rgp1, a Rab6A(A/A')-specific guanine nucleotide-exchange factor, activity. We will utilize a pseudoviral single-round infection assay to define the mechanism by which Rab6A and Rab6A' facilitate HIV-1 replication in primary human macrophages. Our studies may reveal a novel therapeutic pathway to combat HIV-1/AIDS, as well as help dissect the fundamental biological role of Rab6A(A/A') in primary human macrophages.