The role of cellular factors in the HIV-1 capsid assembly pathway

描述(由申请人提供)：本提案的中心假设是，HIV-1 Gag通过一种循序渐进的、能量依赖的中间体途径组装成未成熟的衣壳，其中包含对Gag靶向和组装以及HIV-1基因组RNA封装至关重要的细胞因子。使用ATP结合蛋白ABCE1的抗体，我们可以跟踪和分离这些中间体。ABCE1在组装过程中发挥作用，并存在于这些中间体中。在这里，我们建议使用组装途径作为一个时间和空间框架，以了解人类细胞中的各种细胞因子在GAG靶向和组装以及基因组RNA包装中的作用。在目标1中，我们建议进一步检验ABCE1途径在细胞中GAG组装过程中被普遍使用的假设，方法是检查只包含组装所需的最低结构域的GAG构建物是否利用这一途径。在目标2中，我们将检查在组装途径中HIV-1基因组RNA被特异性包裹的时间点。在目标3和4中，我们将继续我们最近发现的负责将GAG从胞浆重新定位到膜上的组装中间体。我们将研究AP-3Delta，PIP2在这种含有ABCE1的中间体的膜靶向中的作用。提出了一种蛋白质组学方法，用于鉴定该中间体中可能对指导膜靶向很重要的其他因子。免疫金EM将被用来证明细胞因子在含有ABCE1的组装中间体中的共存。最后，在目标4中，我们将进一步测试这一假设，即该中间体的阻断是导致GAG未能在小鼠细胞中组装的原因。初步数据表明，小鼠细胞中的GAG以这种膜靶向中间体的形式被阻止，从而提出了这一假说。质谱学将用于鉴定这种中间体中的细胞因子，这些因子克服了小鼠细胞中的靶向和组装障碍。这些生化和超微结构的方法将为我们提供一个综合的观点，即各种细胞因子如何依次作用于促进HIV-1 Gag在人类细胞中的靶向和组装。

DESCRIPTION (provided by applicant): The central hypothesis of this proposal is that HIV-1 Gag assembles into immature capsids through a stepwise, energy dependent pathway of intermediates that contain cellular factors important for Gag targeting and assembly, as well as HIV-1 genomic RNA encapsidation. Using an antibody to the ATP-binding protein ABCE1, which functions during assembly and is present in these intermediates, we can track and isolate these intermediates. Here we propose to use the assembly pathway as a temporal and spatial framework for understanding the role of a variety of cellular factors in human cells in Gag targeting and assembly as well as in genomic RNA packaging. In aim 1 we propose to further test the hypothesis that the ABCE1 pathway is universally used during Gag assembly in cells by examining whether Gag constructs that contain only the minimum necessary domains for assembly utilize this pathway. In aim 2, we will examine at what point during the assembly pathway HIV-1 genomic RNA becomes specifically encapsidated. In aims 3 and 4, we will follow up our recent identification of the assembly intermediate responsible for relocating Gag from the cytosol to membranes. We will examine the role of AP-3delta, PIP2 in membrane targeting of this ABCE1-containing intermediate. A proteomics approach is proposed for identification of other factors in this intermediate that may be important for directing membrane targeting. Immunogold EM will be utilized to demonstrate colocalization of cellular factors in ABCE1-containing assembly intermediates. Finally, in aim 4, we will further test the hypothesis that a block at this intermediate is responsible for the failure of Gag to assemble in murine cells. This hypothesis is suggested by preliminary data indicating that Gag in murine cells is arrested in the form of this membrane targeting intermediate. Mass spectrometry will be used to identify cellular factors in this intermediate that overcome the targeting and assembly block in murine cells. Together these biochemical and ultrastructural approaches will give us an integrated view of how a variety of cellular factors act sequentially to promote targeting and assembly of HIV-1 Gag in human cells.