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通过一个逐步的、能量依赖的中间体途径组装成未成熟的衣壳，这些中间体含有对Gag靶向和组装以及HIV-1基因组RNA封装很重要的细胞因子。利用一种针对atp结合蛋白ABCE1的抗体，我们可以追踪和分离这些中间体。ABCE1在组装过程中起作用，存在于这些中间体中。在这里，我们建议使用组装途径作为时间和空间框架，以了解人类细胞中各种细胞因子在Gag靶向和组装以及基因组RNA包装中的作用。在目标1中，我们建议进一步验证ABCE1途径在细胞中Gag组装过程中普遍使用的假设，通过检查仅包含组装所需的最小结构域的Gag构建体是否利用该途径。在目标2中，我们将研究在组装途径中HIV-1基因组RNA被特异性封装的时间点。在目标3和4中，我们将继续我们最近鉴定的负责将Gag从细胞质溶胶重新定位到膜的组装中间体。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来鉴定这种中间体中的其他因素，这些因素可能对指导膜靶向很重要。免疫金电镜将用于证明细胞因子在含有abce1的组装中间体中的共定位。最后，在目标4中，我们将进一步验证这一假设，即该中间体的阻断是Gag在小鼠细胞中组装失败的原因。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，这一假设得到了证实。质谱法将用于鉴定该中间体中克服小鼠细胞中的靶向和组装块的细胞因子。这些生化和超微结构方法将使我们对各种细胞因子如何依次作用以促进人类细胞中HIV-1 Gag的靶向和组装有一个综合的看法。

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.