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

描述（由申请人提供）：该提案的中心假设是HIV-1 Gag通过中间体的逐步、能量依赖性途径组装成不成熟衣壳，该中间体含有对Gag靶向和组装以及HIV-1基因组RNA折叠重要的细胞因子。使用ATP结合蛋白ABCE 1的抗体，它在组装过程中发挥作用，并存在于这些中间体中，我们可以跟踪和分离这些中间体。在这里，我们建议使用的装配途径作为一个时间和空间的框架，了解各种细胞因子在人类细胞中的Gag靶向和组装，以及在基因组RNA包装的作用。在目标1中，我们建议通过检查仅含有组装所需的最小结构域的Gag构建体是否利用ABCE 1途径来进一步测试ABCE 1途径在细胞中Gag组装期间普遍使用的假设。在目标2中，我们将研究在组装途径中HIV-1基因组RNA在什么时候变得特异性地被取代。在目标3和4中，我们将跟进我们最近鉴定的负责将Gag从胞质溶胶重新定位到膜的组装中间体。我们将研究AP-3delta，PIP 2在这种含ABCE 1的中间体的膜靶向中的作用。提出了一种蛋白质组学的方法来确定其他因素在这个中间，可能是重要的指导膜靶向。免疫金EM将用于证明细胞因子在含ABCE 1的组装中间体中的共定位。最后，在目标4中，我们将进一步检验该中间体处的阻断是导致Gag在鼠细胞中组装失败的原因的假设。初步数据表明，鼠细胞中的Gag以这种膜靶向中间体的形式被抑制，从而提出了这一假设。质谱法将用于鉴定该中间体中克服鼠细胞中的靶向和组装阻断的细胞因子。这些生物化学和超微结构的方法将为我们提供一个综合的观点，各种细胞因子如何顺序地作用，以促进人类细胞中HIV-1 Gag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.