Biogenesis of HIV 1 in living cells

描述（由申请人提供）：通过发展追踪单个病毒组装的能力，可以识别和表征病毒组装中的离散步骤。这项工作的最初阶段建立了用于确定是否观察到单个病毒粒子（在本例中是HIV-1）的标准。随后建立了确定和表征质膜上各种成分包装的标准，例如Gag的包装，基因组的招募。目前的项目将工作带到了下一个层次，即在病毒组装过程中识别更多的部分反应：基因组何时被招募？基因组和Rev之间的相互作用是什么？基因组和Gag之间的相互作用是什么？是什么决定了膜向外弯曲的能力——仅仅是Gag的包装还是有其他因素？什么时候蛋白酶变得活跃，什么决定了切割步骤的明显特异性？病毒粒子什么时候从细胞中分离出来，这些步骤是如何相互联系的？膜必须弯曲到一定程度以获得足够的接近蛋白酶激活，或者它是否足以使Gag包装到临界间距？宿主ESCRT蛋白的作用是什么？它们只是促进发芽还是加速组装速度？有了这些在单个病毒粒子水平上的组装分析，就有可能最终描述和定义组装过程。

DESCRIPTION (provided by applicant): By developing the ability to follow single viruses as they assemble, it is possible to identify and characterize discrete steps in viral assembly. The earliest stages of this work established the criteria to be used in determining if a single virion, in this case of HIV-1, is being observed. This was followed by establishing criteria for determining and characterizing the packing of various components at the plasma membrane, for example the packing of Gag, the recruitment of the genome. This current project carries the work to the next level of identifying many more partial reactions in the process of viral assembly: When is the genome recruited? What is the interaction between the genome and Rev for packing, between the genome and Gag for packing? What determines the ability of the membrane to bend outward - is it just packing of Gag or are there additional factors? When are does the protease become active, what determines the apparent specificity of cleavage steps? When does the virion septate from the cell and, how do each of these steps inter-relate? Must the membrane bend to a certain degree to attain a sufficient proximity for the protease to activate or is it sufficient for Gag to pack to a critical spacing? What are the role(s) of the host ESCRT proteins? Do they simply facilitate budding or do they accelerate the assembly rate? With these assays of assembly at the level of the single virion, it becomes possible to final describe and define the process of assembly. PUBLIC HEALTH RELEVANCE: Viruses are a major threat to human health. This work has developed the ability to follow single viruses assembling in living cells. By elucidating the procedure of assembly, in this case for HIV-1, it opens the possibility of targeting these steps for disruption.