Biogenesis of HIV 1 in living cells

描述（由申请方提供）：通过开发跟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。这项工作的最早阶段建立了用于确定是否观察到单个病毒体（在本例中为HIV-1）的标准。随后建立了用于确定和表征质膜上各种组分的包装的标准，例如Gag的包装、基因组的募集。目前的这个项目将工作推进到下一个层次，即识别病毒组装过程中更多的部分反应：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--仅仅是填充了Gag还是有其他因素？蛋白酶什么时候变得活跃，什么决定了切割步骤的表观特异性？病毒粒子何时从细胞中分离出来，这些步骤是如何相互关联的？膜是否必须弯曲到一定程度以获得足够的接近度以使蛋白酶活化，或者是否足以使Gag填充到临界间距？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？通过这些在单个病毒体水平上的组装测定，有可能最终描述和定义组装过程。 公共卫生相关性：病毒是人类健康的主要威胁。这项工作发展了跟踪单个病毒在活细胞中组装的能力。通过阐明组装过程，在这种情况下，对于HIV-1，它打开了针对这些步骤进行破坏的可能性。

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.