Biogenesis of HIV 1 in living cells

描述(由申请人提供)：通过发展在单个病毒组装时跟踪它们的能力，有可能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粒子的标准，在这种情况下是HIV-1。随后建立了确定和表征质膜上各种成分的包装的标准，例如GAG的包装、基因组的招募。这项目前的项目将这项工作带入了识别病毒组装过程中更多部分反应的下一个水平：何时招募基因组？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--只是填充了GAG，还是有其他因素？什么时候蛋白酶变得活跃，是什么决定了切割步骤的明显专一性？病毒粒子是什么时候从细胞中分离出来的，这些步骤中的每一个是如何相互联系的？膜是否必须弯曲到一定程度才能获得足够的接近以使蛋白酶激活，或者是否足以使Gag堆积到临界间距？宿主ESCRT蛋白(S)的作用是什么？它们只是促进萌芽，还是加速了组装速度？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。 公共卫生相关性：病毒是对人类健康的主要威胁。这项工作开发了跟踪在活细胞中组装的单一病毒的能力。通过阐明组装程序，在这种情况下是针对HIV-1的，它打开了针对这些步骤进行干扰的可能性。

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.