Dynamics of human immunodeficiency virus (HIV-1) maturation

人类免疫缺陷病毒（HIV-1）颗粒作为未成熟病毒体从宿主细胞释放，其必须经历蛋白水解成熟以变得具有感染性。成熟需要通过病毒体相关蛋白酶切割病毒结构多聚蛋白，伴随着内部颗粒结构的戏剧性重排。尽管对这一主题进行了深入的研究，但关于成熟的时间和动态的一些非常基本的问题仍然没有答案。由于成熟必须受到严格的调控，以确保形成传染性HIV-1，这一信息对于我们理解HIV-1形态发生至关重要。该项目的目的是研究HIV-1成熟过程的动力学，并描述干扰这些动力学的病毒学后果，以便深入了解基本的开放问题。我们计划测试的假设加工动力学的作用来自我们早期的工作与阻断切割位点的HIV-1变异体。此外，我们希望通过开发和应用新的检测系统，为成熟过程的动态方面的调查开辟新的可能性。目前，这样的调查是阻碍了事实，即颗粒的形成和释放是异步的，防止通过批量分析的成熟动力学的研究，并缺乏一个活的读出HIV-1蛋白水解。我们将探索两种旨在在规定条件下同步HIV-1成熟的互补策略。一旦一个合适的系统已经建立，它应该允许我们描绘的时间过程中的病毒内HIV-1蛋白水解成熟和定义形态成熟的中间体。此外，我们计划建立一个实时读数，以便在真实的时间内跟踪HIV-1的成熟。为此，我们将开发和评估两种互补的方法（分别基于双分子荧光互补和荧光共振能量转移），建立在我们关于标记的HIV-1衍生物的产生和使用的长期经验。使用这个系统，结合活细胞显微镜观察个人HIV-1组装网站以前建立了我们的实验室，应该让我们来定义的时间过程中的蛋白水解相对于HIV-1颗粒的形成。

Human immunodeficiency virus (HIV-1) particles are released from the host cell as immature virions, which have to undergo proteolytic maturation to become infectious. Maturation entails cleavage of viral structural polyproteins by the virion associated protease, accompanied by dramatic rearrangements of inner particle architecture. Despite intense research on this topic, a number of very basic questions regarding the timing and dynamics of maturation remain unanswered. Since maturation has to be tightly regulated to ensure formation of infectious HIV-1, this information is crucial for our understanding of HIV-1 morphogenesis. The objective of this project is to study the dynamics of the HIV-1 maturation process and to characterize virological consequences of disturbing these dynamics, in order to provide insight into the fundamental open questions. We plan to test hypotheses about the role of processing kinetics derived from our earlier work on HIV-1 variants with blocked cleavage sites. Furthermore, we want to open up new possibilities for the investigation of dynamic aspects of the maturation process through development and application of novel assay systems. Currently, such investigations are hindered by the fact that particle formation and release is asynchronous, preventing the study of maturation dynamics by bulk analyses, and that a live readout for HIV-1 proteolysis is lacking. We will explore two complementary strategies designed to synchronize HIV-1 maturation under defined conditions. Once a suitable system has been established, it should allow us to delineate the time-course of intravirion HIV-1 proteolytic maturation and define morphological maturation intermediates. In addition, we plan to establish a live readout in order to follow HIV-1 maturation in real time. For this, we will develop and evaluate two complementary approaches (based on bimolecular fluorescence complementation and fluorescence resonance energy transfer, respectively) building on our long-standing experience regarding the generation and use of labelled HIV-1 derivatives. Use of this system, in conjunction with live-cell microscopic observation of individual HIV-1 assembly sites previously established by our lab, should allow us to define the time course of proteolysis with respect to HIV-1 particle formation.