Dynamics of human immunodeficiency virus (HIV-1) maturation

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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.