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Abstract

Human immunodeficiency virus (HIV-1) particles assemble and bud at the host cell plasma membrane and are released as immature, noninfectious particles. Concomitant with release, the viral protease (PR) cleaves Gag and GagProPol polyproteins into single proteins, leading to a major structural rearrangement of the virion. Temporal control of proteolytic cleavage with respect to particle assembly appears crucial for the maturation of infectious particles. The order of Gag processing is tightly regulated by utilization of different proteolytic cleavage sites with specific binding affinities to HIV-1 PR. However, the precise mechanism and kinetics of PR activation, as well as the time course of proteolytic maturation are currently unclear. Since many virus particles are formed in an infected cell and the time course of formation is asynchronous – not only between individual cells but also on the surface of a single cell - bulk biochemical analyses are not suitable for analysis of PR activation. Therefore, the aim of my thesis was to monitor and quantitate the HIV-1 PR activity during virus particle assembly by live-cell microscopy. Live-cell analysis of HIV-1 assembly site formation using fluorescence microscopy was already established in the lab. Previous studies from our and other groups, imaging fluorescently labeled virus assembly by total internal fluorescence microscopy (TIRF), showed that formation of viral assembly sites proceeds over 1-2 h, and Gag assembly for an individual bud is completed within 10-20 min. Here, I designed and explored various approaches for single virus tracking of PR activity in parallel to Gag assembly. For this purpose, I developed and tested different sensors to detect (i) PR enzymatic activity at the nascent assembly site, (ii) Gag polyprotein processing mediated by PR, or (iii) the generation of mature HIV-1 PR by autoprocessing from the GagProPol precursor. To measure PR activity at the assembly site, I designed Förster resonance energy transfer (FRET) reporter molecules which were targeted to nascent assemblies. For this, a FRET pair of autofluorescent proteins was linked via a Gag-derived PR cleavage site and fused to the viral protein r (Vpr) to mediate incorporation into particles. We could show the incorporation, and proteolytic processing of the FRET-based sensor was observed. While sensor co-expression with wild-type HIV-1 PR caused a decrease of the FRET values by two-fold upon proteolytic processing, during time-lapse measurements of the assembly process we could not detect a change in FRET signal over time. In order to detect Gag cleavage as readout for HIV-1 PR activity, I attempted different approaches. Initially, I applied a previously established system for distinguishing immature and mature particles by STED nanoscopy to the analysis of particles formed at the cell surface in live-cell experiments. I then tested two approaches for live-cell measurements of Gag processing. A first attempt to utilize a split fluorescent protein-based method was not successful, since fluorescence was found to be independent of the Gag processing state. In an alternative approach, I explored the use of changes in eCFP fluorescence lifetime depending on fluorophore concentration via homo FRET. Proteolytic processing should release the fluorophore for free diffusion within the virus particle and thereby cause an increase in fluorescence lifetime. Indeed, Gag.eCFP cleavage by HIV-1 PR resulted in changes in fluorescence lifetime measured in purified VLPs and at the plasma membrane of HeLa Kyoto cells. Time-lapse experiments visualized differences in fluorescent lifetime during viral assembly. So far, only few events that may correspond to proteolytic processing were detected. Further adaptation of the system to live cell measurements is under way. Finally, I studied the formation of active HIV-1 PR during assembly with a sensor to fluoresce upon binding to the PR active site, were provided by our collaboration partners who coupled the fluorogenic dye silicone-rhodamine (SiR) to the PR inhibitor Ritonavir (RTV). Specific signal increase upon binding to active PR was shown in vitro and on assembly sites at the plasma membrane of HeLa Kyoto cells. Using this compound, I successful established conditions for time-resolved detection of mature PR at nascent assembly sites. Different SiR recruitment patterns with respect to progression of Gag assembly were observed. Further improvement of signal-to-noise ratio would be beneficial and is currently under way.