(P33) Design and Characterization of Fluorogenic Inhibitors to detect Active HIV-1 Protease during the Viral Assembly Process
Annica Flemming, Jiri Schimer, Aleksandar Salim, Nicolas Lardon, Jan Konvalinka, Kai Johnsson and Barbara Müller
Department of Infectious Diseases, Virology, University Hospital Heidelberg, Germany, UOCHB AV ČR, IOCB Prague, Czech Republic, Max-Planck-Institute for Medical Research, Heidelberg, Germany
Background: Human immunodeficiency virus (HIV-1) assembles and buds at the host cell plasma membrane. Virions are initially released as immature, non-infectious particles, largely consisting of uncleaved structural polyproteins. Concomitant with or shortly after release, the viral protease (PR) cleaves the viral Gag and GagProPol polyproteins into their mature subdomains, leading to structural rearrangement of the virion, which is essential for infectivity. Temporal control of proteolytic cleavage with respect to particle assembly and the order of Gag processing events appear crucial for the virion to gain infectivity. Dimerization of GagProPol polyproteins to form active PR from monomeric PR subdomains encoded in the polyprotein is required to trigger proteolysis, but the precise mechanism and timing of PR activation in relation to virus assembly and release is currently unclear.
Methods: In order to detect formation of active dimeric HIV-1 PR during the course of assembly as a first essential step of maturation, we aimed at developing fluorescent sensors for this process to be used in microscopic studies of virus-producing cells. To this end, we made use of different clinically used PR inhibitors, which were chemically linked to fluorogenic dyes via carbon linkers of different length. We initially tested fluorogenicity and maximum fluorescence of the different compounds in vitro after fluorescence activation. We subsequently validated specific detection of active dimeric PR using purified proteins. Eventually, we applied the most promising compounds in total internal reflection microscopy (TIRF-M) of virus producing cells, using viral constructs carrying another fluorophore in the gag-domain.
Results: In vitro measurements revealed specific binding of the fluorogenic probes to active, dimeric, wild-type HIV-1 PR, yielding a fluorescent signal. In contrast, monomeric PR molecules or inactive dimeric PR carrying a mutation in the PR active site did not induce fluorescence validating specificity of the compounds. TIRF-M of fixed virus-producing cells revealed co-localization of a fluorescent signal for some of the PR probes with bona fide assembly sites, identified as characteristic patches of fluorescent Gag assemblies. Again, no signal was obtained when the active site Asp residue of PR was mutated. Based on these results, we initiated live cell TIRF-M of virus-producing cells expressing fluorescent Gag polyproteins and stained with fluorogenic PR probes. Ongoing analyses attempt to determine the onset of active PR formation with respect to the different stages of HIV-1 assembly.
Conclusion: Active HIV-1 PR was successfully detected using purified proteins and during the assembly process in virus-producing cells.