Abstract
The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.
Highlights
Release and maturation are critical steps in the late phase of the HIV-1 replication cycle
During or shortly after virus release, the mature form of PR generated by autoprocessing from GagProPol mediates proteolytic cleavage of GagProPol and Gag, triggering the morphological rearrangements that yield the characteristic cone-shaped capsid core of the infectious virus [4]
virus-like particles (VLPs), we show that both incorporation of eCFP into the Gag lattice during assembly and its subsequent cleavage from the lattice during maturation result in measurable changes in the fluorescence lifetime of the fluorophore
Summary
Release and maturation are critical steps in the late phase of the HIV-1 replication cycle. Inserting the fluorophore eCFP as an additional domain into the Gag polyprotein allowed us to monitor changes in its fluorescence lifetime as a readout for proteolytic release. This enabled us to detect Gag processing in relation to the assembly process with high time resolution. By tracking individual assembly sites and VLPs, we show that both incorporation of eCFP into the Gag lattice during assembly and its subsequent cleavage from the lattice during maturation result in measurable changes in the fluorescence lifetime of the fluorophore Using this method, we were able to observe the timing and kinetics of Gag processing and rearrangement in relation to the assembly process in individually tracked VLPs
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