Abstract
Upon its entry, HIV-1 replicates and produces new viral particles that are released into the extracellular environment. The crucial step of virus release remains poorly understood because it requires the study at the single cell level. Indeed, quantification of viral production from cell populations with very heterogeneous HIV-1 replication kinetics would give approximate results. This is why we have developed a microfluidic approach that allows the study of HIV-1 release dynamics in real-time at the single cell level. In this study, continuous microfluidics was combined to the virology in order to develop a sensitive and reliable technology to visualize and quantify virus production by a single cell. Three types of chips were fabricated: the trapping chip allowed us to determine the physical and biological parameters that ensure single cell trapping (~10 µm) producing VLPs-GFP. The detection chip, whose performance was compared with the Nanoparticle Tracking Analysis technique, proved to be a valuable tool for accurate and reproducible quantification of fluorescent VLPs (~140 nm) at the single particle scale in cell culture supernatants. The multiplex chip, which combines the two previous chips, allowed us to study in real-time the virus release kinetics at the single cell scale. VLPs-GFP producing HeLa and HEK 293 cell lines were used as study models. For the first time, viral production kinetics could be measured with an average of 50 VLPs / cell / h that was validated by the measurement of viruses produced by the same cells grown in culture dish, confirming the reliability and sensitivity of our approach. Interestingly, the release kinetics profile shows a periodic process (period ~4min) that could be explained by the presence of one or more limiting steps in the virion biogenesis mechanism. The new tools developed here provide novel information on the kinetics of HIV-1 salting-out. They can be used or easily adapted for the study of other pathogens or extracellular vesicles.