Résumé
To date, tremendous progresses have been made to understand the intracellular assembly and budding of HIV-1 using imaging approaches. However, the crucial step of virion release remains incompletely deciphered because it requires adapted techniques. The available data are approximate because they were obtained with cell population whose HIV replication kinetics are heterogeneous. Thus, we are developing a new strategy, that we called “viro-fluidics”, to study in real time the kinetics of HIV-1 release at the single-cell level. We hypothesize that natural selection should favour viruses which maximize the number of progeny produced over the lifespan of the infected cell. Thus, optimal strategy for a virus could be to reproduce as quickly as possible with a production that increases gradually over time. But maximal rate may not be the optimal strategy when the immune response may kill the cell. Thus, for a virus optimal viral production rate should vary with time during infection but also between cells with different lifespans. The viro-fluidics combines virology to the powerful nano/microfluidic strategies to immobilize single cells in a microchannel under continuous medium flow and to measure their viral production in real-time by flow virometry. Up to now, the technique is based on cells producing fluorescent virions. To do so, different PDMS-based microfluidic chips have been designed and fabricated for single cell trapping and single viral particles detection, that ensures a real-time detection of viral release. Practically, inside individual channels, a V shape trap was microfabricated and downstream of this trap another channel with a height in the same order of the depth of field of the objective was designed to serves as a flow virometry system. Single HeLa cells, stably expressing GFP HIV-1 viral-like-particles (GFP-VLP), were trapped, and maintained for several hours in a continuous flow under optimal physiological conditions (37ᵒC, 5% CO2). Their viability was monitored by propidium iodide (PI) staining. The reliability of this new flow virometry system that was implemented downstream to count the GFP-VLP released, was compared to the commercial Nanoparticle Tracking Analysis (NTA) apparatus. The virofluidic system ensures a very sensitive and quantitative analysis of the VLP-GFP at the single particle level. It allows fluorescent viral particles detection in real time: a strategy that enables the study of the dynamics of virion release at the single cell level. Our viro-fluidics system will provide new information about the heterogeneity between cells from same type as well as between different cell types. Moreover, it will allow deep understandings of the production strategy used by the virus to adapt to its host.