Kinetics of HIV-1 latency reversal quantified on the single cell level using a novel flow-based technique

HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes of infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single cell level by flow cytometry. This technique allowed for simultaneous detection of gagpol mRNA, intracellular p24 Gag protein and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with hTNFα/RMD or HIV-1 infection of primary CD4(+) T cells, four cell populations according to their expression levels of viral mRNA and protein were detected. Gagpol mRNA was quantifiable for the first time in J89 cells 3 hrs post-stimulation with hTNFα and 12 hrs post-stimulation with RMD, while p24 Gag protein was detected for the first time after 18 hrs post-stimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2 and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, we here describe a novel approach allowing to quantify the kinetics of HIV-1 mRNA and protein synthesis on the single cells level and to phenotypically characterize HIV-1-infected cells at different stages of the viral life cycle.

IMPORTANCE: Early after infection, HIV-1 establishes a pool of latently infected cells, which are hiding from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells is one of the goals of current HIV-1 cure approaches, however little is known about the HIV-1 reactivation kinetics following stimulation with latency reversing agents. Here we describe a novel approach allowing for the first time to quantify the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single cells level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently and de novo infected cells dependent on the presence of viral RNA or protein.