Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 T cell model.

Investigating the molecular mechanisms of HIV latency reversal in a proper physiological context can only be done in primary cells. Here, we describe a primary T cell model of HIV latency and a reliable flow cytometry assay to measure latency reversal efficacy by dual immunofluorescence staining for Nef and Tat. We also describe a procedure for identifying latency-reversing agents that effectively induce the biogenesis of P-TEFb, an obligate host transcription factor for HIV, while monitoring their effects on T cell activation.

Structural rearrangements in the nucleus localize latent HIV proviruses to a perinucleolar compartment supportive of reactivation.

Using an immunofluorescence assay based on CRISPR-dCas9-gRNA complexes that selectively bind to the HIV LTR (HIV Cas-FISH), we traced changes in HIV DNA localization in primary effector T cells from early infection until the cells become quiescent as they transition to memory cells. Unintegrated HIV DNA colocalized with CPSF6 and HIV capsid (CA, p24) was found in the cytoplasm and nuclear periphery at days 1 and 3 post infection. From days 3 to 7, most HIV DNA was distributed primarily in the nuclear intermediate euchromatic compartment and was transcribed.