HIV-1 can integrate into the genomes of quiescent CD4+ T cells

William J. Swiggard¹, Clifford Baytop²,Jenny Yu² and Una O’Doherty², Departments of Medicine¹ and Pathology and Laboratory Medicine², University of Pennsylvania (USA)

Background: Although resting T cells were initially thought to be resistant to HIV-1, in vivo and in vitro studies reveal low level production and hence integration in apparently resting CD4+ T cells. These cells receive subtle signals including cytokine stimulation from their environment, so they may not be truly quiescent. We now describe in vitro studies using the first fully quantitative integration assay sensitive enough to detect provirus in HIV-1 infected clinical samples. Material and Methods: Resting (HLADR-, CD69-, CD8-, CD14-, CD16-, CD20-) and activated CD4+ T cells (hypersusceptible T cell line) were spinoculated with HIV-1, and then cultured for up to 4 days in medium supplemented with 30% autologous donor serum and the protease inhibitor saquinavir (to inhibit spreading infection). The numbers of reverse transcripts and proviruses formed per initially-bound virion were measured at multiple time points after exposure using quantitative real-time PCR. The amount of viral DNA was also measured in simultaneously spinoculated endogenously activated T cells (HLA-DR+, CD69+, CD8-, CD14-, CD16-, CD20-). Results: Using this assay, we show for the first time that integration occurs in resting CD4+ T cells in the absence of cytokines at low levels. While the number of bound virions was similar for both cell types, the efficiencies of both reverse transcription and integration were much lower in the resting T cells. The integration signal detected in resting CD4+ T cells cannot be attributed to contaminating activated T cells based on the amount of viral DNA present in simultaneously sorted endogenously activated T cells. Thus, HIV-1 can integrate (albeit inefficiently) into the genomes of resting T cells without immunologic activation. This finding has multiple implications as to the mechanism(s) whereby reservoirs of treatment-resistant virus accumulate and are maintained in HIV-1 infected individuals.