Background:  Resting CD4⁺ T cells comprise the best defined reservoir of HIV-1 latent

infection, but how these reservoirs are formed is unclear. One hypothesis is that latent reservoirs form when activated T cells are infected as they return to a resting state. Another hypothesis is that CD4⁺ T cells receive some transitory subtle stimuli that allow integration to occur. A third hypothesis is that integration and latent infection can occur in resting CD4⁺ T cells without any stimuli. While integrated DNA has been demonstrated in resting CD4⁺ T cells from HIV-1-infected individuals, it has not been reported in resting CD4⁺ T cells isolated from blood that have been inoculated with HIV-1 in vitro in the absence of exogenous stimuli. This has lead to the assumption that HIV-1 cannot integrate into quiescent (G₀) T cells without stimulation.

Methods:  We tested whether integration can occur after in vitro inoculation of resting (G₀) CD4⁺ T cells isolated from blood in the absence of exogenous stimuli using a highly specific assay for HIV-1 integration. To increase the frequency of rare events such as integration, an inoculation technique was used that deposits large numbers of virions onto the cell surface with centrifugal force. In addition, we determined the fraction of cells capable of productive infection by stimulating the cells 4 days after inoculation. Production was assessed by staining for intracellular p24 3 days after stimulation.

Results:  Under these conditions, a minority (~1 in 10) of reverse transcripts integrates in resting CD4⁺ T cells, while the majority of reverse transcripts integrate into simultaneously sorted activated CD4⁺ T cells. In our model, at least 1% of the CD4⁺ T cells are capable of productive infection.

Conclusions:  These results show that latent HIV-1 infection of resting CD4⁺ T cells can occur without T cell activation and challenge the current dogma of how HIV-1 reservoirs form. HIV-1 inoculated resting CD4⁺ T cells may provide a model for studying HIV-1 latency.

Keywords: latency; reservoirs; resting T cells