Resting CD4 ؉ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4؉ T cells in vitro, albeit at lower levels than in activated CD4؉ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4؉ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4 ؉ T cells. Resting CD4ϩ T cells (rCD4 cells) appear to be a major target of human immunodeficiency virus (HIV) and simian immunodeficiency virus infections in vivo, especially in the gut during acute infection (12,20,42,43,47,48), yet in vitro rCD4 cells resist HIV infection (8,34,35,45). Understanding this paradox is important to HIV pathogenesis. Early in vitro studies showed that reverse transcription and possibly other postentry steps are restricted in rCD4 cells (3,35,37,45,46). However, extended kinetic studies showed that reverse transcription is not completely blocked in resting T cells but rather occurs with slower kinetics than in activated cells (8,29,33,39). The slower kinetics of reverse transcription in resting T cells can be partially overcome by inducing the cells to enter G 1b (11,18,40), by knocking down putative restriction factors (8, 36) and by adding deoxynucleosides (dN) (17), which are present at lower levels in resting cells than in activated cells (9,10,24,26,32).Our previous data (1, 38) and those of Vatakis et al. (41) suggest that, despite this restriction, HIV can integrate directly into rCD4 cells, although this occurs with slower kinetics and to lower levels in resting cells than in activated cells. Given that dN increase reverse transcription in resting T cells (17), we tested the effect of dN addition on HIV integration and 2LTR formation in resting T cells. Here we show that dN increase integration and 2LTR formation in rCD4 cells.Two populations of CD4 ؉ T cells were prepared from PBMCs. To study the restriction of HIV infection by rCD4 cells, we prepared rCD4 cells and activated CD4 ϩ T cells (aCD4 cells) from peripheral blood mononuclear cells (PBMCs; Fig. 1A).Partially purified CD4 ϩ T cells (ppCD4 cells) were prepared by negative selection (Rosette Sep; Stem Cell Technologies) for other cell lineages (CD8, CD16, CD19, CD36, and CD56) (Fig. 1A). rCD4 cells were prepared from the ppCD4 cells by a second negative selection using phycoerythrin-labeled antibodies for ␥␦ T-cell receptor (␥␦TCR) and activation markers (CD69, CD25, and HLA-DR) followed by anti-phycoerythrin magnetic beads (magnetically activated cell sorting; Miltenyi). The resulting cells were 99% pure rCD4 cells (Fig. 1A). Preactivated CD4 ϩ T cells were prepared by stimulating ppCD4 cells with anti-CD3/28-coated beads (Dynal) for 3 days (5, 6). The aCD4 cells uniformly expressed CD25 and CD69 by day 3 (see Fig. 3).dN enhanced integration and 2LT...