Background:  Resting CD4 T cells are the best-defined reservoir of latent HIV-1 infection in vivo, although mechanisms leading to formation of this reservoir are unclear. Recent data have shown that primary resting CD4 T cells without classic activation markers (CD25, CD69, etc.) can be infected in vivo, ex vivo, and in vitro under some culture conditions. Here, we examined productive and latent infection of primary CD4 T cells after cell-to-cell transmission of HIV-1, and the contribution of the endocytic pathway to transmission of replication-competent virus.

Methods:  Primary resting CD4 T cells (target cells) were co-cultured with wild-type (WT) HIV NL4-3- or NL4-3-green fluorescent protein (GFP)⁺-infected Jurkat or CEM-SS cells (effector cells), and compared to mock-infected cells. Expression of HIV Gag or GFP in primary resting CD4 T cells was analyzed by flow cytometry. Intracellular expression of GFP was confirmed by digital immunofluorescence microscopy. In some experiments, the target cells or effector cells were labeled with a fluorescent dye for live cell sorting and functional analysis of highly purified populations of primary CD4 T cells after co-culture.

Results:  After overnight co-culture, 13% to 49% of resting primary CD4 T cells were p24+, while only 2% of the primary T cells expressed GFP. Direct cell-to-cell contact was required. Soluble CD4 or cytochalasin D inhibited both p24 and GFP expression in the resting CD4 T cells, suggesting an actin-dependent transfer of viral particles from effector cell to target cell. When the resting CD4 T cells were pre-treated with Bafilomycin A, an inhibitor of endosomal acidification, the expression of p24 was greatly reduced, suggesting transfer of viral particles via an endocytic pathway. GFP, but not p24 expression, was inhibited by the chemokine receptor CXCR4 antagonist, AMD3100, or the fusion inhibitor T20, suggesting dependence of productive infection on virus-cell membrane fusion.

Conclusions:  These data suggest that an actin-dependent cell-to-cell contact transfers HIV-1 viral particles from effector cells to target cells by 2 independent mechanisms. Transfer of viral particles (i.e., p24-positivity) can occur via an endocytic pathway, leading to degradation of the particles in the endosome. In contrast, transfer of productive infection (i.e., GFP positivity) via the virological synapse requires fusion via CD4 and CXCR4. These data have implications for HIV-1 latency and viral spread.

Keywords: virological synapse; retroviral pathogenesis; virus entry