Background. In contrast to the CD4/chemokine receptor complex that mediates virus-cell fusion and entry, HIV-attachment factors do not mediate fusion of the virus and the host cell membranes, but rather could internalize virus particles by receptor-mediated endocytosis. Such mechanisms of virus accumulation not only have the potential to protect the invading HIV from being degraded, but also create a reservoir of virus that is competent for ultimate transfer to target T-cells. Interactions of HIV-1 with immature dendritic cells (DC) are believed to be multi-factorial and include DC-SIGN, mannose receptors (MR) and heparan sulfate proteoglycans (HSPG). In this study, we assessed the relative contributions of these previously defined virus-attachment factors to HIV binding and accumulation in DC and the subsequent transfer of the bound virus particle to CD4⁺ T-cells.

Methods. Primary human monocyte-derived DC generated by differentiation of CD14⁺ monocytes in the presence of GM-CSF and IL-4 were treated with inhibitors of DC-SIGN (neutralizing antibodies), MR (increasing concentrations of mannan), and HSPG (treatment with heparinases) prior to exposure with lab-adapted and primary strains of HIV-1. Unbound virus was washed away, and the amount of cell-associated virus was quantitated by either p24^gag ELISA or real-time PCR using gag specific primers. Internalization and cellular localization of HIV in the presence of inhibitors was visualized by electron microscopy for intact virus particles, and via immunofluorescence with antibodies were directed against p24^gag protein. Finally, these virus-exposed DC were co-cultured with T-cells (at a 1:10 ratio). The efficiency of viral replication in the co-cultures was determined by measuring the p24^gag content of cell-free supernatants at defined time intervals by ELISA.

Results. Using competitive inhibitors of HIV-1 attachment to DC, our studies have identified the existence of DC-SIGN, MR, and HSPG independent mechanism(s) of HIV attachment and internalization. Intact virus particles were found on the DC surface and in unique sub-cellular vesicular compartments. Furthermore, virus particle bound by DC independently of DC-SIGN, MR, and HSPG was efficiently transmitted to T-cells.

Conclusions. We propose that pathways to HIV-1 internalization and uptake in DC exhibit functional redundancy; that is, they are made up of multiple independent activities that can, at least in part, compensate for one another.