Background:  Dendritic cells (DC) are a primary target for HIV crossing the mucosa, impacting the onset and spread of infection. Virions interact with DC via CD4s, CCRs, and CLRs, but virus spread from DC to T-cells mainly involves CD4 and CCRs on the T cells. Preventive strategies designed to interrupt trans-mucosal infection will benefit from a detailed grasp of the multiple mechanisms used by HIV to enter DC and how DC-captured HIV is transferred to T cells.

Methods:  We exploited the free sulfhydryls on the internal proteins of retroviruses, including the nucleocapsid protein, to chemically inactivate virions with aldrithiol-2 (AT-2 HIV/SIV) or directly fluorescently label SIV virions using a fluorophore tagged N-ethylmaleimide reagent (ALEXA SIV). Virions inactivated or labeled in this way retain structurally and functionally intact envelope glycoproteins. Capture of virions by moDCs, and spread to T cells was monitored (flow cytometry or immunofluorescent/electron microscopy) in the presence and absence of specific inhibitors (e.g., T1249, mannan).

Results:  AT-2 HIV adefovir dipivoxil (ADA) and MN as well as ALEXA SIV E11S were taken up by immature and mature DC. All viruses exhibited the typical intracellular localization we defined previously, with virus positioned at the cell periphery in immature DCs and in a deeper perinuclear location in mature DC. Fluorescent imaging showed that all viruses were in compartments co-staining with the tetraspanin molecule CD81, but not with other markers (CD63, CD68, CD208). As expected, virus capture by immature DCs was more dramatically reduced by the fusion inhibitor T1249, but virions were still internalized by immature and mature DC in the presence of the inhibitor. Electron microscopy verified that captured virions accumulated at the DC-T-cell synapses, moving to the T cell. Preliminary data also suggest that T1249 may restrict the release of virus from DC and that viruses may collect in the DC under these conditions.

Conclusions:  AT-2 inactivated and ALEXA-labeled viruses represent a unique way to monitor DC-virus interplay and DC-driven virus spread. Being non-infectious, we can selectively explore the contribution of this phase of DC-to-T-cell transfer to virus spread. The complex nature of DC-virus interactions and the partial blocking effects of single inhibitors underscore the need to explore a combination of attachment, fusion, and entry inhibitors to efficiently block virus capture by DC and virus growth in the DC-T-cell milieu.

Keywords: dendritic cells; transmission; inhibition