Phases of HIV transfer from MDDCs to CD4 T cells. Both immature MDDCs (A-B) and mature MDDCs (C) were treated with infectious HIV-1_BaL for 2 hours and washed. MDDCs were then cultured in IL-4/GM-CSF media for varying time points. In panel B, MDDCs were cultured in the presence of 10 μM zidovudine. MDDCs at indicated time points were washed, harvested by pelleting, and resuspended in IL-4/GM-CSF media and then cultured with PHA-activated CD4⁺ T cells in a ratio of 1 DC to 4 CD4⁺ T cells. At 24 hours after mixing of the cells, the combined cell pellet was lysed and assayed for full-length HIV-1 DNA by Q-PCR. Pure cultures of MDDCs were also harvested at the same time points to determine DNA levels in DCs alone. The DNA input into the cocultures by DCs was subtracted from coculture HIV-1 DNA and is presented as HIV-1–LTR gag DNA per 1 × 10⁶ cells. HIV-1–LTR gag DNAin 0.2 × 10⁶ DCs is also presented in overlaid graphs in open circles. In panel D, a model of transfer from immature MDDCs to CD4⁺ lymphocytes over time is illustrated on the basis of results in upper panels A-B. In panels Di-ii, the characteristics of both phases of HIV-1 transfer from MDDCs to CD4⁺ lymphocytes are shown. The first phase (i) results from diversion of infectious virus from the endolysosomal pathway of degradation (“trans-infection”). The proposed second phase (ii) of de novo viral production in DCs (“cis” infection) prior to transfer to CD4⁺ lymphocytes. Note that this phase is stable and consistent with R5 HIV-1 strain selection initially observed in in vivo HIV-1 transmission.