Tetherin restricts cell-cell transmission of a dual-tropic HIV-1 from MDMs to T cells.

<p>(A) Schematic outline of the assay used to quantify cell-cell transmission from MDMs to autologous CD4⁺ T cells. (B) R3A-(+), -(−), or -Udel-infected MDMs, or uninfected control MDMs, were co-cultured with autologous CD4⁺ T cells for 6 h, the T cells were washed off the MDMs with PBS and lysed immediately (0 days), or after another two day-incubation (2 days). As a control, 500 nM NVP was added to parallel MDM-T cell co-cultures, and to the T cells after the co-culture (T cells/NVP during and after co-culture). No T cells were added to infected MDMs, and the culture supernatants treated as the T cells, as another control (no T cells). All T cell lysates were analysed by western blotting. (C) T cells were co-cultured with R3A-infected MDM for 6 h, separated from the MDMs, and incubated for another zero to two days (0–2 days), or for two days in the presence of 500 nM NVP (2 days/NVP after co-culture). The T cells were lysed and analysed by western blotting. Note that parallel cultures of the MDMs used for the experiments in (B) and (C) were analysed by western blotting to confirm equal infection levels (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004189#ppat.1004189.s011" target="_blank">Fig. S11</a>). (D) MDMs were infected with R3A and transfected with control (control RNAi) or Tetherin (Tetherin RNAi) siRNA the next day. Six days later, autologous T cells were co-cultured with the infected MDMs for 6 h, separated from the MDMs, and lysed immediately (0 days) or incubated for another two days in the presence of 500 nM NVP (2 days). The T cell lysates (T cells), and lysates of the MDMs (MDMs) used for the experiment, were analysed by western blotting. Note that residual Tetherin levels in the MDMs had to be <5% to see a rescue of the T cell infection with R3A-(−) and -Udel.