Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV
Figure 9
Co-culturing cells leads to a larger proportion of infected target cells and a higher proviral content.
(A) Infection by cell-free or by co-cultures with MT4, Jurkat or primary CD4+ T cells were repeated as in Figure 5B using HEK293 donor cells producing HIVIRES-GFP. Target cells were identified based on their expression of CD2 (MT4 cells) or CD3 (Jurkat and primary CD4 T cells). GFP fluorescence was gated based on the background fluorescence from a corresponding sample treated with 1 µM of efavirenz (EFV). Numbers represent the average percent of GFP-positive cells +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. MFI values correspond to the average mean fluorescence +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. (B) Comparison of the GFP fluorescence intensity of cells infected by cell-free virus or by co-culture from the gated population in panel (A). Histograms represent the combination of 3 measurements. Fluorescence was normalized to the fluorescence from the corresponding efavirenz-controls to account for fluorescence shifts due to sample variability. The grey dashed line represents cell-free infection and solid black line represent co-culture infection. The black vertical dashed line represents the limit of the gates shown in (A). (C) An experiment as in (A) was repeated with wild-type HIVNL4-3 and target cells were stained with CFSE. CFSE-positive cells expressing HIV Gag above the efavirenz-treated control (see Figure S5) were sorted and the number of HIV integration events was analyzed by Alu-PCR. Control samples were treated with 1 µM efavirenz (+EFV). Error bars represent the standard deviation of 3 measurements of integration.