Figure 3

Transmitted/founder viruses display modestly reduced entry efficiency in MDM. (A,B) CEM.NKR-CCR5-Luc cells (CEM) or MDM were infected with equal amounts of p24 of BlaM-Vpr containing viruses for 4 h. Cells were loaded with CCF2/AM dye and fusion events were detected by flow cytometry using BD LSR Fortessa, and gated from 10,000 cells. (A) A representative example of three independent experiments. Percentage in each panel represents virus fusion positive cells (cleaved CCF2). (B) Graph shows a percentage of detected fusion events (blue cells) normalized to YU-2 as a control (100%), and represents an average of three independent experiments, each conducted in triplicate. Black bars represent CEM cells and white bars MDM. (C) MDM were infected with equal amounts of p24 of BlaM-Vpr containing viruses for 4 h. Cells were inspected for virus entry as described above, at the same time cells were harvested at 6 h post-infection for total DNA isolation and total viral DNA was determined using quantitative PCR.% blue cells or copies/100 ng DNA are normalized to YU-2 (100%). (D,E) MDM were infected with equal amounts of p24 of YU-2 (D) or BaL (E) BlaM-Vpr containing viruses for 4 h. Cells were inspected for virus entry as described above, at the same time cells were harvested at 6 h post-infection for total DNA isolation and total viral DNA was determined using quantitative PCR.% blue cells or copies/100 ng DNA are normalized to higher p24 input of virus (480 ng ~ 100%). (F) MDM were infected with different amounts of p24 of BlaM-Vpr containing viruses to achieve equal virus entry for 4 h. Inspected for entry efficiency and also labeled for intracellular p24 48 h post-infection.% blue cells and infected cells/field are normalized to YU-2 (100%). (C-F) Data shown are representative example of at least two independent experiments conducted in duplicates. Error bars represent the standard deviation.