Strategy to detect rare viral fusion events at the cell surface.

<p>(A) Illustration of fusion, recycling and retrograde trafficking of viral particles co-labeled with EcpH-ICAM (pH-sensitive membrane marker, green) and Gag-imCherry (releasable content marker, red). Changes in particle’s fluorescence signal (in false colors) associated with fusion at the plasma membrane and entry/fusion with acidic endosomes is shown in boxes above and on the right of the cartoon. Due to the loss of EcpH reference signal before fusion with acidic endosomes, these events are not detected. (B) HXB2pp co-labeled with Gag-imCherry (red) and extra-viral pH sensor EcpH-ICAM (green) were bound to poly-L-lysine coated coverslips and imaged at room temperature in buffers adjusted to pH 6.3, 6.8 or 7.3. (C) Plot shows the distribution of EcpH-ICAM background-subtracted sum intensity of more than 3,000 virus particles at each pH. Green bar shows median and interquartile range and whiskers contain the middle 80% of the intensity distribution. (D) Comparison of specific infectivity (infectious units per nanogram p24; IU/ng p24) of HXB2pp containing Gag-imCherry and YFP-Vpr or EcpH-ICAM untreated and treated with 5 μM C52L.