Incoming LCMV transiently co-localized with Tsg101 prior to reaching late endosomes.

<p>(A) Co-localization of LCMV WE54 with Tsg101 and Rab7. A549 cells were cooled on ice for 30 min and LCMV WE54 added at an MOI of ∼100. After incubation for 1 hour on ice, unbound virus was washed off, cells shifted to 37°C and fixed at the indicated time points. Cells were then immunostained to detect endogenous Tsg101 or Rab7 and incoming viral particles. Representative images are shown. Left: LCMV (green) and Tsg101 (red) at 20 min after temperature shift; right: LCMV (green) and Rab7 (red) at 60 min after temperature shift. Scale bar = 5 µm. (B) Quantification of co-localization. Ten randomly selected cells per time point were analyzed by confocal microscopy and the percentage of co-localizing viruses determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#s4" target="_blank">Materials and Methods</a>. Data presented are means ± SD (n = 10).