Figure 2

Subcellullar distribution of Ad2, Ad2-ts1 and Ad2-BAC46. A) TEM analyses of subcellular localization of Ad2 (grown at 37°C), Ad2-ts1 (grown at 40°C), Ad2-BAC46 (grown at 32°C), Ad2-BAC46 (grown at 40°C). 10⁵ HeLa cells were incubated with 3 × 10¹⁰ purified virions in the cold (60 min), washed (10⁴ virus particles bound per cell) and warmed to 37°C for 30 min [30]. P-values are from one-sided student t-tests. B) Representative TEM images of Ad2-infected cells at high (a, b) or low magnification (c) 30 min pi are shown, including ruthenium-red stained plasma membrane [32]. Arrows, and back and white arrowheads indicate particles at the plasma membrane, endosomes or the cytosol, respectively. C-E) Kinetics of Ad2 and Ad2-ts1 endocytosis and endosomal escape measured at 37°C. Cold-bound Ad2 or Ad2-ts1 were internalized into HeLa cells for 7, 15, 30, and 60 min, cells processed for TEM, and analyzed for localization at the plasma membrane, endosomes and the cytosol. Mean values of Ad2 (filled squares) and Ad2-ts1 (triangles) are shown from 8-10 cells and 23 to 45 virus particles (v.p.) per cell at the time points indicated above (see table below panel E). Averaged fits of the data points and extrapolation to the 0 min time pointare shown by continuous or dashed lines. In panel E, the time point for half maximal escape of Ad2 from endosomes to the cytosol is indicated by thin lines and calculated to be 15 min based on the cytosolic levels of 58% at 60 min pi. Half maximal escape of Ad2-ts1 is estimated to be 17 min (dashed fine line) based on 34% cytosolic levels at 60 min pi.