Fig. 4.

ESCRT-III adaptor protein ALIX contributes to HSV-1 nuclear egress. a Confocal microscope images of HeLa or HeLa/ALIX-low cells mock-infected or infected with HSV-1 for 22 h and stained with anti-ALIX and anti-UL34 antibodies. Bars, 20 μm. Images are representative of three independent experiments. b Confocal microscope images of HeLa and HeLa/ALIX-low cells treated with control siRNA (siCt) or siRNA to ALIX (siALIX), respectively, for 48 h, and then infected with HSV-1 for 22 h. Bars, 20 μm. Images are representative of three independent experiments. c Percent of cells (50–100 cells in each experiment) with aberrant punctate structures along with the nuclear rim in the experiment in (b). Data are shown as the mean ± SEM of three independent experiments. d Electron microscope images of HeLa and HeLa/ALIX-low cells treated with siCt or siALIX, respectively, for 48 h and infected with HSV-1 for 22 h. C cytoplasm, N nucleus, NM nuclear membrane. Bars, 500 nm. Arrowheads indicate virions defective in the scission steps. Images are representative of three independent experiments. Percent of (e) perinuclear enveloped virions and f capsids in the cytoplasm of 13 cells in the experiment in (d) was determined. Data are shown as the mean ± SEM and are representative of three independent experiments. g–i HeLa and HeLa/ALIX-low cells treated with siRNA as described in (b) were infected with HSV-1 at an MOI of (g) 0.05 or (h) 10, or with i influenza virus at an MOI of 0.01, and progeny virus titers were assayed at the indicated hours post infection (h.p.i.). Data are shown as the mean ± SEM of (g, i) three or (h) four independent experiments. The indicated P-values were obtained using the unpaired Student’s t-test (c, e–i). n.s., not significant