Fig 5.

Induction of autophagy requires viral glycoprotein-mediated membrane fusion. (A) Syncytium formation and intracellular distribution of GFP-LC3 puncta in Vero cells cotransfected with GFP-LC3 and CDV 5804P and MeV Edm glycoprotein expression plasmids. Pictures were taken 20 h after transfection. Phase-contrast images are shown at a 400-fold magnification, and fluorescence images are shown at a 1,000-fold magnification. (B) Proportion of GFP-expressing cells with punctum formation. For each sample, the total number of GFP-expressing foci and the number of foci with GFP puncta were counted in four separate fields. Data from at least four independent experiments were used for the analysis. Error bars represent the standard deviation, and statistically significant differences (ANOVA, P < 0.05) are indicated by asterisks. (C) LC3-I/LC3-II ratios in Vero cells transfected with the CDV and MeV glycoprotein expression plasmids. Fusion-inhibiting peptide (FIP) was used to prevent membrane fusion in MeV glycoprotein-expressing cells. GAPDH was used as a loading control, and viral glycoproteins were detected using rabbit antipeptide antisera against the F- and H-protein cytoplasmic tails, respectively. Mean values of LC3-I/LC3-II ratios of at least three independent experiments are shown, and error bars indicate the standard deviation. Statistically significant (ANOVA, P < 0.05) differences between samples and the control are indicated by asterisks.