Fig. 3: ALIX interaction with HAV capsids.

A–L Co-localization of ALIX and HAV capsids in Huh7-A-I-infected cells. A–F correspond to L0-infected cells, and G–L to HP-infected cells. A Indirect immunofluorescence (IIF) of L0 capsids at 48 hours post-infection (h p.i.). B IIF of ALIX at 48 h p.i. C Co-localization of L0 capsids and ALIX at 48 h p.i.; red and green fluorescence correspond to HAV capsids and ALIX, respectively; orange to yellow fluorescence depicts co-localization of capsids and ALIX. Similarly, D–F depict the same analysis in L0-infected cells at 72 h p.i. (G–I) in HP-infected cells at 48 h p.i., and (J–L) in HP-infected cells at 72 h p.i. The white bar corresponds to 10 µm. M Quantitative confocal microscopy analysis of the proportion of HAV capsids co-localizing with ALIX (M1 coefficient) at 72 h p.i. Dots correspond to the M1 value of each of n = 12 images from n = 3 independent biological replicates. The total number of infected cells counted was 51 and 77 for L0 and HP, respectively. Significant differences between L0 and HP-infected cells are depicted. N Immunoprecipitation of L0 and HP naked capsids, released from exosomes by freeze-thaw cycles combined with sonication, with an anti-HAV antibody (blue) and an anti-ALIX antibody (gray). The percentage of capsids pulled down with the anti-ALIX antibody is expressed relative to the percentage of capsids pulled down with the anti-HAV antibody. As a control, no lysed particles were pulled down with the anti-ALIX antibody (white). Data represent the mean ± standard error of n = 4 independent biological replicates. Significant differences are depicted. O Effect of ALIX-coding gene silencing on HAV egress. The ratio between the total intracellular genomes and the total number of infectious viruses, expressed in TCID₅₀, in the supernatant, is shown. NT-siRNA and siALIX columns represent the effect of silencing using a mixture of non-target siRNAs and a mixture of siALIX RNAs, respectively. A silencing effect on HAV egress should induce an increase in this ratio. Data represent the mean ± standard error from n = 10 biological replicates. Each biological replicate included two technical replicates, whose average is depicted.