An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A.; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

Typically, cells were processed in two ways after the siRNA-mediated knockdown for 48 h. For analyzing endogenous p62 and LC3 puncta accumulation in order to measure autophagy, cells were fixed with 4% PFA and then incubated with the blocking buffer (PBS, 1% bovine serum albumin, and 0.1% saponin) before being stained first with p62 and LC3 antibody and then with Alexa Fluor 568-and 488conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) during incubation with the secondary antibody. To examine virus replication, 50 µl of culture medium was aspired and replaced with 50 µl fresh culture medium containing virus strains carrying the luciferase gene at an MOI of 0.2. Infections were conducted for 6 h for EMCV, VaV, HSV-1, SFV, and MHV and 16 h for IAV before lysing the cells and enzymatically measuring luciferase expression. The virus infection and LC3 puncta assessment screens were run four times, whereas the ones for p62 puncta quantification were run three times.