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

U2OS cells were transfected with control siRNA or ATG13 siRNA for 48 h and infected with CV for 7 h at an MOI of 1. After a 2-h fixation at room temperature in 2% PFA and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4, cells were embedded in epon resin as previously described (Verheije et al., 2008) . Subsequently, 70-nm sections were obtained using an UC7 Leica Ultra-microtome and stained with uranyl acetate and lead citrate as previously described (Verheije et al., 2008) . Cell sections were analyzed using an 80-kV transmission electron microscope (CM100; FEI). Two independent grids were used to perform the counting analysis of 125 cells per condition in total. Three different categories of membranous rearrangements were identified in CV-infected cells and defined as follows: category 1, closely packed, elongated, regular shaped single-, double-, or multimembrane vesicles; category 2, closely packed, irregular shaped, single-membrane vesicles with a light content; and category 3, irregular, collapsed membrane clusters.