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

The customized ON-TAR GETplus SMA RTpool human siRNA library (Table S1 ) and the deconvoluted ON-TAR GETplus SMA RTpools were obtained from GE Healthcare. The screens were run in 96-well plates, and the rows of wells on the limits of the plates were not used to avoid change in the readout caused by a differential in cell growth. 2 pmol siRNA per well was used for single gene knockdown, whereas for multiple gene depletions, total amounts of 3 pmol (double knockdowns), 4.5 pmol (triple knockdowns), or 6 pmol (quadruple knockdowns) were used. Reverse transfection was conducted according to the manufacturer's protocol using 0.1 µl RNAiMax and 3,000 cells (U2OS, HeLa-mCC1 or HEK-mCC1 cells) per well in a final volume of 100 µl DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. These conditions, which were set with pilot experiments, allowed optimal knockdown of the target genes without causing cell death (unpublished data). The customized siRNA library also included probes knocking down genes known to be relevant for the replication of one or more viruses. Runs of the screen where these positive controls gave no change in luciferase activity were discarded and repeated. The genes used for this purpose were GBF1 and ARF1 for MHV (Verheije et al., 2008) , PI4KA for EMCV (Dorobantu et al., 2015) , and ATP6V1A for IAV and SFV (Ochiai et al., 1995; Glomb-Reinmund and Kielian, 1998) .