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 following primary antibodies were used: rabbit anti-LC3 (Novus Biologicals), mouse anti-Flag (Sigma-Aldrich), mouse anti-tubulin (Sigma-Aldrich), rabbit anti-ATG13 (Sigma-Aldrich), rabbit anti-ULK1 (Santa Cruz Biotechnology, Inc.), rabbit anti-FIP200 (Bethyl Laboratories, Inc.), guinea pig anti-p62 (Progen), rabbit anti-phospho-ATG13 (S318; Rockland), mouse antienterovirus (Dako), rabbit anti-CV2bc (a gift from L. Whitton, Scripps Research Institute, La Jolla, CA), rabbit anti-Capsid (EMCV; a gift of A. Palmenberg, University of Wisconsin, Madison, WI), mouse anti-dsRNA (English & Scientific Consulting Bt.), and rabbit anti-ATG7 (Cell Signaling Technology). Goat antimouse and chicken anti-rabbit Alexa Fluor 488-conjugated antibodies, goat anti-mouse and donkey anti-rabbit Alexa Fluor 568-conjugated antibodies, goat anti-mouse antibodies, goat anti-rabbit Alexa Fluor 680-conjugated antibodies, and goat anti-mouse and goat anti-rabbit Alexa Fluor 800-conjugated antibodies were used for the visualization of the primary antibodies. All secondary antibodies and DAPI were from Invitrogen and Thermo Fisher Scientific. Puromycin, guanidine hydrochloride, and cycloheximide were purchased from Sigma-Aldrich, blasticidin was purchased from Invivogen, and doxycycline was purchased from Takara Bio, Inc. GPC-N114 is a nonnucleoside inhibitor that targets the enterovirus RNA-dependent RNA polymerase and exerts broad-spectrum antiviral activity against all enteroviruses (van der Linden et al., 2015) .