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 notion that ATG13 and FIP200 have important autophagy-independent functions is underlined by the fact that although most autophagy-deficient mice, including ULK1 −/− ULK2 −/− animals (Joo et al., 2016) , die shortly after birth, Figure 7 . ATG13 depletion enhances EMCV and CV replication, but not virus cell entry. (A) U2OS cells depleted or not of ATG13 using siRNA for 48 h were transfected with Renilla luciferase EMCV RNA for 7 h. GPC-N114 was added or not 1 h after virus inoculation before measuring luciferase expression (n = 3). (B) Cells prepared as in A were infected with luciferase-expressing EMCV for 7 h. GPC-N114 was added or not 1 h after virus inoculation before assessing luciferase expression (n = 5). (C) Cells prepared as in A were transfected with Renilla luciferase CV RNA for 7 h. Guanidine-HCL (Gn-HCl) was added or not 1 h posttransfection before measuring luciferase expression (n = 4). (D) Cells prepared as in A were infected with luciferase expressing CV for 7 h. Gn-HCl was added or not 1 h after virus inoculation before assessing luciferase expression (n = 4). (E) Cells were transfected with the cap RNA Renilla-luciferase (Rluc) transcript for 7 h and, when indicated, cycloheximide (CHX) was added 1 h after transfection before determining luciferase expression (n = 3). (F) Cells were transfected with the IRES-luciferase RNA transcript for 7 h and when indicated, CHX was added 1 h after transfection before measuring luciferase expression. (n = 3). All data are presented relative to the control (folds). Error bars represent SEM. *, P < 0.05; **, P < 0.01; n.s., not significant. siCtr, scramble siRNA. FIP200 −/− and ATG13 −/− mice die during embryonic development (Joo et al., 2011; Hieke et al., 2015; Kaizuka and Mizushima, 2015) . Our RNA-sequencing analysis supports this observation, as a subset of the genes that we found to be differentially expressed in absence of ATG13 and FIP200 are involved in transcriptional regulation and development (11 out of 225 in uninfected cells and 8 out of 68 in infected cells). These results are in line with the requirement of ATG13 and FIP200 in embryonic development in an autophagy-independent manner (Chen et al., 2016) . A possible link how ATG13 and FIP200 regulate this developmental process could be HSBP1, which we identified as a new interactor of ATG13 and FIP200 (Fig. S4) , and which is also required for embryonic development (Eroglu et al., 2014) .