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

To validate our screening approach and identify unconventional functions of ATG proteins, we focused on ATG13, because its depletion markedly enhanced EMCV replication, whereas knockdown of other ULK complex components (such as ULK1, ULK2, or ATG101) did not. A detailed analysis confirmed that not only ATG13 but also FIP200 decrease EMCV replication independently of their function in autophagy (Fig. 10 C) . Although FIP200 was not among the significant hits in the screen, EMCV replication was strongly up-regulated upon its depletion. In contrast to what was found for UVR AG during IAV infection (Pirooz et al., 2014) , ATG13 and FIP200, possibly in complex, control not virus cell entry but rather virus replication. Interestingly, the unconventional function of ATG13 and FIP200 is not restricted to EMCV but also controls more members of the picornavirus family, i.e., CVB3, CVA21, and EV71. It has been shown that picornaviruses induce autophagy upon infection, and this is beneficial for both enterovirus 71 (Huang et al., 2009; Lee et al., 2014; Fu et al., 2015) and CV (Wong et al., 2008; Kemball et al., 2010; Alirezaei et al., 2012 Alirezaei et al., , 2015 Tabor-Godwin et al., 2012) , whereas it is still a matter of debate whether autophagy has also a proviral role for EMCV Chakrabarti et al., 2012) . Based on the conclusions of these studies, one would expect that depletion of any ATG protein that blocks autophagy should inhibit the picornavirus life cycle as well. Although we blocked autophagy by ATG13 and FIP200 depletion, we did not observe impairment in picornaviral life cycles. On the contrary, viral replication and subsequent viral particle production were strongly enhanced in the absence of these two proteins, even in cells where ATG7 was simultaneously depleted. It has previously been indicated that autophagy is essential for picornavirus infections mostly through the use of pharmacological autophagy inhibitors but also through BEC LIN1, VPS34, LC3, and ATG7 knockdown (Wong et al., 2008; Zhang et al., 2011; Delorme-Axford et al., 2014) . Pharmacological inhibitors or BEC LIN1 and VPS34 depletion affects specific endosomal functions, and this could also have an impact on picornaviral infections, depending on the cell type. We confirmed, to a certain extent, the inhibitory effect of ATG7 knockdown (Figs. 4, 5, 7, and 8) , which would suggest that this protein, like ATG13 and FIP200, influences picornavirus infection through an unconventional role in a different pathway. Alternatively, the fact that picornaviruses can exploit more than one pathway for replication, including autophagy (Alirezaei et al., 2015) , could explain the relevance of ATG proteins in specific cell types, as was shown for pancreatic acinar cells, in which ATG5 deletion reduces CV replication as well as pathogenesis (Alirezaei et al., 2012) .