AUTOPHAGY 2016, VOL. 12, NO. 12, 2502–2503 http://dx.doi.org/10.1080/15548627.2016.1236878
AUTOPHAGIC PUNCTUM
ATG proteins: Are we always looking at autophagy? Mario Mauthea and Fulvio Reggiorib a Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; bDepartment of Cell Biology, University Medical Center Utrecht, Center for Molecular Medicine, Utrecht, The Netherlands
ABSTRACT
ARTICLE HISTORY
Autophagy is an intracellular degradation pathway that is regulated by the autophagy-related (ATG) proteins. For a long time it has been thought that ATG proteins were exclusively required for autophagy, but recent experimental evidence has revealed that these proteins are part of other cellular pathways, individually or as a functional group. To estimate the extent of these so-called unconventional functions of the ATG proteins, we decided to perform an unbiased siRNA screen targeting the entire ATG proteome and used viral replication as the readout. Our results have uncovered that a surprisingly high number of ATG proteins (36%) have a positive or negative role in promoting virus replication outside their classical role in autophagy. With the increasing knowledge about ATG protein unconventional functions and our investigation results, the interpretations about the possible involvement of autophagy in cellular or organismal functions that solely rely on the depletion of a single ATG protein, should be considered cautiously.
Received 6 September 2016 Revised 8 September 2016 Accepted 12 September 2016
Because many of the currently known unconventional functions of the ATG proteins have been discovered by studying host-pathogen interactions, the central strategy of our study has been to explore the relevance of the ATG proteins in the replication of 6 different viruses: Herpes simplex virus-1 (HSV-1), vaccinia virus (VaV), Semliki forest virus (SFV), mouse hepatitis virus (MHV), encephalomyocarditis virus (EMCV) and influenza A virus (IAV). The idea behind this choice was also that virus replication often relies on the host cell pathways and therefore alterations in the host cells could alter virus replication, which in turn can pinpoint unconventional functions of the ATG proteins that are not directly related to the virus life cycle. To have a uniform read-out system, we took advantage of luciferase-expressing viruses and measured luciferase activity to monitor viral replication. This approach allowed us to assess the early steps of virus infection, i.e. cell entry and replication. Since several ATG genes have evolved into gene families in mammalian cells (e.g. ATG2 or ATG4) and some of them have redundant functions in autophagy, i.e., multiple isoforms or all of them have to be depleted to block this pathway, we also simultaneously knocked them down by combining siRNAs. Finally, we performed the screens in 2 different cell lines to exclude tissue-specific effects, which probably account for conflicting results, for example concerning the relevance of autophagy in EMCV replication. We discovered that depletion of 36% of the analyzed ATG proteins significantly influence either positively or negatively the replication of at least one of the examined virus. This number is probably an underestimation because it could be much higher if the same screen is extended to other pathogens or to
KEYWORDS
ATG proteins; ATG13; autophagy; FIP200; picornavirus; replication; unconventional function; virus
different types of cellular insults. Interestingly, using this approach, we did not find that autophagy is essential to sustain or to control the replication of any of the analyzed viruses. However, we found that autophagy has a cell line-specific role in regulating VaV and SFV infection. For these viruses, autophagy has a beneficial role in cell entry and/or replication in U2OS cells, whereas it negatively affects virus propagation in HeLa cells. Overall, we found that in a large number of the observed infections/cases, virus replication is regulated by the unique functions of ATG proteins. To validate our screen, we decided to investigate in more detail the relevance of ATG13 and RB1CC1/FIP200 in EMCV replication. These 2 proteins are part of the ULK complex, which is required for the initiation of autophagy. This complex consists of ULK1 or ULK2, ATG101, RB1CC1 and ATG13. In our screen, we only found that ATG13 and to a lesser extent also RB1CC1 depletion increase EMCV replication, whereas the knockdown of the other ULK complex components or other ATG genes such as ATG7, have no effect. Since EMCV belongs to the picornavirus family, we wondered whether other members of this virus family behave similarly upon depletion of the ULK complex components. Therefore, we also examined coxsackievirus B3 (CV), coxsackievirus A21, enterovirus 71 and a mutant EMCV called EMCVZn; interestingly, we could confirm that only depletion of ATG13 and RB1CC1 increases the production of viral proteins, the number of infected cells and the virus titers. Conversely, overexpression of ATG13 and RB1CC1 pronouncedly reduces EMCV and CV infection. Importantly, simultaneous depletion
CONTACT Fulvio Reggiori [email protected] Department of Cell Biology, University of Groningen, University Medical Center Groningen, Antonious Deusinglaan 1, Bldg. 3215, 7th floor, room 721, 9713 AV Groningen, The Netherlands Punctum to: Mauthe M, Langereis M, Jung J, Zhou X, Jones A, Omta W, Tooze SA, Stork B, Paludan SR, Ahola T, et al. An siRNA screen for ATG depletion reveals the extent of unconventional functions of the autophagy proteome in virus replication. J Cell Biol 2016; 214: 619-35; http://dx.doi.org/10.1083/jcb.201602046 © 2016 Taylor & Francis
AUTOPHAGY
2503
Figure 1. RNA sequencing points to potential unconventional functions of ATG13, RB1CC1 and ATG7 under basal growth conditions and during a CV infection. The Venn diagrams show the number of differentially expressed genes upon ATG13, ATG7 or RB1CC1 depletion compared to control cells during CV infection (A) or under basal conditions (B). Overlaps in the Venn diagram circles indicate common genes where expression is significantly changed compared to control cells.
of ATG13 and RB1CC1 has no additive effect on viral replication indicating that both proteins most probably act in the same pathway to suppress viral replication. To determine whether suppression of picornavirus infection by ATG13 and RB1CC1 is due to an inhibition of either viral cell entry or virus replication, we performed a series of experiments using specific viral inhibitors and direct transfection of viral RNA into the host cells. These analyses have allowed us to show that ATG13, and by extension RB1CC1, are required to inhibit viral replication rather than cell entry. A possible mechanistic connection between ATG13RB1CC1 and virus replication could have been that the proteins directly interfere with the viral replication complexes, but colocalization experiments and mass spectrometry rule out this scenario. To get insights into the ATG13- and RB1CC1dependent cellular pathway(s) that could be involved in controlling picornavirus infection, we performed a RNA sequencing experiment and compared the gene expression upon depletion of ATG13, RB1CC1 or ATG7 with control cells. We found a set of genes that are differentially regulated only upon ATG13 and RB1CC1 depletion but not upon ATG7 knockdown during CV infection (Fig. 1A, purple area), revealing that these 2 proteins could have multiple unconventional functions. Interestingly, the RNA sequencing analysis also revealed that there is a shared set of genes that is specifically expressed differentially upon depletion of ATG13 or RB1CC1 under basal growth conditions (Fig. 1B, purple area), which shows that the shared unconventional functions ATG13 and RB1CC1 are not solely virus infection-related. RNA sequencing
also revealed that ATG13, RB1CC1 and ATG7 have potential unconventional functions that are not shared with the 2 other ATG proteins tested here (Fig. 1A and B, red, blue and yellow circles). This observation further underlines the extent of the unconventional functions of ATG proteins. In summary, the in-depth study of ATG13 and RB1CC1 in controlling picornaviral infection validated our screen as a platform to identify novel unconventional functions of ATG proteins and shed more insights into the specific functions of 3 ATG proteins (i.e. ATG13, RB1CC1 and ATG7) during viral infections but also under basal growth conditions. Because the unconventional functions of the ATG proteins become more and more evident as highlighted by our study, we always need to ask ourselves: Are we really looking at autophagy?
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We thank Muriel Mari, Idil Orhon and Ruben G omez-Sanchez for the critical reading of the manuscript.
Funding F.R. is supported by ALW Open Program (822.02.014), DFG-NWO cooperation (DN82-303), SNF Sinergia (CRSII3_154421) and ZonMW VICI (016.130.606) grants.
AUTOPHAGIC PUNCTUM
ATG proteins: Are we always looking at autophagy? Mario Mauthea and Fulvio Reggiorib a Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; bDepartment of Cell Biology, University Medical Center Utrecht, Center for Molecular Medicine, Utrecht, The Netherlands
ABSTRACT
ARTICLE HISTORY
Autophagy is an intracellular degradation pathway that is regulated by the autophagy-related (ATG) proteins. For a long time it has been thought that ATG proteins were exclusively required for autophagy, but recent experimental evidence has revealed that these proteins are part of other cellular pathways, individually or as a functional group. To estimate the extent of these so-called unconventional functions of the ATG proteins, we decided to perform an unbiased siRNA screen targeting the entire ATG proteome and used viral replication as the readout. Our results have uncovered that a surprisingly high number of ATG proteins (36%) have a positive or negative role in promoting virus replication outside their classical role in autophagy. With the increasing knowledge about ATG protein unconventional functions and our investigation results, the interpretations about the possible involvement of autophagy in cellular or organismal functions that solely rely on the depletion of a single ATG protein, should be considered cautiously.
Received 6 September 2016 Revised 8 September 2016 Accepted 12 September 2016
Because many of the currently known unconventional functions of the ATG proteins have been discovered by studying host-pathogen interactions, the central strategy of our study has been to explore the relevance of the ATG proteins in the replication of 6 different viruses: Herpes simplex virus-1 (HSV-1), vaccinia virus (VaV), Semliki forest virus (SFV), mouse hepatitis virus (MHV), encephalomyocarditis virus (EMCV) and influenza A virus (IAV). The idea behind this choice was also that virus replication often relies on the host cell pathways and therefore alterations in the host cells could alter virus replication, which in turn can pinpoint unconventional functions of the ATG proteins that are not directly related to the virus life cycle. To have a uniform read-out system, we took advantage of luciferase-expressing viruses and measured luciferase activity to monitor viral replication. This approach allowed us to assess the early steps of virus infection, i.e. cell entry and replication. Since several ATG genes have evolved into gene families in mammalian cells (e.g. ATG2 or ATG4) and some of them have redundant functions in autophagy, i.e., multiple isoforms or all of them have to be depleted to block this pathway, we also simultaneously knocked them down by combining siRNAs. Finally, we performed the screens in 2 different cell lines to exclude tissue-specific effects, which probably account for conflicting results, for example concerning the relevance of autophagy in EMCV replication. We discovered that depletion of 36% of the analyzed ATG proteins significantly influence either positively or negatively the replication of at least one of the examined virus. This number is probably an underestimation because it could be much higher if the same screen is extended to other pathogens or to
KEYWORDS
ATG proteins; ATG13; autophagy; FIP200; picornavirus; replication; unconventional function; virus
different types of cellular insults. Interestingly, using this approach, we did not find that autophagy is essential to sustain or to control the replication of any of the analyzed viruses. However, we found that autophagy has a cell line-specific role in regulating VaV and SFV infection. For these viruses, autophagy has a beneficial role in cell entry and/or replication in U2OS cells, whereas it negatively affects virus propagation in HeLa cells. Overall, we found that in a large number of the observed infections/cases, virus replication is regulated by the unique functions of ATG proteins. To validate our screen, we decided to investigate in more detail the relevance of ATG13 and RB1CC1/FIP200 in EMCV replication. These 2 proteins are part of the ULK complex, which is required for the initiation of autophagy. This complex consists of ULK1 or ULK2, ATG101, RB1CC1 and ATG13. In our screen, we only found that ATG13 and to a lesser extent also RB1CC1 depletion increase EMCV replication, whereas the knockdown of the other ULK complex components or other ATG genes such as ATG7, have no effect. Since EMCV belongs to the picornavirus family, we wondered whether other members of this virus family behave similarly upon depletion of the ULK complex components. Therefore, we also examined coxsackievirus B3 (CV), coxsackievirus A21, enterovirus 71 and a mutant EMCV called EMCVZn; interestingly, we could confirm that only depletion of ATG13 and RB1CC1 increases the production of viral proteins, the number of infected cells and the virus titers. Conversely, overexpression of ATG13 and RB1CC1 pronouncedly reduces EMCV and CV infection. Importantly, simultaneous depletion
CONTACT Fulvio Reggiori [email protected] Department of Cell Biology, University of Groningen, University Medical Center Groningen, Antonious Deusinglaan 1, Bldg. 3215, 7th floor, room 721, 9713 AV Groningen, The Netherlands Punctum to: Mauthe M, Langereis M, Jung J, Zhou X, Jones A, Omta W, Tooze SA, Stork B, Paludan SR, Ahola T, et al. An siRNA screen for ATG depletion reveals the extent of unconventional functions of the autophagy proteome in virus replication. J Cell Biol 2016; 214: 619-35; http://dx.doi.org/10.1083/jcb.201602046 © 2016 Taylor & Francis
AUTOPHAGY
2503
Figure 1. RNA sequencing points to potential unconventional functions of ATG13, RB1CC1 and ATG7 under basal growth conditions and during a CV infection. The Venn diagrams show the number of differentially expressed genes upon ATG13, ATG7 or RB1CC1 depletion compared to control cells during CV infection (A) or under basal conditions (B). Overlaps in the Venn diagram circles indicate common genes where expression is significantly changed compared to control cells.
of ATG13 and RB1CC1 has no additive effect on viral replication indicating that both proteins most probably act in the same pathway to suppress viral replication. To determine whether suppression of picornavirus infection by ATG13 and RB1CC1 is due to an inhibition of either viral cell entry or virus replication, we performed a series of experiments using specific viral inhibitors and direct transfection of viral RNA into the host cells. These analyses have allowed us to show that ATG13, and by extension RB1CC1, are required to inhibit viral replication rather than cell entry. A possible mechanistic connection between ATG13RB1CC1 and virus replication could have been that the proteins directly interfere with the viral replication complexes, but colocalization experiments and mass spectrometry rule out this scenario. To get insights into the ATG13- and RB1CC1dependent cellular pathway(s) that could be involved in controlling picornavirus infection, we performed a RNA sequencing experiment and compared the gene expression upon depletion of ATG13, RB1CC1 or ATG7 with control cells. We found a set of genes that are differentially regulated only upon ATG13 and RB1CC1 depletion but not upon ATG7 knockdown during CV infection (Fig. 1A, purple area), revealing that these 2 proteins could have multiple unconventional functions. Interestingly, the RNA sequencing analysis also revealed that there is a shared set of genes that is specifically expressed differentially upon depletion of ATG13 or RB1CC1 under basal growth conditions (Fig. 1B, purple area), which shows that the shared unconventional functions ATG13 and RB1CC1 are not solely virus infection-related. RNA sequencing
also revealed that ATG13, RB1CC1 and ATG7 have potential unconventional functions that are not shared with the 2 other ATG proteins tested here (Fig. 1A and B, red, blue and yellow circles). This observation further underlines the extent of the unconventional functions of ATG proteins. In summary, the in-depth study of ATG13 and RB1CC1 in controlling picornaviral infection validated our screen as a platform to identify novel unconventional functions of ATG proteins and shed more insights into the specific functions of 3 ATG proteins (i.e. ATG13, RB1CC1 and ATG7) during viral infections but also under basal growth conditions. Because the unconventional functions of the ATG proteins become more and more evident as highlighted by our study, we always need to ask ourselves: Are we really looking at autophagy?
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We thank Muriel Mari, Idil Orhon and Ruben G omez-Sanchez for the critical reading of the manuscript.
Funding F.R. is supported by ALW Open Program (822.02.014), DFG-NWO cooperation (DN82-303), SNF Sinergia (CRSII3_154421) and ZonMW VICI (016.130.606) grants.
