Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate.
ABSTRACT: Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.
Project description:Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.
Project description:Hepatitis C virus (HCV) infection has been shown to induce autophagy but the mechanisms underpinning this process remain to be elucidated. Induction of autophagy requires the class III phosphatidylinositol 3-kinase, Vps34, which produces phosphatidylinositol 3-phosphate (PI3P) within the endoplasmic reticulum (ER) membrane. This recruits proteins with PI3P binding domains such as the double-FYVE-containing protein 1 (DFCP1). DFCP1 generates cup-shaped protrusions from the ER membrane, termed omegasomes, which provide a platform for the production of autophagosomes. Here we present data demonstrating that both Vps34 and DFCP1 are required for HCV genome replication, in the context of both a subgenomic replicon and virus infection, but did not affect virus entry or initial translation. Using live cell fluorescence microscopy we demonstrated that early during HCV infection the nascent viral genome replication complexes (identified by using non-structural protein NS5A as a marker) transiently colocalize with DFCP1-positive punctae (omegasomes), before the two structures move apart from each other. This observation is reminiscent of the transient association of LC3 and DFCP1 during omegasome formation, and therefore we propose that omegasomes are utilized by HCV to generate the double-membrane vesicles which are the hallmark of HCV replication complexes.
Project description:Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as 'omegasomes,' associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.
Project description:Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.
Project description:BACKGROUND:Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. METHODS:LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. RESULTS:Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. CONCLUSIONS:The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process.
Project description:Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.
Project description:Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.
Project description:Cells govern their homeostasis through autophagy by sequestering substrates, ranging from proteins to aggregates and organelles, into autophagosomes for lysosomal degradation. In these processes cells need to coordinate between substrate remodeling and autophagosome formation for efficient engulfment. We found that in Parkin-mediated mitophagy, mitochondria to be turned over first become grape-like mitoaggregates, followed by their disassembly into smaller pieces via the actinomyosin system. At the disassembly step, we observed spatially-associated, synchronous formation of circular F-actin and BATS-labeled autophagy initiation sites near mitochondria, suggesting coordination between substrate downsizing and autophagosome formation during mitophagy. Interestingly, PtdIns(4,5)P2, instead of PtdIns(3)P, regulates this mitophagy-associated formation of circular F-actin and BATS-sites. Selective depletion of PtdIns(4,5)P2 near omegasomes, the endoplasmic reticulum (ER) subdomains involved in autophagosome formation, impaired mitoaggregate disassembly. Our findings demonstrate the presence of a pool of PtdIns(4,5)P2 adjacent to omegasomes, and that they coordinate mitoaggregate disassembly with autophagy initiation during Parkin-mediated mitophagy.
Project description:Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitin-related proteins in this process.
Project description:Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.