Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis

Autophagy is an evolutionarily conserved 'self-eating' process. Although the genes essential for autophagy (named Atg) have been identified in yeast, the molecular mechanism of how Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through ultraviolet irradiation resistance-associated gene (UVRAG) and functions as a positive mediator of the class III PI(3) kinase (PI(3)KC3). In response to nutrient deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as microtubule-associated protein light chain 3 (LC3). Furthermore, loss of Bif-1 suppresses autophagosome formation. Although the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI(3)KC3 and induce autophagosome formation. We also observed that Bif-1 ablation prolongs cell survival under starvation conditions. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumours in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumour suppressor.     

BARgaining membranes for autophagosome formation: Regulation of autophagy and tumorigenesis by Bif-1/Endophilin B1

Autophagy is an intracellular system for the bulk degradation of cytoplasmic components enclosed within double-membrane structures known as autophagosomes. To date, many autophagy-related (Atg) genes have been identified by independent genetic screens for autophagy-defective mutants in yeast; however, the molecular machinery required for the biogenesis of autophagosomes in mammalian systems has yet to be determined.(1,2) Recently, we have reported that Bif-1 interacts with Beclin 1 through UVRAG and promotes the activation of class III phosphatidylinositol 3-kinase (PI3KC3) and the formation of autophagosomes.(3) Moreover, we have found that loss of Bif-1 promotes starvation-induced caspase activation, but prolongs cell survival by suppressing autophagydependent cell death, and enhances spontaneous tumorigenesis in mice. Bif-1 is a member of the endophilin family, which possesses membrane binding and liposome tubulation activities.(4) During nutrient deprivation, Bif-1 accumulates in punctate foci where it co-localizes with LC3, Atg5 and Atg9. Time-lapse microscopy analyses reveal that Bif-1-positive small vesicles expand by recruiting and fusing with Atg9-positive small membranes to form autophagosomes. Taken together, our findings highlight Bif-1 as a potential regulator of autophagosome biogenesis and as a tumor suppressor.     

BARgaining membranes for autophagosome formation: Regulation of autophagy and tumorigenesis by Bif-1/Endophilin B1

Autophagy is an intracellular system for the bulk degradation of cytoplasmic components enclosed within double-membrane structures known as autophagosomes. To date, many autophagy-related (Atg) genes have been identified by independent genetic screens for autophagy-defective mutants in yeast; however, the molecular machinery required for the biogenesis of autophagosomes in mammalian systems has yet to be determined.^1,2 Recently, we have reported that Bif-1 interacts with Beclin 1 through UVRAG and promotes the activation of class III phosphatidylinositol 3-kinase (PI3KC3) and the formation of autophagosomes.³ Moreover, we have found that loss of Bif-1 promotes starvation-induced caspase activation, but prolongs cell survival by suppressing autophagy-dependent cell death, and enhances spontaneous tumorigenesis in mice. Bif-1 is a member of the endophilin family, which possesses membrane binding and liposome tubulation activities.⁴ During nutrient deprivation, Bif-1 accumulates in punctate foci where it co-localizes with LC3, Atg5 and Atg9. Time-lapse microscopy analyses reveal that Bif-1-positive small vesicles expand by recruiting and fusing with Atg9-positive small membranes to form autophagosomes. Taken together, our findings highlight Bif-1 as a potential regulator of autophagosome biogenesis and as a tumor suppressor.

Addendum to: Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, Liang C, Jung JU, Cheng JQ, Mulé JJ, Pledger WJ, Wang H.-G. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biol 2007; 9:1142-51.     

Atg9 vesicles are an important membrane source during early steps of autophagosome formation

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.     

Foot-and-Mouth Disease Virus Induces Autophagosomes during Cell Entry via a Class III Phosphatidylinositol 3-Kinase-Independent Pathway

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

Regulation of Beclin 1 in autophagy

Class III phosphatidylinositol 3-kinase (PI3KC3) plays a pleiotropic role in autophagy and protein sorting pathways. The human core complex of PI3KC3 consists of three major components including PI3KC3/hVps34, p150 and Beclin 1. How the specificity of PI3KC3 complex is derived towards autophagy is not clear. Utilizing a sequential affinity purification coupled with Mass spectrometry approach, we have successfully purified a human Beclin 1 complex and cloned a novel protein we called Barkor (Beclin 1-associated autophagy-related key regulator). The function of Barkor in autophagy has been manifested in several assays, including stress-induced LC3 lipidation, autophagosome formation, and Salmonella typhimurium amplification. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and orients Beclin1 to autophagosomes. Barkor shares considerable sequence homology with Atg14 in yeast, representing an evolutionary conserved autophagy specific regulatory step in early autophagosome formation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Atg9 trafficking in Mammalian cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins(1) localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes.(2) Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway.(3) Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.     

Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3‐kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3‐phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI‐1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

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.     

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

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.     

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34(-/-) animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Regulation of autophagosome formation by Rho kinase

Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that functions at a constitutive level in cells, which may become further activated by stressors such as nutrient starvation or protein aggregation. Autophagy has multiple beneficial roles for maintaining normal cellular homeostasis and these roles are related to the implications of autophagy in disease mechanisms including neurodegeneration and cancer. We previously searched for novel autophagy regulators and identified Rho-kinase 1 (ROCK1) as a candidate. Here, we show that activated ROCK1 inhibits autophagy in human embryonic kidney 293 cells. Conversely, ROCK inhibitory compounds enhanced the autophagy response to amino acid starvation or rapamycin treatment. Inhibition of ROCK during the starvation period led to a more rapid response with the production of larger early autophagosomes that matured into enlarged late degradative autolysosomes. Despite the production of enlarged LC3-positive early autophagosomes, membrane precursors containing WD-repeat protein interacting with phosphoinositides 1 (WIPI1) and mammalian Atg9 were not affected by ROCK inhibition, suggesting that phagophore elongation had been unusually extended. However, the enlarged autophagosomes were enriched in ULK1 which was essential to allow progression of autophagy flux. Our results demonstrate a novel role for ROCK in the control of autophagosome size and degradative capacity.     

Replication of hepatitis C virus RNA on autophagosomal membranes

Previous studies indicated that hepatitis C virus (HCV) perturbs the autophagic pathway to induce the accumulation of autophagosomes in cells. To understand the role of autophagosomes in the HCV life cycle, we established a stable Huh7 hepatoma cell line that contained an HCV subgenomic RNA replicon and also expressed a GFP-LC3 fusion protein. The GFP-LC3 protein is localized to autophagosomes during autophagy and served as a convenient marker for autophagosomes. Our results indicate that the silencing of the expression of LC3 or Atg7, two protein factors critical for the formation of autophagosomes, suppresses the replication of HCV RNA. Confocal microscopy studies revealed the localization of HCV NS5A and NS5B proteins, which are two important components of the HCV RNA replication complex, and nascent HCV RNA to autophagosomes. The association of the HCV RNA replication complex with the autophagosomal membranes was further confirmed by co-immunoprecipitation and immunoelectron microscopy studies. Interestingly, inhibition of Class III PI3K activity had no effect on the autophagosomes induced by HCV. These results indicate that HCV induces autophagosomes via a Class III PI3K-independent pathway and uses autophagosomal membranes as sites for its RNA replication.     

In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.