Nucleocytoplasmic Distribution and Dynamics of the Autophagosome Marker EGFP-LC3

The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol. Microtubule-associated protein light chain 3 (LC3) was the first protein shown to specifically label autophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy. Although LC3 is currently thought to function primarily in the cytosol, the site of autophagosome formation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However, the nuclear pool of EGFP-LC3 has not been specifically studied in previous reports, and mechanisms by which LC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonly studied cell lines, COS-7 and HeLa. Although LC3 contains a putative nuclear export signal (NES), inhibition of active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore, FRAP analysis indicates that EGFP-LC3 undergoes limited passive nucleo-cytoplasmic transport under steady state conditions, and that the diffusional mobility of EGFP-LC3 was substantially slower in the nucleus and cytoplasm than predicted for a freely diffusing monomer. Induction of autophagy led to a visible decrease in levels of soluble EGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-cytoplasmic ratio or diffusional mobility of the remaining soluble pools of EGFP-LC3. We conclude that the enrichment of soluble EGFP-LC3 in the nucleus is maintained independently of active nuclear export or induction of autophagy. Instead, incorporation of soluble EGFP-LC3 into large macromolecular complexes within both the cytoplasm and nucleus may prevent its rapid equilibrium between the two compartments.     

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

The Dynamics of Autophagy Visualised in Live Cells: from Autophagosome Formation to Fusion with Endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5+/+ and atg5-/- MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP1 or LAMP2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP1/LAMP2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP1/LAMP2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualised in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa) - traced by immunoblotting and verified by [3H]ethanolamine labelling - revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counterfluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

Autophagosome supports coxsackievirus B3 replication in host cells

Recent studies suggest a possible takeover of host antimicrobial autophagy machinery by positive-stranded RNA viruses to facilitate their own replication. In the present study, we investigated the role of autophagy in coxsackievirus replication. Coxsackievirus B3 (CVB3), a picornavirus associated with viral myocarditis, causes pronounced intracellular membrane reorganization after infection. We demonstrate that CVB3 infection induces an increased number of double-membrane vesicles, accompanied by an increase of the LC3-II/LC3-I ratio and an accumulation of punctate GFP-LC3-expressing cells, two hallmarks of cellular autophagosome formation. However, protein expression analysis of p62, a marker for autophagy-mediated protein degradation, showed no apparent changes after CVB3 infection. These results suggest that CVB3 infection triggers autophagosome formation without promoting protein degradation by the lysosome. We further examined the role of the autophagosome in CVB3 replication. We demonstrated that inhibition of autophagosome formation by 3-methyladenine or small interfering RNAs targeting the genes critical for autophagosome formation (ATG7, Beclin-1, and VPS34 genes) significantly reduced viral replication. Conversely, induction of autophagy by rapamycin or nutrient deprivation resulted in increased viral replication. Finally, we examined the role of autophagosome-lysosome fusion in viral replication. We showed that blockage of the fusion by gene silencing of the lysosomal protein LAMP2 significantly promoted viral replication. Taken together, our results suggest that the host's autophagy machinery is activated during CVB3 infection to enhance the efficiency of viral replication.     

Quantitation of "autophagic flux" in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3II protein may render possible misinterpretations since LC3II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3II, a technique aptly named the "autophagometer". In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

Quantitation of “autophagic flux” in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure “autophagic flux” in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the “autophagometer.” In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. the addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a “colchicine block.” Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an “in vivo autophagometer” study using colchicine in skeletal muscle.Key words: autophagy, rapamycin, skeletal muscle     

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.     

Monitoring autophagy in wheat living cells by visualization of fluorescence protein-tagged ATG8

Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.     

Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase

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.     

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP-LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome: accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of: cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as: rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G0/G1, S and G2/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

Autophagosome biogenesis in plants: Roles of SH3P2

The autophagy core machinery is essentially conserved in eukaryotic cells for autophagy regulation. However, the underlying mechanisms for autophagosome formation in plant cells remain elusive. We have recently demonstrated that SH3 domain-containing protein 2 (SH3P2), a BAR (Bin-Amphiphysin-Rvs) domain protein, functions as a novel regulator for autophagosome biogenesis in Arabidopsis thaliana. Using SH3P2 and its GFP fusion as probes, we have characterized the dynamics and structures of autophagosome formation in plant cells. The phagophore assembly site, marked by SH3P2, is identified as having a close connection with the ER. SH3P2 also binds to phosphatidylinositol 3-phosphate (PtdIns3P) and functions downstream of the phosphatidylinositol 3-kinase (PtdIns3K) complex. Thus, SH3P2 serves as a novel membrane-associated protein in regulating autophagosome formation in Arabidopsis thaliana.     

Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.