Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages.

Macrophages can undergo M1-like proinflammatory polarization with low oxidative phosphorylation (OXPHOS) and high glycolytic activities or M2-like anti-inflammatory polarization with the opposite metabolic activities. Here we show that M1-like macrophages induced by hepatitis B virus (HBV) display high OXPHOS and low glycolytic activities. This atypical metabolism induced by HBV attenuates the antiviral response of M1-like macrophages and is mediated by HBV e antigen (HBeAg), which induces death receptor 5 (DR5) via toll-like receptor 4 (TLR4) to induce death-associated protein 3 (DAP3). DAP3 then induces the expression of mitochondrial genes to promote OXPHOS. HBeAg also enhances the expression of glutaminases and increases the level of glutamate, which is converted to α-ketoglutarate, an important metabolic intermediate of the tricarboxylic acid cycle, to promote OXPHOS. The induction of DR5 by HBeAg leads to apoptosis of M1-like and M2-like macrophages, although HBeAg also induces pyroptosis of the former. These findings reveal novel activities of HBeAg, which can reprogram mitochondrial metabolism and trigger different programmed cell death responses of macrophages depending on their phenotypes to promote HBV persistence.

Maternal-Derived Hepatitis B Virus e Antigen Alters Macrophage Function in Offspring to Drive Viral Persistence after Vertical Transmission.

In contrast to horizontal transmission of hepatitis B virus (HBV) between adults, which often leads to self-limited acute infection, vertical transmission of HBV from mother to child often leads to chronic infection. However, the mechanisms linking vertical transmission with chronic infection are not known. We developed a mouse model to study the effect of maternal HBV infection on HBV persistence in offspring and found that HBV carried by the mother impaired CD8(+) T cell responses to HBV in her offspring, resulting in HBV persistence. This impairment of CD8(+) T cell responses was mediated by hepatic macrophages, which were predisposed by maternal HBV e antigen (HBeAg) to support HBV persistence by upregulation of inhibitory ligand PD-L1 and altered polarization upon restimulation with HBeAg. Depletion of hepatic macrophages led to CD8(+) T cell activation and HBV clearance in the offspring, raising the possibility of targeting macrophages to treat chronic HBV patients.

Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells.

Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.

Autophagic membranes participate in hepatitis B virus nucleocapsid assembly, precore and core protein trafficking, and viral release.

Hepatitis B virus (HBV) DNA replication takes place inside the viral core particle and is dependent on autophagy. Here we show that HBV core particles are associated with autophagosomes and phagophores in cells that productively replicate HBV. These autophagic membrane-associated core particles contain almost entirely the hypophosphorylated core protein and are DNA replication competent. As the hyperphosphorylated core protein can be localized to phagophores and the dephosphorylation of the core protein is associated with the packaging of viral pregenomic RNA (pgRNA), these results are in support of the model that phagophores can serve as the sites for the packaging of pgRNA. In contrast, in cells that replicate HBV, the precore protein derivatives, which are related to the core protein, are associated with autophagosomes but not with phagophores via a pathway that is independent of its signal peptide. Interestingly, when the core protein is expressed by itself, it is associated with phagophores but not with autophagosomes. These observations indicate that autophagic membranes are differentially involved in the trafficking of precore and core proteins. HBV induces the fusion of autophagosomes and multivesicular bodies and the silencing of Rab11, a regulator of this fusion, is associated with the reduction of release of mature HBV particles. Our studies thus indicate that autophagic membranes participate in the assembly of HBV nucleocapsids, the trafficking of HBV precore and core proteins, and likely also the egress of HBV particles.

Mechanisms of Hepatitis B Virus-Induced Hepatocarcinogenesis.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). There are approximately 250 million people in the world that are chronically infected by this virus, resulting in nearly 1 million deaths every year. Many of these patients die from severe liver diseases, including HCC. HBV may induce HCC through the induction of chronic liver inflammation, which can cause oxidative stress and DNA damage. However, many studies also indicated that HBV could induce HCC via the alteration of hepatocellular physiology that may involve genetic and epigenetic changes of the host DNA, the alteration of cellular signaling pathways, and the inhibition of DNA repair mechanisms. This alteration of cellular physiology can lead to the accumulation of DNA damages and the promotion of cell cycles and predispose hepatocytes to oncogenic transformation.

Autophagy in HCV Replication and Protein Trafficking.

Autophagy is a catabolic process that is important for maintaining cellular homeostasis. It is also known to possess other functions including protein trafficking and anti-microbial activities. Hepatitis C virus (HCV) is known to co-opt cellular autophagy pathway to promote its own replication. HCV regulates autophagy through multiple mechanisms to control intracellular protein and membrane trafficking to enhance its replication and suppress host innate immune response. In this review, we discuss the current knowledge on the interplay between HCV and autophagy and the crosstalk between HCV-induced autophagy and host innate immune responses.

Regulation of Hepatitis C Virus Infection by Cellular Retinoic Acid Binding Proteins through the Modulation of Lipid Droplet Abundance.

Retinoid (vitamin A) is an essential diet constituent that governs a broad range of biological processes. Its biologically active metabolite, all-trans retinoic acid (ATRA), exhibits a potent antiviral property by enhancing both innate and adaptive antiviral immunity against a variety of viral pathogens, such as, but not limited to, HIV, respiratory syncytial virus (RSV), herpes simplex virus (HSV), and measles. Even though the hepatocyte is highly enriched with retinoid and its metabolite ATRA, it supports the establishment of efficient hepatitis C virus (HCV) replication. Here, we demonstrate the hepatocyte-specific cell-intrinsic mechanism by which ATRA exerts either a proviral or antiviral effect, depending on how it engages cellular retinoic acid binding proteins (CRABPs). We found that the engagement of CRABP1 by ATRA potently supported viral infection by promoting the accumulation of lipid droplets (LDs), which robustly enhanced the formation of a replication complex on the LD-associated endoplasmic reticulum (ER) membrane. In contrast, ATRA binding to CRABP2 potently inhibited HCV via suppression of LD accumulation. However, this antiviral effect of CRABP2 was abrogated due to the functional and quantitative predominance of CRABP1 in the hepatocytes. In summary, our study demonstrates that CRABPs serve as an on-off switch that modulates the efficiency of the HCV life cycle and elucidates how HCV evades the antiviral properties of ATRA via the exploitation of CRABP1 functionality.IMPORTANCE ATRA, a biologically active metabolite of vitamin A, exerts pleiotropic biological effects, including the activation of both innate and adaptive immunity, thereby serving as a potent antimicrobial compound against numerous viral pathogens. Despite the enrichment of hepatocytes with vitamin A, HCV still establishes an efficient viral life cycle. Here, we discovered that the hepatocellular response to ATRA creates either a proviral or an antiviral environment depending on its engagement with CRABP1 or -2, respectively. CRABP1 supports the robust replication of HCV, while CRABP2 potently inhibits the efficiency of viral replication. Our biochemical, genetic, and microscopic analyses reveal that the pro- and antiviral effects of CRABPs are mediated by modulation of LD abundance, where HCV establishes the platform for viral replication and assembly on the LD-associated ER membrane. This study uncovered a cell-intrinsic mechanism by which HCV exploits the proviral function of CRABP1 to establish an efficient viral life cycle.

Regulation of Apolipoprotein E Trafficking by Hepatitis C Virus-Induced Autophagy.

Apolipoprotein E (ApoE) plays an important role in the maturation and infectivity of hepatitis C virus (HCV). By analyzing the subcellular localization of ApoE in Huh7 hepatoma cells that harbored an HCV subgenomic RNA replicon, we found that ApoE colocalized with autophagosomes. This colocalization was marginally detected in HCV-infected cells, apparently due to the depletion of ApoE by HCV, as treatment with bafilomycin A1 (BafA1), a vacuolar ATPase inhibitor that inhibits autophagic protein degradation, partially restored the ApoE level and enhanced its colocalization with autophagosomes in HCV-infected cells. The role of HCV-induced autophagy in the degradation of ApoE was further supported by the observations that nutrient starvation, which induces autophagic protein degradation, led to the loss of ApoE in HCV subgenomic RNA replicon cells and that the knockdown of ATG7, a protein essential for the formation of autophagic vacuoles, increased the ApoE level in cells with productive HCV replication. Interestingly, the inhibition of autophagy by ATG7 knockdown reduced the colocalization of ApoE with the HCV E2 envelope protein and the HCV titers released from cells. In contrast, the treatment of cells with BafA1 enhanced the colocalization of ApoE and HCV E2 and increased both intracellular and extracellular HCV titers. These results indicated that autophagy played an important role in the trafficking of ApoE in HCV-infected cells. While it led to autophagic degradation of ApoE, it also promoted the interaction between ApoE and HCV E2 to enhance the production of infectious progeny viral particles.IMPORTANCE Hepatitis C virus (HCV) is one of the most important human pathogens. Its virion is associated with apolipoprotein E (ApoE), which enhances its infectivity. HCV induces autophagy to enhance its replication. In this report, we demonstrate that autophagy plays an important role in the trafficking of ApoE in HCV-infected cells. This leads to the degradation of ApoE by autophagy. However, if the autophagic protein degradation is inhibited, ApoE is stabilized and colocalized with autophagosomes. This leads to its enhanced colocalization with the HCV E2 envelope protein and increased production of infectious progeny viral particles. If autophagy is inhibited by suppressing the expression of ATG7, a gene essential for the formation of autophagosomes, the colocalization of ApoE with E2 is reduced, resulting in the reduction of progeny viral titers. These results indicate an important role of autophagy in the transport of ApoE to promote the production of infectious HCV particles.

HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

Hepatitis C virus (HCV) induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7). Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER). Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication.

Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication.IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells.

HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication.

Hepatitis C virus (HCV) induces autophagy to enhance its replication. However, how HCV regulates the autophagic pathway remains largely unclear. In this report, we demonstrated that HCV infection could induce the expression of Rubicon and UVRAG, which inhibited and stimulated the maturation of autophagosomes, respectively. The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed, resulting in the accumulation of autophagosomes in the early time points of viral infection. The role of Rubicon in inhibiting the maturation of autophagosomes in HCV-infected cells was confirmed by siRNA knockdown and the over-expression of Rubicon, which enhanced and suppressed the maturation of autophagosomes, respectively. Rubicon played a positive role in HCV replication, as the suppression of its expression reduced HCV replication and its over-expression enhanced HCV replication. In contrast, the over-expression of UVRAG facilitated the maturation of autophagosomes and suppressed HCV replication. The HCV subgenomic RNA replicon, which expressed only the nonstructural proteins, could also induce the expression of Rubicon and the accumulation of autophagosomes. Further analysis indicated that the HCV NS4B protein was sufficient to induce Rubicon and autophagosomes. Our results thus indicated that HCV, by differentially inducing the expression of Rubicon and UVRAG, temporally regulated the autophagic flux to enhance its replication.

TNF-α Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism.

Invasion by infectious pathogens can elicit a range of cytokine responses from host cells. These cytokines provide the initial host defense mechanism. In this report, we demonstrate that TNF-α, a pro-inflammatory cytokine, can be induced by hepatitis C virus (HCV) in its host cells in a biphasic manner. The initial induction of TNF-α by HCV was prompt and could be blocked by the antibody directed against the HCV E2 envelope protein and by chemicals that inhibit endocytosis, indicating the specificity of endocytic uptake of HCV in this induction. Further studies indicated that the induction of TNF-α was dependent on toll-like receptors 7 and 8 (TLR7/8) but not on other intracellular pattern recognition receptors. Consistently, siRNA-mediated gene silencing of the downstream effectors in the TLR7/8 signaling pathway including MyD88, IRAK1, TRAF6, TAK1 and p65 NF-κB suppressed the expression of TNF-α. The role of p65 NF-κB in the induction of TNF-α via transcriptional up-regulation was further confirmed by the chromatin immunoprecipitation assay. TNF-α induced by HCV could activate its own receptor TNFR1 on hepatocytes to suppress HCV replication. This suppressive effect of TNF-α on HCV was due to its role in supporting interferon signaling, as the suppression of its expression led to the loss of IFNAR2 and impaired interferon signaling and the induction of interferon-stimulated genes. In conclusion, our results indicate that hepatocytes can sense HCV infection via TLR7/8 to induce the expression of TNF-α, which inhibits HCV replication via an autocrine mechanism to support interferon signaling.

Hepatitis C virus and autophagy.

Autophagy is a catabolic process by which cells remove protein aggregates and damaged organelles for recycling. It can also be used by cells to remove intracellular microbial pathogens, including viruses, in a process known as xenophagy. However, many viruses have developed mechanisms to subvert this intracellular antiviral response and even use this pathway to support their own replications. Hepatitis C virus (HCV) is one such virus and is an important human pathogen that can cause severe liver diseases. Recent studies indicated that HCV could activate the autophagic pathway to support its replication. This review summarizes the current knowledge on the interplay between HCV and autophagy and how this interplay affects HCV replication and host innate immune responses.

Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry.

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.

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.

Enhancement of hepatitis B virus replication by androgen and its receptor in mice.

Hepatitis B virus (HBV) is an important pathogen that chronically infects more men than women. To understand the molecular mechanism of this gender disparity, we analyzed HBV replication in transgenic mice that carried the HBV genome with or without the ability to express the HBV X protein (HBx). We found that gender had no effect on HBV surface antigen (HBsAg), DNA, and RNA levels in mice before puberty, but its effect on HBV after puberty was apparent, with HBV replicating approximately twice more efficiently in male mice than in female mice whether or not HBx was expressed. The castration of male mice resulted in a reduction of HBV HBsAg, DNA, and RNA levels, which could be partially restored by the injection of the androgen agonist R1881, indicating a positive role of androgen in HBV replication. The introduction of HBV genomic DNA and androgen receptor (AR) short hairpin RNA (shRNA) into the liver of naïve mice by hydrodynamic injection revealed that the effect of androgen on HBV was dependent on its receptor, which apparently could also stimulate HBV replication via an androgen-independent pathway. Further studies indicated that the two previously identified androgen response elements (AREs) in the HBV genome could indeed mediate the effect of androgen on HBV RNA transcription and DNA replication in vivo. These effects of androgen and its receptor on HBV thus provide an explanation for why men have a higher risk of HBV infection than women.

Viral-load-dependent effects of liver injury and regeneration on hepatitis B virus replication in mice.

Hepatitis B virus (HBV) is a hepatotropic virus that can cause severe liver diseases. By conducting studies using four different transgenic mouse lines that carry either the wild-type HBV genome or the HBV genome incapable of expressing the X gene, we found that liver injury and regeneration induced by a partial hepatectomy (PHx) could have different effects on HBV replication depending on the mouse lines. Further studies using hydrodynamic injection to introduce different amounts of the HBV genomic DNA into the mouse liver revealed that liver injury and regeneration induced by PHx enhanced HBV replication when viral load was low and suppressed HBV replication when viral load was high. These effects of liver injury and regeneration on HBV were independent of the HBV X protein and apparently due to alpha and beta interferons (IFN-α/ß), as the effects could be abolished by the injection of anti-IFN-α/ß antibodies. Further analysis indicated that PHx could induce the expression of hepatocyte nuclear factor 3 gamma (HNF3γ) when viral load was low and activate the signal transducer and activator of transcription 3 (Stat3) and suppress the expression of the suppressor of cytokine signaling 3 (SOCS3) irrespective of viral load. As both HNF3γ and Stat3 are required to activate the HBV enhancer I to stimulate HBV gene expression and replication, these results provided an explanation to the viral-load-dependent effect of liver injury and regeneration on HBV replication. Our studies thus revealed a novel interaction between HBV and its host and provided important information for understanding HBV replication and pathogenesis during liver injury.

Effects of interferon-α/ß on HBV replication determined by viral load.

Interferons α and ß (IFN-α/ß) are type I interferons produced by the host to control microbial infections. However, the use of IFN-α to treat hepatitis B virus (HBV) patients generated sustained response to only a minority of patients. By using HBV transgenic mice as a model and by using hydrodynamic injection to introduce HBV DNA into the mouse liver, we studied the effect of IFN-α/ß on HBV in vivo. Interestingly, our results indicated that IFN-α/ß could have opposite effects on HBV: they suppressed HBV replication when viral load was high and enhanced HBV replication when viral load was low. IFN-α/ß apparently suppressed HBV replication via transcriptional and post-transcriptional regulations. In contrast, IFN-α/ß enhanced viral replication by inducing the transcription factor HNF3γ and activating STAT3, which together stimulated HBV gene expression and replication. Further studies revealed an important role of IFN-α/ß in stimulating viral growth and prolonging viremia when viral load is low. This use of an innate immune response to enhance its replication and persistence may represent a novel strategy that HBV uses to enhance its growth and spread in the early stage of viral infection when the viral level is low.

The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.

Autophagy is a catabolic process by which cells remove long-lived proteins and damaged organelles for recycling. Viral infections may also induce autophagic response. Here we show that hepatitis B virus (HBV), a pathogen that chronically infects approximately 350 million people globally, can enhance autophagic response in cell cultures, mouse liver, and during natural infection. This enhancement of the autophagic response is not coupled by an increase of autophagic protein degradation and is dependent on the viral X protein, which binds to and enhances the enzymatic activity of phosphatidylinositol 3-kinase class III, an enzyme critical for the initiation of autophagy. Further analysis indicates that autophagy enhances HBV DNA replication, with minimal involvement of late autophagic vacuoles in this process. Our studies thus demonstrate that a DNA virus can use autophagy to enhance its own replication and indicate the possibility of targeting the autophagic pathway for the treatment of HBV patients.