Human hepatitis B virus (HBV) causes chronic hepatitis and is associated with the development of hepatocellular carcinoma. HBV infection alters mitochondrial metabolism. The selective removal of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. Here, we report that HBV shifts the balance of mitochondrial dynamics toward fission and mitophagy to attenuate the virus-induced apoptosis. HBV induced perinuclear clustering of mitochondria and triggered mitochondrial translocation of the dynamin-related protein (Drp1) by stimulating its phosphorylation at Ser616, leading to mitochondrial fission. HBV also stimulated the gene expression of Parkin, PINK1, and LC3B and induced Parkin recruitment to the mitochondria. Upon translocation to mitochondria, Parkin, an E3 ubiquitin ligase, underwent self-ubiquitination and facilitated the ubiquitination and degradation of its substrate Mitofusin 2 (Mfn2), a mediator of mitochondrial fusion. In addition to conventional immunofluorescence, a sensitive dual fluorescence reporter expressing mito-mRFP-EGFP fused in-frame to a mitochondrial targeting sequence was employed to observe the completion of the mitophagic process by delivery of the engulfed mitochondria to lysosomes for degradation. Furthermore, we demonstrate that viral HBx protein plays a central role in promoting aberrant mitochondrial dynamics either when expressed alone or in the context of viral genome. Perturbing mitophagy by silencing Parkin led to enhanced apoptotic signaling, suggesting that HBV-induced mitochondrial fission and mitophagy promote cell survival and possibly viral persistence. Altered mitochondrial dynamics associated with HBV infection may contribute to mitochondrial injury and liver disease pathogenesis. Hepatitis B virus (HBV) chronic infections represent the common cause for the development of hepatocellular carcinoma. Mitochondrial liver injury has been long recognized as one of the consequences of HBV infection during chronic hepatitis. Mitochondria are dynamic organelles that undergo fission, fusion, and selective-autophagic removal (mitophagy), in their pursuit to maintain mitochondrial homeostasis and meet cellular energy requirements. The clearance of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. We observed that HBV and its encoded HBx protein promoted mitochondrial fragmentation (fission) and mitophagy. HBV/HBx induced the expression and Ser616 phosphorylation of dynamin-related protein 1 (Drp1) and its subsequent translocation to the mitochondria, resulting in enhanced mitochondrial fragmentation. HBV also promoted the mitochondrial translocation of Parkin, a cytosolic E3 ubiquitin ligase, and subsequent mitophagy. Perturbation of mitophagy in HBV-infected cells resulted in enhanced mitochondrial apoptotic signaling. This shift of the mitochondrial dynamics towards enhanced fission and mitophagy is essential for the clearance of damaged mitochondria and serves to prevent apoptotic cell death of HBV-infected cells to facilitate persistent infection.

Significance: Persistent hepatitis C virus (HCV) infection is associated with mitochondrial liver injury. Mitochondrial quality control is established as a physiological adaptation to mitochondrial injury. This study provides a new insight into how HCV disrupts mitochondrial dynamics and evades apoptosis and innate immunity to sustain persistent viral infection. HCV promoted dynamin-related protein 1-mediated mitochondrial fission, followed by mitophagy. Interference of HCV-induced mitochondrial fission and mitophagy led to the suppression of virus secretion, a decrease in glycolysis and ATP generation, an increase in interferon synthesis, and an increase in apoptotic death of infected cells via enhanced apoptotic signaling. These observations implicate the functional relevance of altered mitochondrial dynamics in the pathogenesis of chronic liver disease associated with HCV infection.

Hepatic steatosis is a common histological feature of chronic hepatitis C. Hepatitis C virus (HCV) gene expression has been shown to alter host cell cholesterol/lipid metabolism and thus induce hepatic steatosis. Since sterol regulatory element binding proteins (SREBPs) are major regulators of lipid metabolism, we sought to determine whether genotype 2a-based HCV infection induces the expression and posttranslational activation of SREBPs. HCV infection stimulates the expression of genes related to lipogenesis. HCV induces the proteolytic cleavage of SREBPs. HCV core and NS4b derived from genotype 3a are also individually capable of inducing the proteolytic processing of SREBPs. Further, we demonstrate that HCV stimulates the phosphorylation of SREBPs. Our studies show that HCV-induced oxidative stress and subsequent activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt pathway and inactivation (phosphorylation) of PTEN (phosphatase and tensin homologue) mediate the transactivation of SREBPs. HCV-induced SREBP-1 and -2 activities were sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca(2+) chelator 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl) ester (BAPTA-AM), and PI3-K inhibitor (LY294002). Collectively, these studies provide insight into the mechanisms of hepatic steatosis associated with HCV infection.

Significance: N6 -methyladenosine (m ⁶ A) has recently been found to regulate numerous aspects of RNA biology. Similar to methylation of cytosine residues in DNA, eukaryotic RNA is modified by enzymatic addition of methyl groups at adenosines. m ⁶ A modification of RNA affects a wide variety of RNA functions, including mRNA stability, translation, and in the case of viruses, viral replication and production. Our investigation revealed that the adenosine residues present in the known m ⁶ A consensus motif within the 5′ epsilon stem loop of pregenomic RNA and the 3′ ends of all the hepatitis B virus (HBV) transcripts are m ⁶ A modified. We demonstrate here that m ⁶ A modification differentially modulates HBV RNA stability and reverse transcription, thereby playing two distinct regulatory roles in the HBV life cycle.

Hepatitis C Virus (HCV) induces intracellular events that trigger mitochondrial dysfunction and promote host metabolic alterations. Here, we investigated selective autophagic degradation of mitochondria (mitophagy) in HCV-infected cells. HCV infection stimulated Parkin and PINK1 gene expression, induced perinuclear clustering of mitochondria, and promoted mitochondrial translocation of Parkin, an initial event in mitophagy. Liver tissues from chronic HCV patients also exhibited notable levels of Parkin induction. Using multiple strategies involving confocal and electron microscopy, we demonstrated that HCV-infected cells display greater number of mitophagosomes and mitophagolysosomes compared to uninfected cells. HCV-induced mitophagy was evidenced by the colocalization of LC3 puncta with Parkin-associated mitochondria and lysosomes. Ultrastructural analysis by electron microscopy and immunoelectron microscopy also displayed engulfment of damaged mitochondria in double membrane vesicles in HCV-infected cells. The HCV-induced mitophagy occurred irrespective of genotypic differences. Silencing Parkin and PINK1 hindered HCV replication suggesting the functional relevance of mitophagy in HCV propagation. HCV-mediated decline of mitochondrial complex I enzyme activity was rescued by chemical inhibition of mitophagy or by Parkin silencing. Overall our results suggest that HCV induces Parkin-dependent mitophagy, which may have significant contribution in mitochondrial liver injury associated with chronic hepatitis C. Hepatitis C virus (HCV) infection alters host lipid metabolism. HCV-induced mitochondrial dysfunction may promote the metabolic alterations by affecting mitochondrial β-oxidation and oxidative phosphorylation. Dysfunctional mitochondria are detrimental to cell survival and require rapid clearance to sustain cell viability. Here, we investigated the effect of HCV gene expression in promoting selective autophagy of dysfunctional mitochondria, also termed mitophagy. HCV infection stimulated the gene expression of Parkin and PINK1, the two key mediators of mitophagy. Parkin stimulation was also observed in liver biopsies of chronic hepatitis C patients. HCV infection induced the perinuclear clustering of mitochondria and triggered Parkin translocation to mitochondria, a hallmark of mitophagy. Concomitant with the mitochondrial translocation of Parkin, we observed ubiquitination of Parkin and its substrates in HCV-infected cells. We also demonstrate the formation of mitophagosomes and their subsequent delivery to lysosomes in HCV-infected cells. Silencing both Parkin and PINK1 hindered HCV replication, suggesting the functional significance of mitophagy in HCV life cycle. Furthermore, we demonstrate that Parkin-dependent mitophagy is directly associated with HCV-mediated decline in oxidative phosphorylation. Our results implicate the functional significance of Parkin and mitophagy in the persistence of HCV infection and mitochondrial injury commonly seen in patients with chronic hepatitis C.