Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence

Mitochondrial dynamics is crucial for the regulation of cell homeostasis. Our recent findings suggest that hepatitis C virus (HCV) promotes Parkin-mediated elimination of damaged mitochondria (mitophagy). Here we show that HCV perturbs mitochondrial dynamics by promoting mitochondrial fission followed by mitophagy, which attenuates HCV-induced apoptosis. HCV infection stimulated expression of dynamin-related protein 1 (Drp1) and its mitochondrial receptor, mitochondrial fission factor. HCV further induced the phosphorylation of Drp1 (Ser616) and caused its subsequent translocation to the mitochondria, followed by mitophagy. Interference of HCV-induced mitochondrial fission and mitophagy by Drp1 silencing suppressed HCV secretion, with a concomitant decrease in cellular glycolysis and ATP levels, as well as enhanced innate immune signaling. More importantly, silencing Drp1 or Parkin caused significant increase in apoptotic signaling, evidenced by increased cytochrome C release from mitochondria, caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. These results suggest that HCV-induced mitochondrial fission and mitophagy serve to attenuate apoptosis and may contribute to persistent HCV infection.Hepatitis C virus (HCV) infection often leads to chronic hepatitis that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (1). HCV is a hepatotropic, noncytopathic (2, 3), single-stranded, positive-sense RNA virus that replicates its RNA genome on the endoplasmic reticulum (ER)-derived membranous structures (4, 5). HCV stimulates lipogenesis, leading to the accumulation of lipid droplets that facilitate virion assembly and maturation (5–8). HCV infection also induces mitochondrial dysfunction via ER and oxidative stress that results in mitochondrial Ca²⁺ overload, collapse of mitochondrial transmembrane potential (ΔΨm), elevated levels of reactive oxygen species, and disruption of mitochondrial respiration (9–15). Liver tissues of patients with chronic hepatitis C frequently exhibit traits of mitochondrial injury such as swollen, ruptured, and empty mitochondria (16).Mitochondria are dynamic organelles that constantly undergo fission, fusion, and mitophagy to facilitate mitochondrial quality control, which is crucial for maintaining cell viability and bioenergetics (17). Aberrant mitochondrial dynamics are associated with the pathogenesis of several genetic and neurological disorders, cardiac dysfunctions, cancer, and metabolic diseases such as diabetes and obesity (18). Depending on their physiological and cellular context, the balance between mitochondrial fission and fusion processes modulates the mitochondrial morphology (17). Mitochondrial fission/fragmentation is mediated by recruitment of cytosolic Drp1 to the mitochondria, forming spirals that constrict both the inner and outer mitochondrial membranes (19). The mitochondrial fission is modulated by mitochondrial outer membrane proteins, which include mitochondrial fission 1 (Fis1), mitochondria fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kDa. These proteins coordinate to recruit Drp1 to mitochondria (20, 21). Mitochondrial fusion involves mitofusin 1 and 2 proteins and the inner mitochondrial membrane protein optic atrophy 1 (19, 21). More specifically, Drp1 recruitment to mitochondria is regulated by phosphorylation and dephosphorylation of respective serine residues by putative kinases and phosphatases (19). Mitochondrial dynamics is tightly regulated in response to alterations in cellular physiology such as stress, infections, and nutrient supply, and is also shown to play a critical role in apoptosis (18, 22).In this study, we investigated the HCV-induced modulation of mitochondrial dynamics, which plays a crucial role in attenuating apoptosis of infected cells resulting from mitochondrial injury associated with infection. We show that HCV stimulates the gene expression of Drp1 and Mff and promotes Drp1 recruitment to mitochondria by stimulating the phosphorylation of Drp1 (Ser616), leading to mitochondrial fission analyzed by confocal and electron microscopy. By using a dual-fluorescence mito-monomeric red fluorescent protein (mRFP)-EGFP reporter for monitoring complete mitophagy, we demonstrate that HCV-induced Drp1-mediated mitochondrial fission was followed by mitophagy. Interference of HCV-induced mitochondrial fission by silencing either Drp1 or Mff led to the accumulation of swollen mitochondria that resisted mitophagic degradation. Interestingly, interference of mitochondrial fission also suppressed viral secretion and glycolysis paralleled by a concomitant decline in cellular ATP levels and increased IFN synthesis in the HCV-infected cells. More importantly, inhibition of HCV-induced aberrant mitochondrial fission and mitophagy triggered robust apoptosis evidenced by a marked increase in cytochrome C release, caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. These observations unambiguously implicate the functional relevance of mitochondrial dynamics in the maintenance of persistent HCV infection.