No abstract

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome is shown here to induce the activation of NF-B and STAT-3 transcription factors from its cytoplasmic residence via oxidative stress. NS5A causes the disturbance of intracellular calcium. Ca 2؉ signaling triggers the elevation of reactive oxygen species in mitochondria, leading to the translocation of NF-B and STAT-3 into the nucleus. Evidence is presented for the constitutive activation of STAT-3 by NS5A. In the presence of antioxidants [pyrrolidine dithiocarbamate (PDTC), N-acetyl L-cysteine (NAC)] or Ca 2؉ chelators (EGTA-AM, TMB-8), NS5A-induced activation of NF-B and STAT-3 was eliminated. These results provide an insight into the mechanism by which NS5A can alter intracellular events relevant to liver pathogenesis associated with the viral infection.H epatitis C virus (HCV) causes acute͞chronic hepatitis with a significant risk of end-stage cirrhosis and hepatocellular carcinoma (1). The single-stranded RNA genome of the human HCV is a 9.6-kb-long positive-sense molecule, which encodes a polyprotein of about 3,000 aa (2, 3). The polyprotein is posttranslationally cleaved by both viral͞cellular proteases to produce about 10 polypeptides that include structural (core and E1 and E2) and nonstructural (NS2, NS3-NS5A͞B) proteins (2, 3). The single long ORF is preceded by 332 or 342 nt of the 5Ј untranslated region, which harbors an internal ribosome entry site capable of initiating translation at an internal site (4-6). The 3Ј end of the RNA genome contains a unique sequence of the untranslated region that is necessary for initiating RNA replication. Although the infectious cDNA clones that have been generated could infect chimpanzees, they failed to replicate in vitro in cultured cell lines (7). Lohmann et al. (8) described the generation of efficient replicating HCV RNA subgenomic replicons coexpressing a neomycin-resistance marker. A high level of replication of subgenomic replicons was characterized, resulting from adaptive mutations, which were scattered throughout the HCV genes of the replicon (9, 10).The HCV NS5A has generated a significant level of interest as several cellular targets have been identified. NS5A is a serine phosphoprotein, which exists as a polypeptide of p56 or p58 with varying degrees of phosphorylation (11,12). The identity of cellular kinase(s) responsible for NSS5A phosphorylation has not been firmly established. NS4A, an integral membrane protein, has been shown to modulate NS5A phosphorylation at least for some HCV subtypes (13). NS5A is localized to the cytoplasm in the perinuclear reticular network characteristic of the endoplasmic reticulum (ER) membrane (3,14). NS5A came into prominence because of its suggested role in IFN resistance. It was shown that NS5A directly interacted with double-stranded RNA-dependent kinase (PKR) and inactivated its function, thus modulating the IFN-stimulated antiviral response (15). Neither the sites of hyperphosphorylation nor the region designated ISDR (IFN-sen...

The hepatitis B virus (HBV) X gene product trans-activates viral and cellular genes. The X protein (pX) does not bind independently to nucleic acids. The data presented here demonstrate that pX entered into a protein-protein complex with the cellular transcriptional factors CREB and ATF-2 and altered their DNA binding specificities. Although CREB and ATF-2 alone did not bind to the HBV enhancer element, a pX-CREB or pX-ATF-2 complex did bind to the HBV enhancer. Thus, the ability of pX to interact with cellular factors broadened the DNA binding specificity of these regulatory proteins and provides a mechanism for pX to participate in transcriptional regulation. This strategy of altered binding specificity may modify the repertoire of genes that can be regulated by transcriptional factors during viral infection.

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.

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.HCV persistence | innate immunity | autophagy H epatitis 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 Ca 2+ 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), mitocho...