Hepatitis C Virus NS4B Protein Induces Apoptosis

It is estimated that3%of the human population is infected with hepatitis C virus (HCV). Persistent HCV infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC), which is a severe health problem worldwide. HCV is an enveloped, positive-stranded RNA virus with a genome of9,600nucleotides, which consists of a5’untranslated region, a single open reading frame (ORF), and a3’ untranslated region. The ORF encodes a large polyprotein precursor of approximately3,000amino acid residues. The polyprotein is co-and post-translationally cleaved by viral and host cell proteases to yield at least ten different proteins:the core, E1, and E2structural proteins, and the p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B nonstructural proteins.Apoptosis (or programmed cell death), which occurs in multicellular organisms, has been implicated in many different pathologies. Apoptosis is particularly important in the control and elimination of viral infections. The phenotypic features of apoptosis include cell shrinkage, chromatin condensation, DNA fragmentation, apoptotic body formation, cytoplasmic vacuolization, and cell lysis. The signaling cascades that control caspase-dependent apoptosis can be classified into two pathways:the intrinsic (or mitochondrial) death pathway and the extrinsic cell death (or death receptor) pathway. Both of these pathways can activate the executioner caspase3and caspase7. The mitochondrial death pathway can be induced by a large variety of signals and results in the release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c induces the formation of the apoptosome complex, which recruits and activates procaspase-9. Activated caspase9cleaves and activates caspase3which in turn cleaves downstream death events, such as poly (ADP-ribose) polymerase (PARP). Although the exact molecular mechanisms of viral persistence and disease progression are not well understood, there is evidence suggesting that hepatocyte apoptosis plays an important role in HCV pathogenesis. It has been reported that apoptosis is involved in the liver damage that is related to chronic HCV infection. Moreover, HCV infection induces apoptosis through the mitochondrial death pathway. Many HCV proteins have been reported to induce apoptosis, including core, E1, E2, NS4A, NS3, NS5A and NS5B. However, other reports have shown that core, E2, NS2, NS3, and NS5A can inhibit apoptosis.HCV NS4B is a27-kDa hydrophobic, integral membrane protein that contains at least four transmembrane domains. NS4B induces the formation of an intracellular membrane web structure, that is indispensable for the formation of membrane-associated replication complexes with other NS proteins. NS4B not only plays an important role in RNA replication but also contributes to virus assembly and release. However, little is known about the effects of HCV NS4B on cell survival.In this study, we found that NS4B induced apoptosis in293T cells and Huh7cells, as confirmed by Hoechst staining, DNA fragmentation, and annexin V/PI assays. Furthermore, protein immunoblot analysis demonstrated that NS4B triggered the activation of caspase3, caspase7, and poly (ADP-ribose) polymerase (PARP). Further studies revealed that NS4B induced the activation of caspase9, the reduction of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. However, NS4B expression did not trigger XBP1mRNA splicing and increased the expression of binding immunoglobulin protein (BiP, or GRP78) and C/EBP homologous protein (CHOP), which serves as the indicators of ER stress. Taken together, our results suggest that HCV NS4B induces apoptosis through the mitochondrial death pathwayIn this study, we report that HCV NS4B can induce apoptosis through the mitochondrial death pathway. These data may provide a new way for understanding how HCV modifies host cellular functions.