Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. siRNA-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in assembly step without affecting viral entry, HCV IRES-mediated translation, and replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs) and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating phosphorylation of cortactin at tyrosine residue 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration. IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in virus life cycle has not yet been fully understood. The most significant finding is that cortactin strongly interacted with both HCV core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the LDs with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosine 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.
Hepatitis C virus (HCV) is the major causative agent of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The recent development of highly effective direct-acting antivirals (DAAs) has revolutionized the treatment of HCV patients. However, these DAAs are exorbitantly expensive for the majority of HCV patients worldwide. Moreover, these drugs still show genotypic difference in cure rate and have some resistant-associated variants. Tylophorine, a natural compound derived from Tylophora indica plants, is known to have anti-inflammatory and anti-cancerous growth activities. In the present study, we showed that two tylophorine intermediates, 5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl) methyl]-L-proline (O859585) and 2,3,6,7-tetramethoxy-9-phenanthrenecarboxylic acid (T298875), displayed anti-HCV activity with an EC 50 of 38.25 µM for T298875 and 29.11~35.3 µM for O859585 in various HCV genotypes. We demonstrated that O859585 efficiently blocked HCV attachment by neutralizing free viral particles without affecting other stages of the HCV life cycle and interferon stimulation. O859585 interrupted binding between HCV E2 and CD81. Of note, co-treatment of O859585 with either interferon alpha (IFNα) or sofosbuvir exerted either an additive or synergistic antiviral activity in HCV-infected cells with no measurable effect on cell viability. Most importantly, O859585 in combination with IFNα and sofosbuvir exhibited synergistic effects on anti-HCV activity in primary human hepatocytes. Collectively, these data suggest that O859585 may be a novel antiviral agent for HCV therapy.
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