Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly

Abstract

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 Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity

“…Myc-tagged NS5A and GFP-tagged NS5A were described previously ( Nguyen et al, 2020 ). Substitutions of serine to alanine at 2194, 2197, 2201, and 2204 hyperphosphorylation sites in NS5A were performed using a QuikChange ® II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to the manufacturer’s instructions.…”

“…Full-length IRF3 was amplified by PCR using cDNA prepared from HEK293T cells and was subcloned into the pcDNA3.1/Myc-His vector (Invitrogen, USA). All DNA transfections were performed using a polyethyleneimine reagent (Sigma-Aldrich) as we described previously ( Nguyen et al, 2020 ).…”

“…Luciferase and β-galactosidase assays were performed using either IFN-β-luciferase reporter plasmid or NF-κB-luciferase reporter plasmid and β-galactosidase plasmid as described previously ( Nguyen et al, 2020 ). Huh7, Huh7.5, HepG2 cells were transfected with the reporter plasmid and β-galactosidase.…”

Impact of direct-acting antiviral regimens on hepatic and extrahepatic manifestations of hepatitis C virus infection

“…HCV uses cortactin (an actin-binding protein at the cell periphery) for its assembly to promote viral proliferation and controls cortactin phosphorylation to facilitate cell invasion. Cortactin may be involved in hepatic cell migration, so it may be a potential target to interfere with the HCV cellular pathogenesis[ 19 ].…”

TheHelicobacter pyloritypeIVsecretion system upregulates epithelial cortactin expression by aCagA‐ andJNK‐dependent pathway

“…Cortactin can directly bind the Arp2/3 complex or via N‐WASP, and promote actin polymerisation by enhancing Arp2/3 interaction with actin filaments (Uruno et al, 2001). Interaction of cortactin with more than 15 known protein partners makes it an attractive target for multiple pathogens, including viruses, bacteria, fungi and protozoa (Bonfim‐Melo et al, 2015; Jouvenet et al, 2006; Moreno‐Ruiz et al, 2009; Nguyen et al, 2020; Selbach & Backert, 2005). Upon infection with H. pylori , cortactin undergoes tyrosine dephosphorylation at Y‐421 and Y‐486 in a CagA‐dependent manner (Selbach et al, 2003; Tegtmeyer et al, 2011).…”