Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

Abstract

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR. Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (HCC) (2). While the generation of the HCV pseudoparticle (HCVpp) and infectious cell culture (HCVcc) models have resulted in a significant increase in our understanding of HCV entry, the molecular mechanisms involved in viral internalization and fusion still remain unclear. HCV entry occurs through the coordinated interactions between the E1-E2 HCV glycoproteins and at least four essential cellular entry factors: CD81 (42), scavenger receptor B type I (SR-BI) (47), occludin (OCLN) (43), and claudin 1 (CLDN1) (11). The E2 glycoprotein has been demonstrated to bind CD81 (42) and SR-BI (47), and antibodies that bind to highly conserved residues 412 to 423 on the E2 glycoprotein possess broad neutralization capabilities against multiple HCV genotypes by inhibiting HCV-CD81 interactions (40). Although HCV is known to enter hepatocytes via clathrinmediated endocytosis (1), the host-virus interactions that govern HCV internalization are not well understood. Only one of the HCV entry factors, SR-BI, enhances HCV entry by mediating the selective uptake of cholesterol esters from HDL (8). Although HCV was recently demonstrated to induce CD81 and CLDN1 endocytosis (14), the molecular interactions important for HCV internalization still remain unclear.Multiple RNA and DNA viruses have evolved to induce a variety of receptor-mediated signaling events that are critical for different aspects of viral entry (7,12,16,53). HCV regulates multiple intracellular signaling pathways, some of which been implicated in the progression of HCV-related HCC (23, 53). HCV interaction with CD81 has been demonstrated to activate multiple downstream signaling pathways, including Rho GTPase family members, Cdc42, mitogen-activated protein kinase pathways, and members of the ezrin-radixin-moesin (ERM) family of proteins (3, 6, 13). In addition, CD81 binding by HCV primes the E1-E2 heterodimer complex for low...

Identification of transferrin receptor 1 as a hepatitis C virus entry factor

“…Antibodies to CD81, TfR1, or isotype control IgG were added to parallel cultures before virus binding or after virus binding at hour intervals after the temperature shift. Exactly as previous groups have observed (31,32), when normalized to the IgG control at each time, anti-CD81 lost its inhibitory effect by 2 h postbinding. In contrast, addition of anti-TfR1 inhibited HCV by more than 50% until 4 h after the temperature shift, indicating that TfR1 functions in HCV entry at a step after CD81 (Fig.…”

“…To determine when TfR1 acts during entry relative to other HCV entry factors, we used a previously published antibody time-of-addition strategy (23,31,32). The strategy is based on the principle that blocking antibodies lose their inhibitory activity when applied after the targeted protein has already served its function.…”

Molecular Signaling and Cellular Pathways for Virus Entry

“…More recently, Diao and colleagues demonstrated that direct hepatitis C virus (HCV) binding to CD81 can induce EGFR activation and internalization [62]. HCV interaction with CD81 can also activate multiple downstream signaling pathways including Rho GTPase family members, Cdc42, MAPK pathways, and members of the ezrin-radixin-moesin (ERM) family of proteins [63][64][65].…”

Strategies to Preclude Hepatitis C Virus Entry

“…Specifically, CD81 forms a coreceptor complex with the tight junction protein claudin-1 (CLDN1) [49,50] and is engaged in late events of HCV entry [51]. This re-localization and virusreceptor complex association with CLDN1 involves multiple signaling pathways (e.g., Rho GTPases, PI3K/AKT, and ERK/MAPK) [52,53], includes the activation of host cell kinases such as the epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) [54,55], and is influenced by the absence of the CD81-associated partner EWI-2wint on the hepatocytes [56,57]. The EWI-2wint molecule is normally bound to CD81 on most cell type surfaces and inhibits its diffusion which is required to promote HCV entry; however, it is not expressed in the hepatocytes, and hence its absence has been suggested to contribute to the restricted tropism of the virus [56].…”