Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

Abstract

Hepatitis C virus (HCV) or HCV-low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm 3 ), intermediate-density particles (1.12 to 1.18 g/cm 3 ), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4°C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of ␣-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of ␣-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Approximately 85% of people infected with HCV remain persistently viremic, and approximately 20% to 50% of these individuals ultimately develop cirrhosis (12,21,50). Of those with HCV-related cirrhosis, approximately 5% develop hepatocellular carcinoma (21, 50). In the United States, an estimated 4 million people are infected, and HCV is the leading cause of liver transplantation (21). Extrahepatic manifestations, including cryoglobulinemia and B-lymphocyte proliferative disorders, which are characterized by polyclonal B-cell activation and autoantibody production, are also associated with HCV infection (3,13,14,39). Hepatocytes represent the primary site of HCV replication in vivo. Although explanted peripheral blood mononuclear cells (PBMCs) contain HCV RNA (4, 30, 40), it is unclear if HCV replication occurs in PBMCs in vivo (1,16,23,52). No efficient cell culture system has been described for HCV, but in vitro studies have shown that several human cells including primary PBMC cultures (10) and cell lines of hepatocyte and lymphoid origin (42-45) are permissive for HCV replication.Currently, the mechanism of HCV cell entry is not clear. Two cell surface receptors interact with HCV or HCV E2 protein in vitro, leading to speculation t...

Correlation between beta-lipoprotein levels and outcome of hepatitis C treatment

“…Molecular studies of HCV replication and infection have shown entry of the virus into the cell may occur via LDL and CD 81 receptors. [3][4][5] Binding and endocytosis of the virus is limited by LDL particles via competitive inhibition in vitro. 3 We evaluated the possibility of such inhibition in vivo and examined the influence of LDL levelson treatment outcome.…”

“…Two molecules are proposed to function as HCV receptors, namely, the low-density lipoprotein receptor (LDLR) and CD81. [3][4][5] Experiments in vitro showed competitive inhibition of binding between HCV and LDLR by purified LDL. 3 If similar inhibition occurs in vivo, the serum concentration of beta-lipoproteins may influence HCV proliferation because cell infection is required for replication of the virus.…”

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

“…The ability of E2 to bind to CD81 has been widely documented; however, widespread distribution of CD81 on nonpermissive cells (30) led to the view that other liver-specific molecules must also be involved in facilitating entry (19). Several other cell surface molecules have been reported to bind to native HCV particles or recombinant E2, including the low-density lipoprotein receptor (1,51), dendritic-cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver/lymph nodespecific intercellular adhesion molecule 3-grabbing integrin (L-SIGN or DC-SIGNR) (20,32,42), glycosaminoglycans (4), and scavenger receptor class B type I (SR-B1) (6,44). Despite early doubts about the function of CD81 in HCV entry, data using recently developed assays of retroviral pseudoparticles (HCVpp) and in vitro infectious clones support a central role for CD81 in mediating infection (6,11,29,31).…”

Cellular models for the screening and development of anti-hepatitis C virus agents

“…Tetraspanin CD81 has been well characterized as a major factor for HCV cellular entry (Bartosch et al, 2003;Buck, 2008;Cormier et al, 2004;Hsu et al, 2003;Kapadia et al, 2007;Koutsoudakis et al, 2007;Molina et al, 2008;Petracca et al, 2000;Pileri et al, 1998;Wunschmann et al, 2000; (Fig 1, step 3). E2 protein has been shown to exhibit a very high affinity (K d ~10 -9 M) for the large extracellular loop of CD81 (Petracca et al, 2000;Pileri et al, 1998).…”