CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers

Abstract

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.Hepatitis C virus (HCV) infection is a major health problem affecting an estimated 160 million people worldwide (36). It is a major cause of chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (53), and mixed cryoglobulinemia, a B-lymphocyte proliferative disorder (reviewed in references 6 and 49). HCV is a small enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (33). Its genome encodes a single ϳ3,000-amino-acid polyprotein that is co-and posttranslationally processed by viral and cellular proteases to yield the mature structural and nonstructural proteins (33, 37). The structural proteins-the core protein and envelope glycoproteins E1 and E2-are believed to be the major constituents of HCV particles. The E1 and E2 envelope proteins are N glycosylated in their large N-terminal ectodomains and are anchored into membranes by their hydrophobic C-terminal transmembrane domains (TMDs) (39). These domains have been shown to be endoplasmic reticulum (ER) retention signals (10,12,20,23). E1 and E2 associate to form two types of complexes: properly folded E1E2 heterodimers stabilized by noncovalent interactions and misfolded disulfide-linked aggregates (for a review, see reference 39).The E1E2 noncovalent heterodimer, comprising the viral envelope (reviewed in reference 39), is involved in viral entry (3, 30); however, the mechanism of HCV cell entry is not clear. Several putative cell surface receptors of HCV or recombinant E2 proteins have been identified (1,25,34,45,46,50,51). Among these receptors, human CD81 has been repeatedly shown to interact with recombinant soluble E2, the E1E2 comple...

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

“…Mutations were introduced in the context of full-length E1E2 because the E1E2 heterodimer exhibits superior CD81 binding compared to soluble monomeric E2 (9). To recover E1E2, the cells have to be lysed with a mixture of detergents, which precludes their use in cell binding assays.…”

Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes

“…Plasmid (pShuttle/E1 FLAG E2) 21 encoding the HCV E1 and E2 proteins-in which highly variable region 1 was exchanged with a FLAG epitopewas a generous gift from Shoshana Levy. Properly folded HCV envelope E1 and E2 heterodimers were expressed and purified as described by Cocquerel et al 21 Briefly, COS-7 cells (American Type Culture Collection) were transiently transfected using the Mammalian Transfection Kit (Stratagene, La Jolla, CA) with pShuttle/ E1 FLAG E2, then washed and lysed in 1% Triton X-100. Clarified cell lysates were preadsorbed onto a protein A agarose column (Sigma) and then applied to a calciumdependent anti-FLAG M1 affinity column (Sigma).…”

Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

“…Various E2 fusion proteins have been cloned as models of the native E2 protein. The conformation of the E2 polypeptide is sensitive to various microenvironmental factors, exemplified by changes in its reaction with various Abs (35,57). Of the two E2 fusion protein tested in the present study for FLAG-E2 and GST-E2, the former likely approximates more closely the native protein structure.…”

“…Noncovalent E2-E1 complexes as well as disulfide-linked oligomers have been identified on the pseudovirion particles, the closest approximation of infectious HCV available for biochemical studies (43,44). Soluble recombinant E2 versions linked to various polypeptide tags have been proposed as models for native E2 (35,45). We studied two E2 fusion proteins as substrates for hybrid Abs.…”

“…After PvuI linearization, pcDNA3/E1 FLAG E2 was electroporated into Chinese hamster ovary cells and transformants were selected with geneticin as above in Complete Dulbecco's modified Eagle's medium supplemented with non-essential amino acids. Lysates containing FLAG-E2 were prepared by treating the cells with 50 mM Tris, 150 mM NaCl, pH 7.4 (Tris-buffered saline), containing 1% Triton X-100, 10 mM CaCl 2 , 20 mM iodoacetamide, and a protease inhibitor mixture (35). The lysate (40 ml from ϳ2 ϫ 10 8 cells) was fractionated using anti-FLAG M1 Ab conjugated to agarose (0.5 ml of gel; Ͼ0.6 mg of FLAG peptide binding capacity/ml of gel; Sigma).…”