Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2

Abstract

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).CD81 is a member of the tetraspanin family. Tetraspanins are membrane proteins containing four transmembrane domains, short intracellular domains and two extracellular loops (8). Tetraspanins have been shown to be involved in cell activation, proliferation, motility, and metastasis, as well as in cell fusion (14). Although CD81 is widely expressed, its level of expression varies in specific cell lineages and during differentiation. Moreover, its association with cell surface proteins differs in cell types of various lineages. In B cells CD81 is a component of the CD19-CD21-CD81-Leu-13 molecular complex, which plays a role in B-cell activation (2), whereas in T cells the molecule is associated with T-cell-specific molecules, including CD4 and CD8 (5, 15). In addition to its association with lineage-specific proteins, CD81 is associated with integrins and other tetraspanins (8). As a consequence of such protein associations, it is possible to activate multiple adhesion/signaling pathways in different cell types by engaging CD81 at their surface. For example, treatment of B-cell lines with a monoclonal antibody (MAb) specific for CD81 induces changes in cell adhesion and inhibits proliferation, whereas treatment of T-cell lines affects cell adhesion but not proliferation (10).CD81 was recently reported to interact with the hepatitis C virus (HCV) envelope glycoprotein (gp) E2 and hypothesized to act as a putative viral receptor (12). We have confirmed this observation and have shown that cell surface-expressed human CD81, but not murine or monkey (Chlorocebus aethiops, African green monkey [AGM]) CD81, binds E2 661 , a truncated soluble version of the E2 gp. Furthermore, interaction of E2 661 with the B-cell line Daudi had effects on cell adhesion and proliferation similar to the effects induced by anti-CD81 antibodies (3). This observation is of particular interest since in addition to causing acute and chronic liver disease, HCV is the major cause for mixed cryoglobulinemia...

Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection

“…14 In particular, the large extracellular loop (LEL) of the CD81 molecule appears to be involved in the binding between HCV envelope glycoprotein E2 and CD81. 5,15 Thus, CD81 is thought to play a role in HCV endocytosis, although receptor downregulation after binding has not been observed.…”

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

“…A 100 µl aliquot of a 3 µg/ml solution of glutathione S-transferase (GST) fusion protein expressing the second extracellular region (EC2) of the LEL of human CD81 (GST-hLEL) [19] was used to coat each well of a Nunc F96 Maxisorp, 96 well-plate and incubated overnight at room temperature. Unbound protein was discarded, and the wells were blocked with 2% non-fat dry milk in PBS/0.05% Tween 20 for 2 h at room temperature.…”

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

“…key role in recognition for CD81 binding to HCV E2. Binding site of HCV E2 for hCD81 is a conformational structure naturally involving aa 480 to 493 and 544 to 551 within the E2 protein [3,7,26]. Thus it is difficult for a linear peptide library to mimic the discontinuous epitopes of HCV E2 protein.…”

“…It has been demonstrated that HCV envelope glycoprotein 2 (E2) could bind to host cells by interacting with the CD81 molecule [2]. CD81 is a membrane protein of 26 000 KD, and with four transmembrane (TM) and two extracellular (EC) domains, it belongs to the tetraspanin superfamily [3]. The larger extracellular loop (LEL) of CD81 molecule contains four conservative cysteine residues forming two disulfide bounds that maintain natural configuration of the protein.…”