Structure-Function Analysis of Hepatitis C Virus Envelope-CD81 Binding

Abstract

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K d of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10 ؊9 M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.Hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridiae family (7). It has been estimated that 170 million people worldwide are chronically infected with HCV (18). Generally, HCV infection becomes chronic and may have very serious outcomes such as hepatitis, cirrhosis, and hepatocarcinoma. Although HCV was identified molecularly more than a decade ago (5), the virus has not been isolated nor have reliable in vitro systems for viral propagation been described, reverse transcription-PCR (RT-PCR) being the only way to detect HCV. Recently, we have shown that a bona fide HCV particle, i.e., HCV RNA associated with envelope, specifically binds human CD81 as demonstrated by quantitative PCR (14).CD81 is a membrane-associated protein belonging to the family of tetraspanins (10). Like all tetraspanins, CD81 is organized in four highly hydrophobic transmembrane domains, which force the protein to traverse the membrane four times, creating two hydrophilic domains, a small one and a large one, protruding out of the cells. We have found that the large extracellular loop (LEL) of CD81 is sufficient to bind HCV via interaction with the major virus envelope protein E2 (14). Remarkably, chimpanzee sera containing antienvelope antibodies, which are capable of preventing HCV infection in vivo, inhibit the binding of HCV to CD81 in vitro (14, 16), supporting the idea that CD81 represents a cellular receptor for the virus.In this work we have studied the HCV-CD81 interaction in more detail. First, we determined the affinity constant for binding of soluble CD81 LEL and monomeric HCV E2 by using highly purified recombinant LEL and E2 proteins. Second, we assessed the binding of recombinant E2 on fresh hepatocytes and hepatocarcinoma cell lines. Third, we quantitated the ability of cell s...

Monoclonal Antibodies with Broad Specificity for Hepatitis C Virus Hypervariable Region 1 Variants Can Recognize Viral Particles

“…Current evidence suggests that mammalian cell-derived E2 glycoproteins (35) and, to a lesser extent, HCV-LP 1a (M. Triyatni and T. J. Liang, unpublished observations) can bind human CD81, a candidate receptor molecule for HCV. Yet HCV-LP can penetrate into HepG2 cells via alternative receptor(s) which are constitutively expressed on hepatocytes (M. Triyatni and T. J. Liang, unpublished observations), supporting the hypothesis that HCV requires a second receptor molecule for internalization (35). Preliminary evidence suggests that HVR1 is not involved in binding to CD81 (36,37), although no information is yet available on HCV envelope regions interacting with the putative alternative receptor(s) expressed on susceptible cells.…”

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

“…49 It thus seems doubtful that CD81 is the only cell surface receptor for HCV and indeed, it has been shown that CD81 has a poor capacity to mediate virus entry into hepatocytes. 50 In summary, it appears that variants in the LDLR gene, which influence the pathogenesis of FH and related diseases, may contribute to variation in severity of fibrosis, inflammation and response to treatment in hepatitis C. There is a complex interaction between IFN-␣ and LDLR activity, which could play an important role in these processes. LDLR activity appears to be increased by IL-1, 51 which is downregulated by its receptor antagonist, IL-1RA.…”

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

“…The specific interaction with the human tetraspanin CD81-LEL region has been regularly used to monitor the functional properties of HCV E2 glycoprotein [20,21]. Using a recombinant human CD81-LEL (residues 116-202) fusion protein with GST (Glutathione S-Transferase),…”