Rice, and J. 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 genus in the family (33). Its genome encodes a single 3,000-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 proteinsthe core protein and envelope glycoproteins E1 and E2are Rabbit polyclonal to PIWIL3 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, Cethromycin 46, 50, 51). Among these receptors, human CD81 has been repeatedly shown to interact with recombinant soluble E2, the E1E2 complex, HCV-like particles, and HCV particles from infectious plasma (3, 8, 17, 22, 27, 30, 31, 35, 42, 45, 48, 55, 59). CD81 is a member of the tetraspanin family, which contains four TMDs, short intracellular domains, and two extracellular loops designated the small extracellular loop and the large extracellular loop (LEL). CD81 is widely expressed and is associated with other membrane proteins that vary in different cell lineages (32). The CD81 binding site for E2 has been localized within the LEL (45), and specific LEL amino acid residues essential for this interaction have been identified (17, 29). By contrast, the E2 Cethromycin regions involved in CD81 interaction are not well defined. The E2 glycoprotein is one of the most variable HCV proteins and is Cethromycin characterized by hypervariable region 1 (HVR1) and HVR2. Previous reports have shown that HVR2 and/or adjacent residues are involved in the interaction with CD81 (22, 48, 60). Cethromycin In contrast, E2 lacking or containing HVR1 binds equally well to CD81 (24, 43). A more recent study demonstrated that a region comprising residues 613 to 618 is essential for binding to CD81 and that a complex interplay between HVR1 and HVR2 may modulate E2-CD81 interactions (48). The exact mechanisms whereby HCV establishes and maintains its persistence, which, in turn, leads to liver damage and immune modulation, are not well understood. Nevertheless, it has been shown that CD81 engagement by a truncated form of E2 (E2661) provides a costimulatory signal for human T cells (57) and an inhibitory effect on natural killer functions (14, 56). Because of the lack of a suitable cell culture system for propagation of HCV in vitro and the unavailability of virions in sufficient quantities, truncated, secreted versions of E2 have been used as surrogates for native virus particles. Indeed, the identification of CD81 as a putative cellular receptor for HCV is based on its binding to E2661 (45). Intriguingly, intracellular forms of truncated E2, enriched for the presence of monomeric, nonaggregated E2, were found to bind CD81 with greater affinity than the secreted forms (21, 28), suggesting that structural differences exist between the intracellular and secreted forms of the E2 glycoprotein. Indeed, several monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes.