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...