Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Hepatitis

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.

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...

Angiopoietin-like (ANGPTL) proteins are secreted proteins showing structural similarity to members of the angiopoietin family. Some ANGPTL proteins possess pleiotropic activities, being involved in cancer lipid, glucose energy metabolisms, and angiogenesis. ANGPTL7 is the less characterized member of the family whose functional role is only marginally known. In this study, we provide experimental evidences that ANGPTL7 is over-expressed in different human cancers. To understand the role played by ANGPTL7 in tumor biology, we asked whether ANGPTL7 is endogenously expressed by malignant cells or in response to environmental stimuli. We found that ANGPTL7 is marginally expressed under standard growth condition while it is specifically up-regulated by hypoxia. Interestingly, the protein is secreted and partially associated with the exosomal fraction, suggesting that it could be found in the systemic circulation of oncologic patients and act in an endocrine way. Moreover, we found that ANGPTL7 exerts a pro-angiogenetic effect on human differentiated endothelial cells by stimulating their proliferation, motility, invasiveness, and capability to form capillary-like networks while it does not stimulate progenitor endothelial cells. Finally, we showed that ANGPTL7 promotes vascularization in vivo in the mouse Matrigel sponge assay, thereby accrediting this molecule as a pro-angiogenic factor.