Claudin-1, a new junction in the hepatitis C virus entry pathway


  • Potential conflict of interest: Nothing to report.

Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 2007;446:801-805. (Reprinted with permission.)

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, implicated, at least one factor critical to this process remains unknown (reviewed in refs 13). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.


With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. Research into HCV molecular biology and pathogenesis of related liver disease has progressed rapidly over the past decade; however, one area that has been thwarted is our understanding of the early events in HCV infection such as binding and internalization. Defining this process is fundamental to our understanding of the HCV life-cycle and pathogenesis, because receptor-mediated virus/host cell interactions are critical in determining tissue tropism and the outcome of infection. Studies of early events in the HCV life-cycle have been constrained by the lack of appropriate model systems; however, several in vitro models have been developed: these include the production of virus-like particles (HCV-VLPs) in insect cells, HCV envelope glycoproteins pseudotyped onto retroviral core particles (HCVpp), and most recently, the development of an HCV cell-culture system that produces infectious HCV virions (HCVcc) allowing the complete life-cycle of the virus to be studied (reviewed in Cocquerel et al.1 and Barth et al.2). Compounding these difficulties is the fact that many viruses require sequential interactions between viral proteins and multiple cellular factors. Nevertheless, based on use of soluble forms of the HCV glycoprotein E2 and a combination of the abovementioned in vitro model systems, it is widely accepted that the tetraspanin molecule CD81 and scavenger receptor class B type 1 (SR-B1) play a key role in HCV entry. However, some cell lines that express CD81 and SR-B1, either endogenously or via enforced expression systems, are not permissive; this suggests additional molecule(s) are necessary for HCV entry.

Enter Evans and colleagues who have identified Claudin-1 (CLDN-1) as the third factor critical for HCV entry.3 CLDN-1 is a 211-amino-acid protein with 4 transmembrane spanning domains that is highly expressed in the liver and is a key component of tight junction strands. The tight junction is a specialized membrane domain at the most apical region of polarized epithelial cells, including hepatocytes (Fig. 1). They function not only to create a primary barrier to prevent paracellular transport of solutes (barrier function) but also to restrict the lateral diffusion of membrane lipids and proteins, thereby maintaining the cellular polarity (fence function). With no particular leads on the identity of additional factor(s) required for HCV entry, Evans and colleagues used a purely iterative approach in which a lentivirus-based complementary DNA expression library approach derived from the human hepatoma cell line, Huh-7.5 (these cells are highly permissive for HCV infection and replication) was used to identify cellular factors that confer HCVpp entry in the nonpermissive 293T cell line (human renal epithelial cell line). The 293T cells are CD81+, SR-B1+ and thus express 2 of the necessary factors for HCV binding. No doubt to their delight, the researchers identified CLDN-1 as a factor that rendered 293T and SW-13 (adrenal carcinoma) cells permissive for HCVpp and HCVcc infection when it was overexpressed. Furthermore, the hepatoma cell line HepG2 that is SR-B1+, CLDN-1+ but CD81 became permissive for HCVpp or HCVcc only when CD81 was expressed exogenously. These results suggest that in these cell lines, a similar mechanism for HCV entry is being used that involves these 3 factors. However, this does not appear to be the only mechanism and/or factor(s) involved. HeLa cells, a cell line that supports HCV replication after direct transfection of HCV RNA (thus bypassing receptor-mediated mechanisms)4 and HepH cells (both CD81+, SR-B1+ CLDN-1) both did not support HCVpp entry when overexpressing CLDN-1. Moreover, murine CLDN-1 (90% amino acid identity to human CLDN-1) efficiently supports HCV entry. Thus, CLDN-1 is clearly not a determinant of species host range, and the above data strongly imply that other HCV entry factors remain to be discovered.

Figure 1.

Model of Hepatitis C virus (HCV) binding and entry. (I) HCV present in human blood interacts with known receptors such as CD81 and SR-B1 present on the basolateral surface of hepatocytes. (II) Whether binding of the viral envelope glycoprotein to tetraspanin CD81 or SR-B1 individually or in combination triggers signaling that causes (a) plasma membrane relocation of claudin-1 or (b) disruption of tight junctions, and therefore exposure of claudin-1, remains unknown. (III) Binding of viral envelope glycoprotein to surface receptors triggers clathrin-dependent endocytosis, mediating viral particle entry. Endocytosis is followed by transport of the viral particle to the endosome. Acidification of the endosome facilitates membrane fusion and penetration of the virus into the cytoplasm. Abbreviations: HCV, hepatitis C virion; SEC, sinusoidal endothelial cells; SR-B1, scavenger receptor B1.

Overall, the data of Evans and colleagues strongly suggest that a direct interaction between the first extracellular loop of CLDN-1 and the hepatitis C virion is likely to be involved. Using both mutagenesis and antibody-blocking strategies, they identified that discrete binding residues for HCVpp in the first extracellular loop of CLDN-1 were the crucial determinants for CLDN-1–mediated HCV entry. Despite these findings, evidence for direct binding is lacking. Although it is possible to envisage interactions between HCV, CD81, SR-B1, and CLDN-1 in an in vitro cell culture system, one question arises as to how HCV interacts with all 3 in the HCV-infected liver. CLDN-1 is normally sequestered between adjacent hepatocytes and is not accessible to the external environment (basolateral sinusoidal surface) where HCV and other known cellular receptors, such as CD81 and SR-B1, are found (Fig. 1). Antibody-blocking chase experiments demonstrate a temporal pattern for the CLDN-1 and CD81 interaction with HCVpp. Like CD81, CLDN-1 interacts with the virion downstream of the initial binding-entry event; however, CLDN-1 interaction may occur after that of CD81. However, the authors show HCVpp-mediated membrane fusion is CLDN-1 dependent. Taken together, these data fit the concept that CLDN-1 is a co-receptor and not the primary receptor for HCV entry. It is tempting to speculate that exposure of CLDN-1 to the virion can only take place after a prior event such as CD81–HCV binding, thereby triggering a colocalization event where interaction with HCV and uptake into the host cell can occur. A similar event has been demonstrated for coxsackievirus B, whose co-receptor is a tight junction associated protein.5 There is no doubt future studies delineating the precise function of CLDN-1 and the sequence of events leading to claudin-1 and HCV interaction will ensue, preferably using polarized hepatocyte-derived cells that mimic the in vivo situation. The authors also suggest that CLDN-1 is a potential therapeutic target to block HCV entry. Although this is an attractive proposition, CLDN-1 is also highly expressed in the kidney, and systemic disruption of tight junctions in both the liver and kidney may have severe physiological consequences. Only time will tell.