The Function Of Tight Junction Protein Claudin-1 In HCV Infection

Hepatitis C virus (HCV) is a serious global health threat. In most infected individuals, HCV establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. There is no vaccine and current medical treatment options are limited. Viruses initiate infection by attaching to molecules or receptors on the cell surface. Thus, major research efforts are focused on HCV receptors. Strong evidence exists for the involvement of the HCV E2-binding proteins-CD81 and SR-BI in HCV entry. However, these molecules are insufficient for productive viral entry because some cell lines express CD81 and SR-BI, but do not support HCV entry. Most recently, claudin-1 was found to be required in HCV entry and confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Claudin-1 is one of the major proteins forming backbone of tight junctions. It has four transmembrane helices, two extracellular loops (EL). The EL1 of claudin-1 is critical for HCV entry and antibodies directed against an epitope inserted in the claudin-1 EL1 block HCV infection. In addition, HCV cell-cell transmission is dependent on claudin-1 expression, suggesting a novel route for HCV evading neutralizing antibodies, which may contribute to persistent infection of HCV. Once the function of claudin-1 in HCV infection are better understood, it might be possible to reveal the mechanisms of HCV infection and pathogenesis and arm us with new strategies to antiviral therapy.Researchers have been hamepered by the lack of a robust cell-culture system yielding infectious virus until 2005, cell culture model of HCV was established in the lab of HCV center led by Charles M. Rice, Rockefeller University. This cell culture system of HCV generously provided by Prof. Rice facilitates our study of claudin-1 in HCV infection. Our study was as follows:1 Cell-culture derived HCV (HCVcc)Plasmid encoding the full length HCV chimaeric J6/JFH genome was linearized and used as template for in vitro transcription. RNA transcripts were produced by T7 RNA polymerase and electroporated into Huh-7.5 cells. The transfected cell culture media were collected and incubated with na?ve Huh-7.5 cells.2 The influence of the claudin-1's expression on the susceptibility to HCVccThe gene of claudin-1 was cloned from HepG2 cells. The eukaryotic expression vector pEGFP-claudin-1 was constructed by introducing claudin-1 DNA fragment into pEGFP-C3 vector. The plasmid was transfected into 293T cells with lipofectamine and the expression of claudin-1 was investigated by immunofluorescence and Western blot. Then the susceptibility to HCVcc was analysed after the expression of claudin-1 in 293T cells. Chemically synthesized siRNA of claudin-1 was transfected into Huh-7.5 cells to silence the expression of claudin-1 and the susceptibility to HCVcc was tested.3 The effect of Interferon-γon the tight junction and the susceptibility to HCVccInterferonγis a proinflammatory cytokine which influence the tight junction. It is critical in anti-HCV infection. We did primitive research to study the correlation between these factors. The polarized colorectal adenocarcinoma Caco-2 cells grown on Transwell PET membrane were similar to hepatocytes. The expression of CD81, SR-BI, and claudin-1 were evaluated by qRT-PCR and Western blot. The barrier function of tight junction was monitored by measurement of TER and FITC-labeled dextran. The distribution of CD81, SR-BI, and claudin-1 were examined by confocal microscopy after incubation with IFN-γ. The susceptibility of Caco-2 cells to HCVcc was tested.Results:1 The full length chimeric J6/JFH HCV transcripts were transcribed in vitro and electroporated into Huh-7.5 cells. The infectious HCV virus particles were generated.2 The claudin-1 gene was cloned from HepG2 cells and eukaryotic expression vector pEGFP-claudin-1 was constructed successfully. The plasmid was transfected into 293T cells using lipofectamine. The expression of claudin-1 confered susceptibility to HCV infection when expressed in 293T cells.3 The claudin-1 expression was reduced by chemically synthesized siRNA in Huh-7.5 cells. Susceptibility to HCVcc was markedly reduced in claudin-1- silenced Huh-7.5 cells compared with cells treated with an irrelevant siRNA.4 Caco-2 cells express CD81, SR-BI, and claudin-1, which were required for HCV entry. Caco-2 cells support HCV entry and replication.5 The down-regulation of claudin-1 induced by IFN-γtreatment resulted in disruption of barrier function. IFN-γtreatment also led to significant changes in the distribution of claudin-1, CD81 and SR-BI. Moreover, infection assays revealed that IFN-γ-treated Caco-2 cells showed decreased susceptibility to HCVcc infection.Conclusions: 1 The tight junction protein claudin-1 is one of the most important receptors of HCV. The expression of claudin-1 is important determinants of a cell's susceptibility to infection and of virus tropism for particular tissues.2 IFN-γtreatment-induced reduction in claudin-1 expression and redistribution of HCV receptors may play important roles in the inhibition of HCV infection.