Inhibition of CD81-CLDN1 coreceptor interaction was specific as shown by the unchanged FRET between

CD81-CD81 and CLDN1-CLDN1 following preincubation with anti-CLDN1 serum. Taken together, these data suggest that anti-CLDN1 antibodies interfere with CD81-CLDN1 heterodimer association. For the first time, we report the genesis and characterization of antibodies Fostamatinib solubility dmso directed against the extracellular loops of human CLDN1 that inhibit HCV infection. CLDN1 showed no evidence for a direct association with the viral envelope E1E2 glycoproteins, and yet anti-CLDN1 serum inhibited E2 association with the cell surface and disrupted CD81-CLDN1 interactions. These data suggest a role for CD81-CLDN1 complexes in viral entry and highlight new antiviral strategies targeting coreceptor complex formation. CLDN1 is an essential cofactor conferring HCV entry9; however, the precise role of CLDN1 in the multistep entry process remains poorly understood.

Using antibodies directed against CLDN1 EL, we demonstrate a dose-dependent inhibition of viral envelope association with HCV permissive cell Compound Library supplier lines. Using transfected CHO cells expressing human HCV entry factors, we demonstrate that in contrast to CD81 and SR-BI, CLDN1 does not directly interact with envelope glycoprotein E2 at the cell surface. Using a recent FRET-based system to study CD81-CLDN1 coreceptor association,17 we demonstrate that neutralizing anti-CLDN1 antibodies specifically disrupt CD81-CLDN1 FRET (Fig. 8). These data suggest that CD81-CLDN1 coreceptor complexes are critical for HCV entry, and CLDN1 may potentiate CD81 association with HCV particles by way of E2 interactions. The functional relevance of the CD81-CLDN1 coreceptor complex for HCV entry is further corroborated by kinetic studies demonstrating that CD81 and CLDN1 act at a similar time point during HCV entry (Fig. 5).

Although the magnitude of antibody-mediated inhibition of HCVcc infection was medchemexpress slightly different, the kinetics of inhibition by anti-CLDN1 and anti-CD81 antibodies were similar (Fig. 5C-F, Table 2). Using an HCVpp kinetic assay in 293T cells expressing Flag-tagged CLDN1 and anti-Flag antibody, Evans et al.9 observed anti-Flag antibody inhibition of HCVpp infection at a later time point than anti-CD81, suggesting that CLDN1 has a role in late stages of the viral internalization process. Evans et al. reported that the inhibitory activity of anti-CD81 antibody was lost much earlier than the anti-Flag antibody (half-maximal inhibition at 18 and 73 minutes post–temperature shift, respectively). However, we observed a loss of anti-CLDN1 and anti-CD81 inhibitory activity at similar times (half-maximal inhibition for both antibodies at +30 and +33 minutes post–temperature shift, respectively). Comparable results using HCVpp infection of 293T/CLDN1 cells (Fig. 5F) suggest that the differences between the two studies relate to the inserted Flag epitope in CLDN1 sequence or the use of an anti-Flag antibody.