HCV entry is a multistep event involving a number of host factors, including heparan sulfate proteoglycan, LDLR, SR-BI, CD81, CLDN1, and OCLN,8,18
all of which are located on the plasma membrane of permissive cells. These cellular factors now offer promising targets for novel antiviral treatments because viral entry is necessary for disease initiation, spreading, and transmission. For example, antibodies against CD81, SR-BI, and CLDN1 extracellular regions have been shown to block viral entry.19,20
Further, compounds such as ITX 5061, a SR-BI antagonist, or atriazine compound called EI-1 also inhibit HCV entry at a postbinding step.21
More strikingly, a novel peptide derived from the N terminus of HCV NS5A protein exerts a broad spectrum of virocidal effect on HCV and several other enveloped viruses.22
Through peptide library screening and rational design, we obtained a novel peptide, CL58, derived from human CLDN1, which potently inhibited HCV entry at a postbinding step. Together, our findings provide a proof of principle that a new class of inhibitors that block virus-host interactions may be developed.
The finding that CL58 inhibits HCV entry is interesting for two reasons. First, a number of peptides derived from OCLN ELs have been reported to induce endocytosis of TJ proteins and interfere with TJ integrity.13–16,23
Similarly, addition of a CLDN1 EL1 peptide (residues 53–80) to polarized cells interferes with epithelial barrier function.17
These findings make CLDN1 a relatively less attractive target for anti-HCV therapies, because reagents targeting CLDN1/OCLN ELs will likely cause leakage of important cellular barriers due to disrupted TJs. In sharp contrast, CL58 contains the first 18 aa of the CLDN1 N terminus but has no effect on CLDN1/OCLN distribution and is noncytotoxic at doses that exert potent antiviral activity. Thus, CL58 can potentially be a lead peptide for further design of useful therapeutics.
Second, the observed inhibitory kinetics of CL58 suggests that CL58 acts at a postbinding stage of virus entry. Interestingly, another study has also implicated CLDN1 in a late step during in HCV entry, perhaps after viral engagement of CD81.6
In this study, CL58 retained its inhibitory activity when added at even later time points than anti-CD81 antibody. Interestingly, Flag-tagged CL58 immunoprecipitated with HCV E1E2. Therefore, it is possible that CL58 readily penetrates lipid membrane owing to its small size and hence becomes capable of interacting with HCV E1E2. However, what this interaction means to CL58-mediated inhibition remains unclear. It will be interesting if such interaction disrupts the yet-to-be confirmed interactions between HCV glycoproteins and endogenous CLDN1 or the CLDN1-CD81 complex.24,25
Although we are unable to nail down either possibility (data not shown), the observation that CL58 also inhibited cell-cell fusion mediated by HCV glycoprotein and CLDN1 warrants further investigation in its ability to inhibit intracellular fusion between HCV and cellular membranes. It is noteworthy that TJ was first depicted as a fusion of the outer lipid leaflets of adjacent cell membrane bilayers (hemifusion).26
Regardless of its direct target, the anti-HCV activity is unique to CL58, but not those peptides derived from the respective region of CLDN6, CLDN7, and CLDN9.
In conclusion, the identification of CL58 now adds new tools in developing novel antiviral drugs that target HCV entry. This reagent will also aid to dissect the molecular mechanisms of HCV entry. Although most small molecule inhibitors that have advanced to the clinic target viral components, the peptide inhibitor described here may offer advantages, because it targets cellular proteins that are required for HCV infection and hence reduce the likelihood of developing resistance. By virtue of its distinct mechanisms of inhibition, CL58 may be used in combination with other anti-HCV drugs for potential synergistic effects in treating HCV infections.