Structural characterisation of cd81-claudin1 hepatitis c cirus receptor complex

Johnson & Johnson (J&J) invests in five therapeutic areas where there continues to be significant unmet need for novel drug development. One of these is anti-viral therapies, where it is now recognised that there is an urgency to design drugs that inhibit the early steps of viral infection. This contrasts with the current generation of protease and polymerase inhibitors which rapidly select viral variants in the clinic. Indeed, the rapidity at which these variants appear in treated subjects suggests viruses such as hepatitis C virus (HCV) are infecting new cells. The proposed collaboration addresses the company's strategic interest in developing new drugs targeted at infectious diseases such as HCV infections. By gaining an understanding of the structural and cell biology of key receptor molecules, novel discovery pipelines will emerge from this project. We propose that structural and functional characterisation of two membrane proteins each with 4 transmembrane domains, CD81 and Claudin-1 (CLDN1), will enable the mechanism underlying their protein-protein interactions to be elucidated. CLDN1 and CD81 form a receptor that enables HCV to infect cells. The oligomerisation status of these two co-receptor components is critical to this process. We have shown that CD81 oligomerises at the plasma membrane, with the identification of CD81-CD81 homodimers and CD81-CLDN1 heterodimers suggesting a recruitment of CLDN1 to CD81-enriched domains. Perturbation of CD81-CLDN1 complexes inhibits HCV entry, suggesting a critical role in the viral entry process. Furthermore, we have recently identified the amino acid residues in the first extracellular loop (EC1) of CLDN1 that define association with CD81 and viral co-receptor activity. The Bill laboratory contributes by providing access to suitable recombinant membrane protein targets and mutants of varying oligomeric states. The McKeating laboratory provides expertise in biological assays for these proteins, as well as having access to a range of unique antibody reagents. Bill and McKeating have previously published together, and have a number of additional manuscripts in preparation. Understanding these molecules will aid the design of therapeutic agents targeting viral infection. Our specific aims will address the following questions: A. What is the oligomeric status of CD81? To date, because of the lack of appropriate tools, the oligomeric status of CD81 and its family members (the tetraspanins) is largely unknown and the relationship between protein conformation and biological activity is poorly defined. We hypothesise that HCV uses dimeric CD81 to enter cells. We will test this hypothesis and identify protein motifs defining tetraspanin oligomerisation and receptor activity. Our ongoing crystallisation trials of monomeric and oligomeric CD81 (we have diffracting crystals of monomeric CD81) will complement this work, since there is no structural information available for any full-length member of either family. B. What is the oligomeric status of CLDN1? We recently reported CLDN1 dimerisation based on fluorescence resonance energy transfer (FRET) between tagged molecules, suggesting that dimers are the primary building block(s) of tight junction strands. Biophysical and structural characterisation will identify the oligomeric status and conformation used by HCV. C. What is the structural organisation of the CD81-CLDN1 co-receptor complex? We recently demonstrated that yeast-expressed CD81 and CLDN1 can bind HCV. Yeast does not have CD81 or CLDN1 homologues, allowing us to study (rather than indirectly in mammalian cells) the structural basis of CD81 and CLDN1 association and viral co-receptor activity. This collaborative research programme in membrane protein biochemistry and cell biology, in the context of the drug discovery pipeline, provides an exiting opportunity for all partners and especially a PhD student.