Function and mechanism of the HCV p7 channel and its therapeutic potential

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 DESCRIPTION (provided by applicant): Many viral genomes encode small integral membrane proteins that form channels in membrane, and they conduct protons, cations, and other molecules across the membrane barrier to aid various steps of viral entry and maturation. These viral proteins, collectively named viroporins, are crucial for viral pathogenicity and have been pursued as antiviral targets. The viroporin p7 encoded by the Hepatitis C virus (HCV) genome is one of the more important viral channels that has been validated as a target for developing drugs for treating HCV infections. p7 is an integral membrane protein that oligomerizes to form channels with cation selectivity, for Ca2+ over other cations; it has been shown to facilitate assembly and egress of infectious virions. The name "viroporin" assigned to p7 suggests a simple pore until our lab showed in Ouyang et al Nature 2013 that p7 adopts a rather sophisticated mode of hexameric assembly. The novel architecture implies a new channel mechanism developed by the HCV, but at present, the structural bases for Ca2+ selectivity and ion conduction are unknown. It has been shown that blocking p7 channel activity reduces production of infectious viral progeny, and several compounds have already been identified. These drug interactions should provide useful information for rational drug development, but there is no information on how and where these compounds act in the p7 channel. Furthermore, despite the consensus that p7 channel activity is important for virus production, it remains unclear in which of the steps of assembly and release is the role of channel activity required. We will capitalize on the recent p7 channel structure and utilize a multidisciplinary approach involving structural biology, channel recording, molecular virology, and medicinal chemistry to address these key questions. In Aim 1, we will determine the structure of the p7 channel in bicelles, identify key residues important for cation binding and conduction, and investigate conformational exchange relevant to the conduction mechanism. In Aim 2, we will separate the channel activity of p7 from its role in protein-protein interaction through mutagenesis to understand the role of p7 channel activity during virus infection. Finally in Aim 3, we will identiy the inhibitor binding sites, delineate mechanism of inhibition, and explore strategies to develop new inhibitors. The knowledge to be gained from the proposed research may give rise to new opportunities for developing compounds for treating HCV infections.