Structure and Dynamics of Membrane-Associated Hepatitis C Virus Protein P7 by NMR Spectroscopy

Abstract

P7 is a 63-residue viroporin encoded by human hepatitis C virus (HCV). It has ion channel activity and plays essential roles in viral proliferation. The NMR results show that the protein consists of two transmembrane spanning regions connected by a cytoplasmic loop. The solution NMR structural data that has been acquired includes: hydrogen/deuterium exchange, paramagnetic relaxation enhancement, residual dipolar couplings, and bicelle ‘q-titration.’ These data demonstrate that the protein has a range of dynamic properties, and interestingly two segments in each of the membrane spanning helices display local motions, possibly related to function. Oriented Sample (OS) solid-state NMR spectra of p7 in aligned phospholipid bilayers provided the tilt angles of the helical segments, 25° and 10° to the membrane normal. Recent magic angle spinning (MAS) solid-state NMR data of p7 in liposomes are being used to determine the three-dimensional structure. It has been shown that known channel-blocking compounds inhibit the ion channel activity. This and the fact that knockout models of this protein inhibit infectivity of the virus indicate it may be an ideal target for future drugs. There are six major genotypes and over 100 relevant subtypes. Our current studies aim to investigate the structure and dynamic differences among the genotypes. In addition to studying different genotypes, specific mutations to the HCV J4 genotype were made in order to modulate the dynamics of the protein and obtain additional secondary structural information. These studies have the potential to identify the reasons why different genotypes have varying activity and drug binding affinities.