Determinants of Hepatitis C Virus p7 Ion Channel Function and Drug Sensitivity Identified In Vitro

Abstract

Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two transmembrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.Hepatitis C virus (HCV) chronically infects 170 million individuals and is a major cause of severe liver disease such as cirrhosis and hepatocellular carcinoma. Acute HCV infection is asymptomatic which, combined with the lack of an available vaccine, means that the majority of carriers are unaware of their positive status. Thus, clinical intervention takes place upon the presentation of symptoms when liver damage is already extensive and when the virus is well established. Current therapy comprises a combination of pegylated alpha interferon (IFN-␣) with ribavirin (Rib), which is effective in only 50% of cases and is both expensive and poorly tolerated by patients. This relatively low success rate is due to the highly prevalent, IFN-resistant genotype 1 viruses; other genotypes generally respond well to treatment (27). As IFN-Rib acts primarily via stimulation of the immune system, improving current therapy relies on the development of new, virus-specific drugs. A small number of polymerase and protease inhibitors are at late stages of development, but progress has been hampered by the inability until recently to culture HCV in vitro (21, 40, 45). The highly variable nature of HCV, however, means that new drugs will most likely have to be used in combination, making expansion of available drug targets and the development of new inhibitors a major research focus.HCV is the prot...

Viroporins: structure, function and potential as antiviral targets

“…However, genotype 1a (H77) channels retain His17 and are not pH-activated (Atkins et al, 2014;Chew et al, 2009;Li et al, 2012). Ser/Tyr21, Trp30 and Tyr/His31 have also been shown to modulate channel activity and/or infectious virion production in various studies (Brohm et al, 2009;Steinmann et al, 2007a;StGelais et al, 2009). A Phe25Ala mutation generates hyper-conductive genotype 1b and 2a channels in vitro (Foster et al, 2011), consistent with channel models based upon the PDB ID: 3DZ0 1b monomer structure where it forms a hydrophobic 'gate-like' constriction (Foster et al, 2014).…”

“…The stoichiometry of p7 channel complexes has been reported as both hexameric and heptameric in membrane-mimetic detergents and lipid bilayers, with some studies reporting mixtures of both forms (Clarke et al, 2006;Griffin et al, 2003;Luik et al, 2009;OuYang et al, 2013;StGelais et al, 2009;Whitfield et al, 2011). Molecular dynamics confirms that both species are theoretically viable, although both display a degree of metastability (Chandler et al, 2012).…”

“…Furthermore, high efficiency particle-producing chimaeric HCV strains yield measurable infectivity despite carrying p7 basic loop mutations (albeit with *1000-fold reduction in titre); mutant-derived virions possessed equivalent specific infectivity to that of WT chimaeric HCV (Steinmann et al, 2007a). However, loop mutations likely disrupt p7 channel activity indirectly rather than by the formation of inactive channel complexes, via effects upon protein processing/stability and membrane insertion (Bentham et al, 2013;Pérez-Berná et al, 2008;StGelais et al, 2009). Thus, it is possible that the low level of infectious virions produced in this scenario in fact retain intact channel complexes.…”

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

“…Single-particle electron microscopy recently allowed the three-dimensional structure of the hexameric p7 ion channel to be determined at 16 Å resolution, revealing a flower-shaped protein architecture with six protruding petals oriented toward the endoplasmic reticulum lumen (23). However, no detailed structure of p7 monomers has been reported hitherto, and currently proposed p7 models are based on secondary structure predictions (23)(24)(25)(26). Electrophysiology experiments indicate that the predicted N-terminal helix lines the p7 oligomer pore (27,28), a finding consistent with proposed p7 oligomer models.…”

“…5A, the hairpin motif features a doubleinteraction formed by three stacked aromatic residues (i.e. Phe 26 , Tyr 45 , and Trp 30 ) (see supplemental Fig. S4).…”