Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel

Abstract

The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of highresolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. Conclusion: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies. (HEPATOLOGY 2011;54:79-90) H epatitis C virus (HCV) infects over 3% of the population, causing severe liver disease. Current therapy comprising pegylated interferon (IFN) and ribavirin (Rib) is inadequate, which, combined with high cost and poor patient compliance, has driven the demand for new virus-specific drugs.1 Future standard of care will replace IFN/Rib with combinations of specific inhibitors, such as seen for human immunodeficiency virus (HIV) therapy. However, extensive HCV variability raises concerns over the ability of relatively few compounds to suppress resistance. Thus, great effort focuses on expanding the repertoire of HCV drug targets, expedited by the availability of the Japanese fulminant hepatitis clone 1 (JFH-1) infectious isolate.

Viroporins: structure, function and potential as antiviral targets

“…p7 channels also adopt multiple conductance states and exhibit 'burst activity', with a strong influence afforded by the membrane environment, potentially via effects on the overall channel structure. p7 from a variety of genotypes has also been shown to conduct small molecules, such as the pH-sensitive fluorophore HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) (Wozniak et al, 2010) and carboxyfluorescein (StGelais et al, 2007), indicative of channel-pore dualism; one study recently questioned the relevance of such behaviour (Gan et al, 2014), yet indirect systems are widely utilized in viroporin studies, including by these same authors , and results faithfully and consistently reproduced those obtained for infectious HCV culture (Foster et al, 2011(Foster et al, , 2014Griffin et al, 2008;Wozniak et al, 2010). In this regard, the ability of p7 to mediate proton conductance within infected Huh7 cells remains the only activity for which a biologically relevant function has been assigned within the HCV life cycle (Wozniak et al, 2010), although roles for other observed conductances cannot be ruled out.…”

“…Viable full-length HCV containing IRES elements inserted between E2 and p7 or p7 and NS2 argued against a functional role for p7 precursors (Jones et al, 2007). Both early-and late-acting defects in virion production have been described where p7 was (partially) deleted, mutated at specific residues or treated with inhibitors (Bentham et al, 2013;Foster et al, 2011Foster et al, , 2014Jones et al, 2007;Steinmann et al, 2007a;Vieyres et al, 2013;Wozniak et al, 2010). This is now known to result from p7 performing multiple functions within infected cells, comprising distinct protein-protein interactions as well as its channel-forming activity.…”

“…p7 channel activity appears to influence a late-acting phase of the HCV life cycle, distinct from that concerning protein-protein interactions; whereas p7 deletions and deleterious point mutations abrogate infectivity in all compartments (Atoom et al, 2013;Bentham et al, 2013;Brohm et al, 2009;Jones et al, 2007;Steinmann et al, 2007a;Wozniak et al, 2010), small-molecule p7 inhibitors prevent the accumulation of secreted, but not intracellular infectivity (Foster et al, 2011(Foster et al, , 2014. Point mutations recapitulating the p7 inhibitor-induced phenotype have not been identified, yet unlike (partial) deletion mutants (Brohm et al, 2009), infectivity of HCV carrying mutations to the basic loop region (to either Ala or the less hydrophobic Glu) can be partially restored by trans-complementation with IAV M2 or by treating cells with the V-type ATPase inhibitor bafilomycin A (Bentham et al, 2013;Wozniak et al, 2010).…”

“…As the majority of intracellular HCV infectivity is known to reside in the pH-neutral ER (Gastaminza et al, 2008), p7 likely controls a secretory 'bottleneck' with relatively few virions passing through acidic compartments at a particular time. Hence, secreted rather than the bulk of cell-associated infectivity is sensitive to p7 inhibitors (Foster et al, 2011(Foster et al, , 2014. However, HCV cell-to-cell spread appears less sensitive to the effects of p7 inhibitors (Meredith et al, 2013), suggesting that this pathway may be less dependent on channel activity, albeit with genotype variability.…”

“…In support of this notion, p7 influences the acid stability of secreted particles (Atkins et al, 2014) and non-infectious intracellular particles are present within cells harbouring loop mutant JFH-1 (Bentham et al, 2013), although these may also retain envelopment defects . However, a conclusive answer to this question should be achievable in the near future, given recent advances in the purification of infectious HCV particles (Catanese et al, 2013) and the identification of p7 inhibitors resistant mutants (see below) (Foster et al, 2011).…”