The low fitness cost for these mutations observed in culture implies that a minimal genetic barrier to their selection would exist in vivo, explaining the perceived lack of efficacy for p7 inhibitors in clinical trials. HCV IFN/Rib resistance is a multifactorial phenomenon, involving virus and host-associated factors. This is distinct to resistance against direct-acting STAT-C antivirals, which are host-independent and mediated through single HCV point mutations. According to quasispecies theory, all possible single variants exist within Staurosporine ic50 an HCV-infected individual,

with selection dependent on fitness. Generation of dual, triple, and further variants becomes exponentially less Saracatinib molecular weight likely and forms the basis for the successful application of combination therapies. Combination of IFN/Rib with single STAT-C molecules targeting replication therefore suppresses HCV replication through distinct mechanisms. As such, IFN/Rib-resistant HCV will rapidly become resistant to a third STAT-C drug,

depending on fitness cost and drug potency, because it is essentially a monotherapy. For virus assembly inhibitors, resistance would be expected to arise all the more rapidly in IFN/Rib-resistant viruses as no suppression of genome replication occurs. Combinations of assembly inhibitors, however, can suppress RNA virus resistance.37 Our demonstration of distinct, specific antiviral effects for two classes of p7 inhibitor therefore

supports that combination with STAT-C therapies, rather than IFN/Rib, may enhance patient responses, because the genetic barrier to dual resistance would be significantly raised. Given that prototype p7 inhibitors have been trialed in patients (amantadine, rimantadine, UT-231b [IS] and BIT225 [amiloride]), these could be rapidly deployed alongside other STAT-C compounds. Our approach was necessarily Dipeptidyl peptidase based on molecular modeling of p7 ion channel complexes. Models comprised a lumenal N-terminal helix with a conserved His17 proton sensor, analogous to M2 His37. Cu2+-mediated inhibition confirms His17 as lumenal,35 and lowered pH activates GT1b p7.33 Accordingly, modeling p7 under acidic conditions where His17 is protonated induced an opening of the structure (Fig. 1A). We recently showed that p7 induces vesicle alkalinization, protecting intracellular virions from reduced pH.19 Because low pH induces the fusogenic action of HCV glycoproteins,38 p7 may act analogously to M2 from certain influenza A virus strains, where it prevents such change in hemagglutinin.39 Interestingly, secreted HCV virions are acid resistant,19, 40 meaning that an as-yet unidentified maturation event occurs at a late stage of virion production where particles are acid-stabilized. Accordingly, p7 inhibitors do not reduce intracellular infectivity (Fig. 2D), supporting a post-assembly role for p7 proton channel function.