Simeprevir is a novel NS3/4A protease inhibitor (PI) of hepatitis C virus (HCV). The baseline polymorphism NS3-Q80K is frequently observed in genotype (gt) 1a HCV and often associated with treatment failure in simeprevir-containing regimens. We aimed to elucidate mechanisms of treatment failure due to NS3-Q80K. We included a Q80R mutation in our study and generated a series of Huh-7.5 cells, each of which harbored either wild-type gt 1a H77S.3 or one of the variants, Q80K or Q80R. The cells were cultured with increasing concentrations of simeprevir, and NS3 domain sequences were determined. The mutations identified by sequence analyses were subsequently introduced into H77S.3. The sensitivity of each mutant to NS3/4A PIs simeprevir, asunaprevir, grazoprevir, and paritaprevir was analyzed. We introduced the mutations into gt 1b N.2 and compared the sensitivity to simeprevir with that in gt 1a H77S.3. While simeprevir treatment selected mutations at residue D168, such as D168A/V in the wild-type virus, an additional mutation at residue R155, R155K, was selected in Q80K/R variants at simeprevir concentrations lower than 2.5 μM. Sensitivity analyses showed that simeprevir concentrations of less than 1 μM significantly boosted the replication of Q80K/R+R155K variants. Interestingly, this boost was not observed with the other NS3/4A PIs, and not in Q80R+R155Q/G/T/W variants or gt 1b. The boosted replication of Q80K+R155K by simeprevir could be related to treatment failure in simeprevir-containing antiviral treatments in gt 1a HCV-infected patients with the NS3-Q80K polymorphism. This result provides a new insight into how resistance-associated variants can cause treatment failure.
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