Through cell splitting, recombinants were further passaged with PIs and 10 clones of each culture sequenced to identify variants with low and high copy numbers. To determine whether identified substitutions conferred resistance to the corresponding PI, they were introduced into the original recombinant by site-directed mutagenesis. Recombinants containing single mutations were then assessed for their susceptibility towards PIs as described above. No contribution to the study design, collection or analysis of data, writing, or publication
decision was made by the funding source (BBSRC). The intra- and intergenotypic chimeric recombinant viruses J1b1b, J2a2a-T1066S, J3a3a, J4a4a-19, J5a5a-Q1247L, and J6a6a-V1040L were chosen to represent each major genotype. Naïve Huh7.5 cells were infected with infectious virus or alternatively electroporated with synthetic RNA and the reduction in frequency of NS5A-positive cells upon Tyrosine Kinase Inhibitor Library order PI treatment assessed (Fig. 2A; individual median inhibitory concentration [IC50] values and a comparison with those from BILN
2061 are listed in Table 1). Danoprevir showed a similar efficacy pattern of genotype susceptibility we determined for BILN 2061, a structurally similar cyclic PI.16 The chimeras J1b1b, J4a4a-19, and J6a6a-V1040L showed a 100- to 400-fold greater susceptibility postelectroporation than J2a2a-T1066S, J3a3a, and J5a5a-Q1247L (Fig. 2A). The susceptibility of the infectious clones J2a2a-T1066S, 上海皓元医药股份有限公司 J5a5a-Q1247L, and J6a6a-V1040L to danoprevir as determined by measurement of selleck chemicals llc infectivity reductions closely matched results from the replication assays. In particular, genotype 6a showed a similar
greater susceptibility to danoprevir treatment postinfection than genotypes 2a, 3a, and 5a (Fig. 3A). Genotype-associated differences in susceptibility to telaprevir were also observed. This linear PI showed the greatest efficacy posttransfection in genotype 1b and 6a-infected cells (Fig. 2B, Table 1; IC50 values of 840 and 650 nM). Genotypes 2a and 3a showed intermediate susceptibility to telaprevir posttransfection (1,100 and 1,410 nM), whereas genotypes 4a and 5a were resistant (2,300 and 2,700 nM). Similarly, genotype 6a infectivity reductions were over 5 times greater than those of genotypes 2a, 3a, and 5a (Fig. 3B). To identify mutations conferring resistance to the PIs, Huh7.5 cells, transfected with the different recombinants, were passaged by cell splitting under subinhibitory concentrations of the individual PI as described.16 The addition of increasing concentrations of PI did not result in any visual cytopathic effects. Substitutions indicative of resistance development were those that occurred in both replicas of the experiment but not in the control, many of which corresponded to those previously observed in treated patients.