HPI does not appear to bind like most peptidomimetic inhibitors because neither the Q526A nor H528A substitutions influenced the ability of HPI to inhibit the NS3 protease. subgenomic dengue computer virus replicon,22 and no antiviral activity, and no effect on cell viability were observed (Fig. 2A). To test HPI on a wider variety of HCV genotypes, genotype 3a and 4a hepatitis C computer virus replicons23 were also used to examine the antiviral activity of HPI. About half the concentration of HPI was needed to lower RNA levels of both the genotype 3a and 4a replicons by 50% than was needed to lower the concentration of the genotype 1b replicon to the same extent (Fig. 2B). When colony-formation assays were used to compare the effect of HPI on HCV genotype 1b and 2a replicons, some antiviral activity was noted against genotype 2a (Fig. 2C). Open in a separate window Physique 2 HPI specificity. (A) The ability of HPI to reduce cellular content of luciferase tagged subgenomic replicons made from HCV genotype 1b (HCVsg 1b(con1), circles), HCV Ro 90-7501 genotype 2a (HCVsg 2a(JFH1), squares) and dengue computer virus strain 2 (DENVsg 2, triangles) (B) Effect of numerous HPI concentrations on relative levels of subgenomic replicon RNA, as measured by quantitative reverse-transcriptase PCR, with data normalized to RNA levels seen in cells treated with DMSO only. (C) Colony formation models (CFU) of Huh7.5 cell cultures harboring the HCVsg 1b(con1) or the HCVsg 2a(JFH1) replicon. Cells were in the beginning plated at 2 105 cells/dish, and G418-resistant colonies were stained with crystal violet after 3 weeks of antibiotic selection. Note CFUs for the HCVsg 2a(JFH1) replicon were about 10 occasions higher than CFUs observed with HCVsg 1b(con1) in the absence of HPI or telaprevir. (D) Unique residues in genotype 2a(JFH1) are highlighted around the scNS4A-NS3 structure in which HPI is usually docked. Residues pesent in 2a(JFH1) NS3 but not genotypes 1a(H77), 1b(con1), 3a(S52), Ro 90-7501 or 4a(ED42) are highlighted as spheres with unique amino acids within 5 ? of HPI noted with arrows. Sequence alignments are shown in Physique S1 (Supporting Information). To understand why HCV genotype 2a seems to be less sensitive to HPI than HCV genotypes 1b, 3a, and 4a, we aligned the replicon sequences (Fig. S1, supporting information) and examined the location of amino acids present in genotype 2a but not the other HCV genotypes (Fig. 2D). Forty-one amino acids in genotype 2a NS3 are not conserved in the other three genotypes, and these are evenly distributed throughout each NS3 domain name. While any of these substitutions could explain the resistance of genotype 2a to HPI, three unique genotype 2a residues are within 5 ? of the site in which HPI can bind NS3 in a computer-generated model Rabbit Polyclonal to Cytochrome P450 2A6 (observe below). For example, Ala482 replaces a proline in the other genotypes. In the model, Pro482 appears to contact the fluorinated end of HPI. Two conserved threonines near HPI in the model are similarly not present in genotype 2a. Thr295 contacts the other end of HPI, and Thr435 contacts the center of HPI in the model (Fig. 2D). HPI has higher barrier to resistance than the protease inhibitor telaprevir Ro 90-7501 To better understand how HPI might interact with NS3, we next attempted to select for HCV alleles encoding HPI resistance. Even after continued incubation of numerous replicon-bearing cell lines with HPI, no noteworthy resistance to HPI could be detected. For example, when HCVsg 1b(con1) Huh7.5 cells were incubated with telaprevir for 3 weeks, the cells became resistant to telaprevir (Fig. 3A). In contrast, when the same cells were incubated twice as long with HPI, the sensitivity of the cell collection to HPI did not change more than 2-fold (Fig. 3B), and no mutations Ro 90-7501 could be detected in the NS3 region. Cells that become resistant to telaprevir upon incubation retained sensitivity to HPI, and cells that were incubated with HPI retained sensitivity to telaprevir (data not shown). Open in a separate windows Physique 3 Development of HCV resistant to telaprevir and HPI. (A) Sensitivity of the HCVsg 1b(con1)-luciferase activity remaining after exposure to numerous amounts of each telaprevir or HPI for 3 days. We next examined if HPI was able to reduce cellular replicon levels if the replicons contained the telaprevir-resistant mutations R155K24 and V36A.25 In control experiments, 4.2 times more telaprevir was required to inhibit replication of HCVsg 1b(con1) replicons harboring a R155K by 50% than was needed to.