A systematic in vitro investigation of the NS5A sequence of subject P identified substitutions that delivered the antiviral response observed. Hybrid replicons containing the entire NS5A sequence or the N-terminal 129 amino acids of NS5A- or NS5A-specific
amino-acid substitutions from subject P were analyzed. The studies revealed that the BL NS5A variant, E62D, did not confer any detectable resistance to BMS790052, but when combined with Q30R, the double substitution variant (Q30R-E62D) conferred a very high level of resistance. Although Q30 is not present in either of the published crystal structures,20, 21 these structures show that residue E62 is located adjacent to Zn++ coordinate residues C57 and C59. In fact, we predict that the E62D substitution may affect Q30R resistance by influencing Zn++ binding. Studies Selleckchem Tofacitinib to determine if this substitution, when combined with Q30R, would affect Zn++ binding are in progress. Hybrid replicons with the entire H77c NS5A that were replaced with NS5A derived from either BL or day 14 specimens of subject P had decreased, but measurable, replication ability (replication window 3-32; Table 2A). However, little or no replication signal was detected when the first 129 amino acids of the NS5A replicon were Small molecule library solubility dmso replaced with sequence from clinical specimens, suggesting
that the mixing of domains from different NS5A proteins (convenient for cloning) may impair the formation of a proper replication
complex. Our ability to isolate replication-competent cell lines containing the first 129 amino acids of NS5A from clinical specimens indicated that compensatory mutation(s) must have been selected to enhance replication ability. We did not attempt to identify these mutations, because the primary aim was to resolve the discrepancy between the in vitro and in vivo resistance profiles. When two specific amino acids (Q30R-E62D) were substituted, the replication ability of the replicon was preserved (Table 5) selleck kinase inhibitor and a reliable EC50 value was determined. Unlike the HCV-infected population, both lab-strain replicons (H77c and Con1) were constructed from consensus sequences.22, 23 Isolated replicon cell lines have adaptive mutations that enhance HCV RNA replication ability and differ under different selective pressures. In general, the genomic sequences of these cell lines are more homogenous at both the RNA and amino-acid sequence level than clinical isolates. This study indicates that the heterogeneity of HCV sequences in infected specimens with no detectable level of previously identified resistant substitutions has a minimal effect on the potency of BMS-790052, a conclusion similar to that made from observations of compensatory mutations. However, the effect of the polymorphism, E62D, present in the heterogeneous BL sequence of subject P significantly affected the emergence of resistance.