Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

KAY-2-41, a Novel Nucleoside Analogue Inhibitor of OrthopoxvirusesIn VitroandIn Vivo

“…If KAY-2-41 in its triphosphate metabolite form ultimately targets the viral DNA polymerase, our data suggest that the presence of the A314V or T831I mutation in the DNA polymerase gene (E9L) did not impact its ability to accept this substrate, whereas these changes clearly influenced the acceptance of acyclic nucleoside pyrimidine and purine analogues [i.e., cidofovir and (S)-HPMPDAP]. In fact, while these mutations were associated with different ANP resistance profiles when present in a CMLV or in a VACV backbone (in line with our published observations [35]), KAY-2-41 remained equally active against WT and mutant viruses bearing the A314V or T831I mutation. Interestingly, the CML14 and VACV A314V plus A684V double mutants appeared to be more sensitive to KAY-2-41 inhibition, suggesting that through these conformational changes, E9L might become more prone to accept the thymidine analogue triphosphate metabolite.…”

“…We therefore wondered whether KAY-2-41 could inhibit OPVs bearing mutations within E9L that are known to confer resistance to certain ANPs. In previous studies, we identified such amino acid substitutions in the exonuclease domain of E9L (A314V), in the polymerase domain (T831I), or in both domains (A314V plus A684V) (35). Recombinant CML1, CML14, and VACV harboring these amino acid substitutions were used, and the two ANPs cidofovir and (S)-HPMPDAP were included as control drugs to confirm the resistance phenotype of each strain ( Table 3).…”

“…Antiviral and cytostatic assays were performed as previously described (35). Briefly, confluent monolayers of HEL cells (in 96-well plates) were infected at a multiplicity of infection (MOI) of 0.01 PFU/cell for 2 h. Residual virus was removed and replaced with medium containing serial dilutions of the test compounds (in duplicate).…”

“…Sequencing of A48R (TMPK), E9L (viral DNA polymerase), D4R (uracil DNA glycosylase), B1R (Ser/Thr kinase), F10L (Ser/Thr kinase), and J2R (TK) of wild-type (WT) and drug-resistant clones was performed as follows: DNA was extracted from virus-infected HEL cells using a QIAamp DNA blood minikit (Qiagen Benelux B.V, Venlo, Netherlands) following the manufacturer's instructions. The genes A48R, D4R, B1R, F10L, and J2R were PCR amplified as one amplicon, and the full-length E9L gene was amplified as five (CMLV) or four (VACV and CPXV) overlapping amplicons using FastStart high-fidelity DNA polymerase (Roche Applied Science, Mannheim, Germany) (35). Sequencing reactions and data assembling were performed as previously reported (35).…”

“…The genes A48R, D4R, B1R, F10L, and J2R were PCR amplified as one amplicon, and the full-length E9L gene was amplified as five (CMLV) or four (VACV and CPXV) overlapping amplicons using FastStart high-fidelity DNA polymerase (Roche Applied Science, Mannheim, Germany) (35). Sequencing reactions and data assembling were performed as previously reported (35).…”