Fitness landscape of human PKR mutations antagonizing vaccinia virus K3L A110G and A140G variants
This study systematically characterizes the fitness landscape of adaptive mutations in human PKR (hsPKR) in response to two dominant escape variants of the vaccinia virus K3L protein, A110G and A140G. We constructed a large-scale random mutagenesis library of hsPKR and used PacBio HiFi long-read sequencing to accurately associate each variable barcode (vBc) with its corresponding hsPKR genotype. The hsPKR mutant library was then introduced into yeast strains expressing either K3LA110G or K3LA140G. Competitive growth assays were performed in these two distinct genetic backgrounds to map the adaptive fitness landscape of hsPKR against variant-specific K3L inhibition. Population samples were collected at the start (S) and end (E) of the competition, and Illumina PE150 deep sequencing was used to quantify changes in mutation frequencies, from which fitness scores were inferred.