Fitness landscape of the vaccinia virus K3L H47R mutants in the context of the human PKR I378S mutant

Under selection pressure from the human PKR I378S variant, this study employed deep mutational scanning to systematically characterize the fitness effects of mutations in the vaccinia virus K3L protein carrying the H47R (A140G) substitution. We constructed a comprehensive mutant library of K3L-H47R, with each variant paired to a variable barcode (vBc). An initial Illumina PE250 sequencing mapped these barcodes to their corresponding mutations. Mutants from the library were then integrated into the genome of Saccharomyces cerevisiae strains co-expressing human PKR I378S. Competitive growth assays were performed with four replicate populations. Genomic DNA was harvested at the start (Tstart) and end (Tend) points. The vBc regions were amplified and sequenced (Illumina PE150), enabling us to quantify frequency changes for each mutant and calculate fitness scores.