Fitness landscape of vaccinia K3L mutants under human PKR selection

This study employs deep mutational scanning to systematically characterize the fitness effects of mutations in the vaccinia virus K3L protein under selection pressure from human PKR (protein kinase R). We constructed a comprehensive mutant library of vacK3L and paired each variant with a unique barcode (vBc). Initial Illumina PE250 sequencing mapped these barcodes to specific mutations. The mutant library was then integrated into Saccharomyces cerevisiae genomes co-expressing wild-type human PKR. Four replicate populations underwent competitive growth assays. Genomic DNA was harvested at start (Tstart) and end (Tend) points, with vBc regions amplified and sequenced using Illumina PE150. This enabled quantification of each mutant's frequency change, from which fitness scores were calculated.