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.

本研究系统表征了人源PKR（human PKR，简称hsPKR）针对痘苗病毒K3L蛋白的两种优势逃逸变异株A110G与A140G的适应性突变适合度景观。我们构建了hsPKR的大规模随机诱变文库，并采用PacBio HiFi长读长测序技术，将每个可变条形码（vBc）与其对应的hsPKR基因型精准关联。随后将该hsPKR突变文库导入分别表达K3L A110G与K3L A140G的酵母菌株中。在这两种不同的遗传背景下开展竞争性生长实验，以绘制hsPKR针对变异株特异性K3L抑制作用的适应性适合度景观。分别在竞争实验的起始阶段（S）与结束阶段（E）收集群体样本，并通过Illumina PE150深度测序量化突变频率的变化，进而推导得到适合度得分。