Figure 4. Validation of top experimentally adaptive mutations.

The polymerase activity of selected PB2 mutants as measured using minigenome assays in A549 (A) and HEK293T (B) human cells. The mutations chosen for characterization include previously known human adaptive mutations, top adaptive mutations identified by our deep mutational scanning (orange = human adaptive, green = avian adaptive), and additional mutations differentially selected in human (light orange) or avian (light green) cells. E627E is a synonymous mutation at site 627 used as a negative control. Minigenome activity is represented as percent of transfected cells that expressed a viral GFP reporter. The gray horizontal line indicates the mean value measured for the wild type avian PB2. Minigenome assays were performed in biological triplicate. Mutations that have significantly different minigenome activity from wild type are indicated by asterisks (unpaired t-test, p<0.05). (C) Competition of virus bearing the indicated mutant PB2 against virus with wild-type PB2. For each competition, human A549 and avian CCL-141 cells were infected with mutant and wild-type viruses mixed at a 1:1 ratio of transcriptionally active particles, and the frequency of each variant after viral replication was measured by deep sequencing viral RNA. For samples collected at 10 hr post infection, we infected cells at MOI of 0.1, and sequenced vRNA from cellular extract. For samples collected at 48 hr post infection, we infected cells at MOI of 0.01, and sequenced vRNA from the supernatant. The plots show the ratio of the mutant over wild-type variant in A549, divided by the same ratio in CCL-141 cells. A ratio >1 indicates that a viral mutant grows better in human than avian cells. Competition assays were performed in biological duplicate; circle and cross represent replicate experiments. Flow data for minigenome activity and and mutation counts for viral competition are provided in Figure 4—source datas 1 and 2.