Figure 6.

Substitutions in PA and PB1 increase the production of full-length PB2 vRNA. (A) Schematic diagram of the full-length PB2 (top) and the PB2-derived defective viral genome (DVG, bottom). In the DVG schematics, regions shared with the full-length segment are colored blue, and the regions containing deletion junction sites are colored orange. Nucleotide positions of the most proximal and most distal junction sites, as determined by deep sequencing, are indicated. Positions of PB2e and PB2i amplicons are depicted, as well as the location of the Uni12 RT primer annealing site (all nucleotide positions are numbered from the full-length vRNA 3′ end). (B) Relative levels of total PB2-derived genomic segments (light blue) and the full-length PB2 vRNAs (blue) were measured by RT-qPCR using PB2e and PB2i primer pairs, respectively. (C) PB2i to PB2e ratios calculated from (B) were plotted. (B,C) Error bars represent standard deviation (n = 3 independent biological replicates). * p-value < 0.05, paired Student’s t test.