Generation of mutagenesis library of PA, PB1, and PB2.

All three gene segments (PA, PB1, and PB2) of influenza virus A/WSN/33 underwent Mu transposon-mediated mutagenesis (MGS kit; Finnzymes) according to the manufacturer's instructions. This resulted in three mutant libraries, with each gene containing a randomly positioned 15-nt insertion that includes the 10-nt unique sequence 5′-TGCGGCCGCA-3′. The mutated segment and seven wild-type (WT) plasmids were cotransfected into HEK293T cells for virus packaging (17), and virus then was collected and used to infect MDCK cells for 24 to 48 h for each of the three consecutive passages. The 2nd and 3rd passages in MDCK cells were achieved through subsequent inoculation of the viral pool at a multiplicity of infection (MOI) of 0.1 from the previous passage. Treatment at an MOI of 0.1 still maintains genetic diversity and does not introduce artificial stochastic bottlenecks. To ensure that there was no contamination with DNA used for transfection, the medium was replaced 12 h postinfection. Total RNA of the mutant pool from all passages of virus was isolated using TRIzol and converted to cDNA with iScript (Life Technology). The mutants were genotyped by PCR amplification with gene-specific forward primers and a Vic-labeled reverse primer against the 10-nt insertion. To minimize PCR bias, we designed forward primers to hybridize 300 to 500 nt apart to ensure complete coverage of each gene (see Table S1 in the supplemental material). The final product was sequenced using a 96-capillary DNA Analyzer with the size standard Liz 500 (3730xl DNA Analyzer; Applied Biosystems) at the UCLA GenoSeq Core facility. Sequencing data were analyzed for clarity using the ABI software with the following criteria: (i) all data passed the standard default detection level; (ii) the first 70 nt were removed due to nonspecific background noise; (iii) all data were aligned to the nearest nucleotide or amino acid position in the specific gene; and (iv) all genotyping experimental data were normalized with WT WSN, nontransfected cells, and a different gene library as controls. This eliminated nonspecific data from the PCR, primers, and DNA analyzer.