Functional analyses of top candidate PB2 interactors.

(A) Secondary screening of proteomic hits by siRNA treatment and reporter virus infection. After knockdown, 293T cells were infected with human (PB2-627K; MOI, 0.01) or avian-adapted (PB2-627E; MOI, 0.05) WSN NLuc virus for 24 h. Viral supernatants were titered and normalized to an NT control. Control NXF1 (gray) and validated proteins highlighted (hnRNP UL1, cyan; MECR, yellow). Data are mean ± SEM of n = 2–3 biological replicates. (B) Concordance of virus titer for PB2-627E vs. PB2-627K virus infections in siRNA-treated cells (from (A)). Statistical analysis performed with a two-tailed Pearson correlation coefficient. (C) Screening as in (A), except A549 cells were infected with viruses as above for a single cycle of infection (MOI, 0.1; 8 h) and virus gene expression in infected cells normalized as above. (D) Concordance of gene expression (data from (C), analyzed as in (B)). Individual quantitative observations that underlie the data summarized here can be located under the Supporting information file as S1 Data. hnRNP UL1, heterogenous nuclear ribonuclear protein U-like 1; MECR, mitochondrial enoyl CoA-reductase; NLuc, nanoluciferase; NT, nontargeting. (EPS)