Functional analysis of top candidate PB2 interactors reveals important roles for hnRNP UL1 and MECR.

(A) Secondary screening of proteomic hits by siRNA treatment and reporter virus infection. After knockdown, A549 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 outliers hnRNP UL1 (cyan) and MECR (yellow) are highlighted. (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) Multicycle virus replication of WT virus was measured 24 h postinfection in A549 cells treated with the indicated siRNAs. Knockdown efficiency was analyzed by western blot. Asterisk indicates nonspecific band. (D) Knockdown impacts viral gene expression of divergent influenza viruses. siRNA-treated A549 cells were infected with reporter viruses based on WSN (pre-2009 H1N1; MOI, 0.1), CA04 (pandemic 2009 H1N1; MOI, 0.5), or B/Bris (Victoria-lineage; MOI, 1). Viral gene expression was measured 8 h postinfection and normalized to NT controls. (E) A549 cells stably expressing hnRNP UL1 (top) or MECR (bottom) were infected with WSN PB2-FLAG (MOI 3, 8 h). Protein localization was detected by immunofluorescence, and nuclei were visualized with DAPI. Arrow indicates minor PB2 population consistent with previously reported patterns of mitochondrial localization. Scale bars, 20 μm. Data in (A) are mean ± SEM of n = 3 biological replicates. Comparisons were performed with two-way ANOVA with post hoc Fisher LSD test. For (C) and (D), data are mean ± SD of n = 3. Comparisons were performed with a two-tailed Student t test (C) or a two-way ANOVA with post hoc Dunnett multiple comparisons test (D); *, P P P P S1 Data. Uncropped images can be found in the Supporting information file as S1 Raw Images. hnRNP UL1, heterogenous nuclear ribonuclear protein U-like 1; MECR, mitochondrial enoyl CoA-reductase; NLuc, nanoluciferase; NT, nontargeting; WT, wild-type.