The influenza A virus endoribonuclease PA-X usurps host mRNA processing machinery to limit host gene expression

Lea Gaucherand; Brittany K. Porter; Summer K. Schmaling; Christopher Harley Rycroft; Yuzo Kevorkian; Craig McCormick; Denys A. Khaperskyy; Marta Maria Gaglia

To identify cellular proteins that bind both the X61 and X41 X-ORFs, we collected protein 300 lysates from HEK293T cells expressing the BirA*-X-ORF fusion proteins. We prepared affinity-301 purified biotinylated proteins for quantitative mass spectrometry, using reductive dimethylation 302 to introduce stable epitope tags. We performed three independent replicates of the experiment, 303 two with BirA*-X61 and one with BirA*-X41, comparing them in each case to the BirA* alone 304 and BirA*-X61(4A) controls ( Figure 6A ). We identified 156 candidate X-ORF interacting 305 proteins that were represented by at least two unique peptides (Table S5) , and were identified in 306 all runs ( Figure 6B ). Among the 156 candidates, we identified 29 high-confidence interacting 307 proteins based on relative peptide abundance in the test conditions (BirA*-X61 or X41) vs. the 308 control conditions (BirA*-X61(4A) or BirA* alone) ( Figure 6D , Table S5 ). These high-309 confidence hits were enriched more than two-fold over controls in at least two experiments or 310 more than 1.5-fold in all three experiments. Figure 6C depicts the relative peptide abundance of 311 proteins in X61 samples compared to the X61(4A) control or BirA* alone control, with black 312 and red open circles identifying the high confidence hits. The red circles indicate two proteins 313 All rights reserved. No reuse allowed without permission.