Figure 3

The C-terminal NoLS of the NS1 protein in H3N2 subtype influenza A viruses binds nucleolar nucleolin, B23 and fibrillarin in a GST-NS1 pull-down experiment. (A) N- and C-terminal fragments of the NS1 gene encoding for amino acids 1–73 and 203–237 in A/Udorn/72 and wt genes, encoding for amino acids 1–237 in A/Udorn/72 and amino acids 1–230 in A/Brevig Mission/1/18 and in A/Mallard/Netherlands/12/00, were inserted into the GST expression vector pGEX 2 T(+) to express GST-NS1 fusion proteins. Mutations were created into the N-terminal NLS1 and C-terminal NLS2/NoLS in the A/Udorn/72 virus NS1 protein are as indicated in the figure. (B) The cell extracts of cultured A549 cells were prepared, and proteins in cell extracts were allowed to bind to E. coli-expressed and Sepharose-immobilized GST, GST-NS1 A/Udorn/72 wt, GST-NS1 A/Udorn/72(1–73), GST-NS1 A/Udorn/72(1–73)R38A, GST-NS1 A/Udorn/72(1–73)K41A, GST-NS1 A/Udorn/72(203–237), GST-NS1 A/Udorn/72(203–237)K219A,R220A + R231A,R232A, GST-NS1 A/Brevig mission/1/18 wt and GST-NS1 mallard/Netherlands/12/00 wt at +4 °C for 1 h. Sepharose-bound proteins were dissolved in Laemmli sample buffer and separated on 8 % SDS-PAGE. Western blots were stained with anti-nucleolin, -B23 and -fibrillarin immunoglobulins. As a control, A549 cell extract, containing 10 μg of proteins, was stained in lane C. M indicates the Sepharose matrix-bound proteins. A similar gel was also stained with Coomassie Brilliant Blue to visualize the amount of Sepharose-immobilized GST and GST-NS1 fusion proteins.