The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication
Figure 5
Mapping of ADAR1-NS1 interaction sites.
(A) Subcellular localization of ADAR1 (green), HA and NS1 (red) 8 h post infection of A549 cells. (B) Schematic representation of full-length and truncated ADAR1 proteins. Zα DNA-binding domain (grey boxes), dsRNA binding domain (DRBD, purple boxes), adenosine deaminase domain (yellow boxes). (C) ADAR1 interaction mapping. Yeast diploids co-expressing full length A/H1N1/Puerto Rico/8/34 virus NS1 protein fused to the Gal4 DNA-binding domain and full-length ADAR1 or ADAR1 truncated mutants fused to Gal4 activating domain were spotted onto a plate containing medium without histidine. STAU1 is a positive control for interaction with NS1 [10]. Controls with empty vectors (Gal4-AD and Gal4DB) show no auto-activation induced by the different constructs. (D) GST pull-downs were performed with GST alone or GST fused to NS1 after co-expression of a 3×Flag-tagged DRBD1 peptide in HEK293T cells and incubation of cell lysates with glutathione-Sepharose beads. Cell lysates and pull-downs were analyzed by western blot using antibodies against GST or 3×Flag. (E) Schematic representation of the NS1 protein with its two domains: the N-terminal RNA-binding domain (RBD, 1-73) and the effector domain (74-230). (F.) NS1 interaction mapping. GST alone or GST fused to NS1, NS1 RBD or NS1 effector were co-expressed with 3×Flag-tagged ADAR1 in HEK293T cells and their interaction was assessed after co-affinity purification with glutathione sepharose beads and immunoblotting.