P107 Identification of adaptive mutations in the nucleoprotein of pandemic influenza A viruses required to evade restriction from the interferon induced MxA protein

The interferon induced human MxA protein is a potent restriction factor against avian influenza A virus infections. We therefore predicted that zoonotic transmission of these viruses into the human population is accompanied by the acquisition of adaptive mutations allowing evasion from MxA restriction. The H5N1 polymerase reconstitution assay was utilized to identify adaptive mutations in the nucleoproteins (NP) of pandemic viruses required for resistance against human MxA. Highly pathogenic H5N1 viruses harboring the identified adaptive mutations in NP were generated and their growth properties were determined in cell culture. The evolutionary pressure to maintain the MxA resistance-contributing amino acids was assessed using the NCBI Influenza Virus Sequence Database. We identified the MxA-adaptive mutations in the nucleoprotein (NP) of the pandemic strains A/Brevig Mission/1/1918(H1N1) (1918) and A/Hamburg/4/2009(H1N1) (pH1N1). Intriguingly, the amino acids conferring resistance towards MxA differ in both strains, but cluster to the same area of the body domain of NP. Sequence analysis revealed that the amino acid cluster required for MxA resistance in the 1918 strain remained highly conserved in all descendant seasonal and pandemic strains. However, the amino acid cluster in NP of the pH1N1 strain contains three unprecedented adaptive mutations, indicating an independent evolution of MxA resistance in this porcine-derived virus. Introduction of either the 1918 or the pH1N1 amino acid cluster into NP of an H5N1 virus mediated resistance to MxA but also decreased viral fitness. Vice versa, mutation of the corresponding amino acid cluster in pH1N1 NP to avian signature impaired MxA resistance, while viral growth was increased. Taken together, the acquisitions of NP mutations required for MxA resistance emerged in both the 1918 and the 2009 pandemic strain independently and were most likely accompanied by compensatory mutations to overcome the associated strong attenuation.