Identification of potential virulence determinants associated H9N2 avian influenza virus PB2 E627K mutation by comparative proteomics

Some highly pathogenic H5N1, H7N9, and H10N8 isolated from China carried six internal genes from H9N2 avian influenza viruses (AIV) and the key amino acids at 627 in PB2 of these viruses had mutated to K. To investigate the mechanism of increased pathogenicity for H9N2 AIV PB2 627K, we analyzed the difference in mouse lung proteins expression response to PB2 K627E. By iTRAQ method, we found that the mutated K627E contributed to a set of differentially expressed lung proteins, including five upregulated proteins and nine downregulated proteins at 12 h postinfection; ten upregulated proteins and 25 downregulated proteins at 72 h postinfection. These proteins were chiefly involved within the cytoskeleton and motor proteins, antiviral proteins, regulation of glucocorticoids signal-associated proteins, pro- and anti-inflammatory proteins. Alteration of moesin, FKBP4, Hsp70, ezrin, and pulmonary surfactant protein A (sp-A) may play important roles in increasing virulence and decreasing lungs antiviral response. Further, three upregulated proteins (moesin, ezrin, and sp-A) caused by PB2 K627E were also confirmed in A549 cells. Moreover, overexpression of sp-A in A549 inhibited virus replication and downregulation promoted virus replication. In this study, sp-A as a potential virulence determinant associated H9N2 AIV PB2 E627K mutation was identified using comparative proteomics.