Residue substitutions in PA and PB2 contribute to variations in viral polymerase activity

Abstract

Several substitutions in PA and PB2 of influenza A virus (IAV) have been determined to be associated with virus pathogenicity. It was found the recently emerged avian IAV of human isolates, including H7N9 and H5N6 in 2015, presented residues 37S/61T/63I/100A in PA N-terminal domain (PA-Nter), which were different from the majority of the IAV isolated from humans, such as H7N7 and H5N1. Meanwhile, most IAV isolates from human infections carried residues PB2-389R and -598T, and several human isolates of H7N9 and H9N2 harbored residue PB2-598I, which were different from those of most recently emerged avian IAV. Here, I investigated effect of PA-Nter substitutions at positions 37, 61, 63 and 100 on viral replication and pathogenicity in the backbone of H7N7 IAV, and effect of PB2 substitutions at positions 389 and 598 in the backbone of H7N9 IAV. The reverse genetics of strain A/Anhui/1/2013 (H7N9) and A/Netherlands/219/2003 (H7N7) were established to introduce desired substitutions into the viruses. The resulting mutant viruses were evaluated and the results showed that, firstly, V63I and the combinational substitutions, i.e. I61T/V63I and Mfour (A37S/I61T/V63I/V100A), exhibited substantially increased viral growth and transcription/replication, which were attributed to the enhanced polymerase activity and endonuclease activity. Notably, V63I and Mfour strongly increased viral pathogenicity in mice. These findings demonstrated that the PA-Nter V63I alone and the combined substitutions could enhance the virulence of IAV through the modification of viral polymerase activity and endonuclease activity. Secondly, decreased effect was identified in A37S and the related substitutions (A37S/I61T and A37S/V63I) on virus replication and pathogenicity in vitro and in vivo through reducing viral polymerase activity and endonuclease activity. Thirdly, V100A may up-regulate viral transcription but down-regulate the subsequent replication, and the related substitutions (I61T/V100A and V63I/V100A) decreased the transcription and replication, which were attributed to the altered endonuclease activity. Collectively, PA-Nter substitutions at positions 37, 61, 63 and 100 contributed to variations in viral pathogenicity. The results on the biological significance of substitutions PB2-K389R and -V598T/I showed that these substitutions enhanced viral growth capacity in human cells. Moreover, these substitutions exhibited substantially increased virus transcription and replication. Furthermore, the substitutions PB2-K389R and -V598T/I contributed to higher virus polymerase activity, which might be responsible for the higher virus activity. Notably, the substitutions PB2-V598T/I significantly increased virus replication and virulence in mice. The result provided evidences that the substitutions PB2-K389R and -V598T/I, especially for PB2-V598I, which had been identified in some H7N9 human isolates, contributed to higher replication and virulence. Collectively, PA-Nter substitutions at positions 37, 61, 63 and 100 in the background of H7N7 IAV, and PB2 substitutions at positions 389 and 598 in the background of H7N9 IAV contributed to variations in the viral replication and pathogenicity in vitro and in vivo. The study may help the understanding of the pathogenicity of IAV subtypes carrying these substitutions, facilitate the assessment of pandemic risks and benefit the combating against the virus infection.