PAN substitutions A37S, A37S/I61T and A37S/V63I attenuate the replication of H7N7 influenza A virus by impairing the polymerase and endonuclease activities.

Substitutions in the PA(N)-terminus (PA(N)) of influenza A viruses are associated with viral pathogenicity. During our previous study, which identified PA(N)-V63I and -A37S/I61T/V63I/V100A substitutions as virulence determinants, we observed a severe decrease in virus growth and transcription/replication capacity posed by PA(N)-A37S/V100A substitution. To further delineate the significance of substitutions at these positions, we generated mutant H7N7 viruses bearing the substitutions PA(N)-A37S, -A37S/I61T, -A37S/V63I, -V100A, -I61T/V100A and -V63I/V100A by reverse genetics. Our results showed that all mutant viruses except PA(N)-V100A showed a significantly reduced growth capability in infected cells. At the same time, the PA(N)A37S, -A37S/I61T and -A37S/V63I mutant viruses displayed decreased viral transcription and replication by diminishing virus RNA synthesis activity. Biochemical assays indicated that the substitutions PA(N)-A37S, -A37S/I61T and -A37S/V63I suppressed the polymerase and endonuclease activities when compared with those of the wild-type. Together, our results demonstrated that the PA(N)-A37S, -A37S/I61T and -A37S/V63I substitutions contributed to a decreased pathogenicity of avian H7N7 influenza A virus.