Influenza A virus: host restriction of the viral polymerase and a mechanism for vRNP nuclear export

Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. However, an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase increases the activity of avian-derived virus polymerases in mammalian cells. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that do not require ribonucleoprotein (RNP) assembly. To reconcile this conflict, a nucleoprotein(NP)-independent cell based transcription/replication assay was used to assess viral polymerase activity. This indicated that PB2 627E polymerase inhibition in mammalian cells is independent of NP expression, but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase could be overcome by mutations to the viral RNA template promoter sequence. Taken together, these results suggest that PB2 627 affects interaction between the viral RNA promoter and the viral polymerase in human cells. Due to influenza viruses replicating and transcribing their genomes within the nucleus of infected cells, progeny virus production is dependent on the nuclear export of genomic RNA, in the form of viral ribonucleoprotein complexes (vRNPs). This function is carried out by the viral nuclear export protein (NEP) but the mechanism whereby this process is regulated remains unclear. Experiments measuring NEP nuclear export demonstrate that phosphorylation of a highly conserved serine-rich motif specifically up-regulates nuclear export of NEP in a manner that is conserved across viral genera. Further biophysical investigations revealed that modification of this site alters the backbone dynamics of NEP, shifting it towards an extended state that has the potential to be preferentially recognised by the cellular nuclear export machinery.