Conserved polymerase structural features and ubiquitination regulate the influenza virus RNA replication machinery

The influenza A virus polymerase is essential for the virus life cycle. The polymerase is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher order ribonucleoprotein (RNP) complexes. In the context of these RNPs, the polymerase expresses viral genes and replicates the viral genome. The polymerase is also a major determinant of influenza virus host tropism and pathogenicity. It is a target for species-specific restriction of influenza viruses in mammals. Polymerases encoded by avian influenza viruses do not function efficiently in mammals. This restriction has been mapped to position 627 in the PB2 subunit, which is normally a glutamic acid in avian isolates. Conversely, a lysine is present at position 627 in most mammalian viral isolates and creates a basic face on the domain surface that confers high activity in mammals. In addition to species-specific regulation, the polymerase is also regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including post-translational modifications, however the mechanisms are not fully understood.;We created a series of mutants in the 627 domain of the PB2 subunit to alter the conserved "P[F/P]AAAPP" sequence motif and a number of conserved basic residues that give the domain surface a basic face. Mutating the basic face or the P[F/P]AAAP motif impaired polymerase activity, assembly of replication complexes and viral replication. We found that the P[F/P]AAAP motif residues were important for polymerase function in both human and chicken cells, suggesting that they play a structural role and are essential for overall polymerase function. We also identified PB2 positions 586 and 589 on the basic surface of the 627 domain to be species-specific determinants of polymerase function that are preferentially required for function in human versus avian cells. Thus, we identified new residues in the 627 domain that regulate overall polymerase function and those that function in a species-specific fashion. This work highlights the importance of the surface charge and structure of the PB2 627 domain for virus replication and host adaptation.;We assessed the ubiquitination status of the RNP complex and investigated the effect of ubiquitin expression on polymerase function. We show that all protein subunits in the RNP complex are ubiquitinated and that their levels are not significantly affected, despite the well known activity of ubiquitin-mediated protein degradation. Instead, we found that ubiquitination and an active proteasome enhance polymerase activity. Ubiquitin expression up-regulates polymerase function causing increased accumulation of vRNA, cRNA and mRNA and enhanced viral gene expression during infection. We show that ubiquitin expression enhances polymerase activity independent of NP or RNP assembly. Ubiquitination and the proteasome pathway play multiple roles in the influenza virus cycle, and we now demonstrate that ubiquitination also modulates polymerase activity independent of protein degradation.;Overall, we describe here key features of the influenza virus polymerase that regulate its function in a species-specific fashion and a new way in which a host cell process can be co-opted by the virus to enhance its polymerase function.