title: Targeting of the influenza A virus polymerase PB1-PB2 interface indicates strain-specific assembly differences.
abstract: Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication. The conserved protein-protein interaction sites have been suggested as potential drug targets. To characterize the PB1-PB2 interface, we fused the PB1-binding domain of PB2 to green fluorescent protein (PB2(1-37)-GFP) and determined its competitive inhibitory effect on the polymerase activity of influenza A virus strains. Coexpression of PB2(1-37)-GFP in a polymerase reconstitution system led to substantial inhibition of the polymerase of A/WSN/33 (H1N1). Surprisingly, polymerases of other strains, including A/SC35M (H7N7), A/Puerto Rico/8/34 (H1N1), A/Hamburg/4/2009 (H1N1), and A/Thailand/1(KAN-1)/2004 (H5N1), showed various degrees of resistance. Individual exchange of polymerase subunits and the nucleoprotein between the sensitive WSN polymerase and the insensitive SC35M polymerase mapped the resistance to both PB1 and PA of SC35M polymerase. While PB2(1-37)-GFP bound equally well to the PB1 subunits of both virus strains, PB1-PA dimers of SC35M polymerase showed impaired binding compared to PB1-PA dimers of WSN polymerase. The use of PA(SC35M/WSN) chimeras revealed that the reduced affinity of the SC35M PB1-PA dimer was mediated by the N-terminal 277 amino acids of PA. Based on these observations, we speculate that the PB1-PA dimer formation of resistant polymerases shields the PB2(1-37) binding site, whereas sensitive polymerases allow this interaction, suggesting different assembly strategies of the trimeric polymerase complex between different influenza A virus strains.
authors.affiliation: Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Reuther
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mänz
authors.forename: Linda
authors.initials: L
authors.lastname: Brunotte
authors.forename: Martin
authors.initials: M
authors.lastname: Schwemmle
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wunderlich
mesh_terms: D019677:Artificial Gene Fusion; D001665:Binding Sites; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C061290:PA protein, influenza viruses; C502893:PB2 protein, influenza virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D049452:Green Fluorescent Proteins; D012324:RNA-Dependent RNA Polymerase