Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1 and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation for influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In conjunction, our studies also revealed that PA plays a critical role in enhancing PB1-PB2 interaction while PB2 in modulating PA-PB1 interaction, which could be important targeting sites for anti-flu intervention. Collectively, these findings can not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex, but also provide new tool to investigate the exquisite mechanism of PPIs.
IMPORTANCE:
Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1 and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silicon modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method used to investigate PPIs of multi-subunit complex effectively and to identify new molecules inhibiting influenza virus polymerase assembly.