title: Detection and characterization of influenza A virus PA-PB2 interaction through a bimolecular fluorescence complementation assay.
abstract: The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.
authors.affiliation: Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA.
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Hemerka
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Yuejin
authors.initials: Y
authors.lastname: Weng
authors.forename: Wuxun
authors.initials: W
authors.lastname: Lu
authors.forename: Radhey S
authors.initials: RS
authors.lastname: Kaushik
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Harmon
authors.forename: Feng
authors.initials: F
authors.lastname: Li
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005453:Fluorescence; D053118:Influenza A Virus, H1N1 Subtype / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase