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.Transcription and replication of the influenza A viral genome involves a complex set of enzymatic reactions catalyzed by a heterotrimeric complex that is composed of three subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) (7,25,37). The PB1 subunit plays a central role in both polymerase and endonuclease activity (2, 26), the PB2 subunit has cap-binding activity and is responsible for the initiation of transcription (6,7,11), and the PA subunit has been suggested to function in both transcription and replication (4,5,30,33), but its exact role remains undetermined.In order for the individual polymerase proteins to perform their various functions, the three subunits need to come together to form a viral RNA polymerase complex; however, a significant proportion of polymerase dimers and oligomers can be detected in vitro (23). Our knowledge of the viral RNA polymerase complex assembly has increased over the past several years primarily by characterizing protein-protein interactions among the individual polymerase subunits (4,5,10,30,36,38,39,45,48). It is believed that the PB1 subunit forms the core of the polymerase complex. PB1 utilizes its N-terminal region to interact with PA, while PB1 binds PB2 through its C-terminal region. A specific interaction between PA and PB2 subunits has yet to be detected (36).Two models have been proposed to explain the assembly pathway and nuclear import of the polymerase complex (PA-PB1-PB2) (4,...

Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

“…PB2 interacts with PB1, PA, and NP (49)(50)(51)(52). To examine whether PB2-S1 also binds to these viral proteins, we performed a coimmunoprecipitation assay.…”

Reassortment and Mutation of the Avian Influenza Virus Polymerase PA Subunit Overcome Species Barriers

“…The N terminus of PA contains the endonuclease domain responsible for the cleavage of host mRNA during cap snatching and transcription and associates weakly with PB2, while the C terminus binds PB1 (7,16,17,32,52). PA amino acid 552 maps to a loop that projects away from the core of the C-terminal domain.…”

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

“…Therefore, we analyzed the PB2-NP interaction in the presence or absence of Mx1 by coimmunoprecipitation after transfection of all the components of the minireplicon system in HEK293T cells. We were unable to pull down unmodified PB2 from the complex using a monoclonal anti-PB2 antibody (monoclonal antibody 170-3D5; kindly provided by J. Yewdell, NIH, Bethesda, MD), presumably because the N-terminal 170-3D5 epitope in PB2 was shielded by interaction with PB1 or PA (17,39,48). Therefore, we used V5-tagged PB2 (PB2V5) and unmodified NP to assess whether they coexist in one complex.…”