Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay
Autor: | Aaron F. Harmon, Dan Wang, Joseph N. Hemerka, Jing Jin, Feng Li, Yuejin Weng, Radhey S. Kaushik, Wuxun Lu |
---|---|
Rok vydání: | 2009 |
Předmět: |
viruses
Protein subunit Immunology Orthomyxoviridae RNA-dependent RNA polymerase medicine.disease_cause Microbiology Fluorescence Viral Proteins Bimolecular fluorescence complementation Influenza A Virus H1N1 Subtype Transcription (biology) Virology Chlorocebus aethiops Protein Interaction Mapping Influenza A virus medicine Animals Protein Interaction Domains and Motifs Polymerase biology virus diseases biochemical phenomena metabolism and nutrition RNA-Dependent RNA Polymerase biology.organism_classification Molecular biology Genome Replication and Regulation of Viral Gene Expression respiratory tract diseases Cell biology Viral replication Insect Science COS Cells biology.protein Protein Binding |
Zdroj: | Journal of Virology. 83:3944-3955 |
ISSN: | 1098-5514 0022-538X |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |