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Journal of Virology, April 2009, p. 3944-3955, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02300-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay{triangledown}

Joseph N. Hemerka,1,{dagger} Dan Wang,1,{dagger} Yuejin Weng,2,3 Wuxun Lu,1 Radhey S. Kaushik,1,2,3 Jing Jin,4 Aaron F. Harmon,2,3 and Feng Li1,2,3*

Department of Biology and Microbiology,1 Department of Veterinary Science,2 Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings, South Dakota 57007,3 Section of Microbial Pathogenesis, Yale University School of Medicine, 295 Congress Ave., New Haven, Connecticut 065364

Received 3 November 2008/ Accepted 26 January 2009

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.

* Corresponding author. Mailing address: Department of Veterinary Science, South Dakota State University, Brookings, SD 57007. Phone: (605) 688-6036. Fax: (605) 688-6003. E-mail: feng.li{at}sdstate.edu

{triangledown} Published ahead of print on 4 February 2009.

{dagger} J.N.H. and D.W. were equal contributors to this work.

Journal of Virology, April 2009, p. 3944-3955, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02300-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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