title: Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.
abstract: 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 of 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 addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs.IMPORTANCEFormation 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 combiningin silicomodeling 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 to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Li
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.|Collaborative Innovation Center of Cancer Medicine, Department of Experimental Medicine, State Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou, China.
authors.forename: Zining
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Center of Growth, Metabolism and Aging, Key Lab of BioResources and EcoEnvironment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.|Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Lulan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Jingyun
authors.initials: J
authors.lastname: Ji
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Zhigao
authors.initials: Z
authors.lastname: Chen
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Deng
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Taijiao
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China GCheng@mednet.ucla.edu fqin1@foxmail.com.|Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Genhong
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, and Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China GCheng@mednet.ucla.edu fqin1@foxmail.com.
authors.forename: F Xiao-Feng
authors.initials: FX
authors.lastname: Qin
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000201:enzymology*; D007251:Influenza, Human / Q000188:drug therapy / Q000821:virology; D061985:Madin Darby Canine Kidney Cells; D025941:Protein Interaction Mapping; D055503:Protein Multimerization / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D017361:Viral Nonstructural Proteins
keywords: BiLC; influenza inhibitor screening; influenza virus polymerase; protein-protein interactions