title: Functional Genomics Reveals Linkers Critical for Influenza Virus Polymerase.
abstract: Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis.
  
  
  OBJECTIVE
  The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA.
authors.forename: Lulan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Aiping
authors.initials: A
authors.lastname: Wu
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA.
authors.forename: Yao E
authors.initials: YE
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Quanquin
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Li
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: Jingfeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Faculty of Medicine, and Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Hsiang-Wen
authors.initials: HW
authors.lastname: Chen
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA.
authors.forename: Suyang
authors.initials: S
authors.lastname: Liu
authors.affiliation: Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China.
authors.forename: F Xiao-Feng
authors.initials: FX
authors.lastname: Qin
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China Institute of Biophysics, Chinese Academy of Sciences, Beijing, China taijiao@moon.ibp.ac.cn gcheng@mednet.ucla.edu.
authors.forename: Taijiao
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Center of System Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, Jiangsu, China Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA taijiao@moon.ibp.ac.cn gcheng@mednet.ucla.edu.
authors.forename: Genhong
authors.initials: G
authors.lastname: Cheng
mesh_terms: D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000502:physiology; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase