title: Phosphorylation controls the nuclear-cytoplasmic shuttling of influenza A virus nucleoprotein.
abstract: The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication.
  
  
  OBJECTIVE
  It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China.
authors: Weinan
authors: W
authors: Zheng
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors: Jing
authors: J
authors: Li
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors: Shanshan
authors: S
authors: Wang
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China College of Life Sciences, Anhui University, Hefei, China.
authors: Shuaishuai
authors: S
authors: Cao
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors: Jingwen
authors: J
authors: Jiang
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China.
authors: Can
authors: C
authors: Chen
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: Chan
authors: C
authors: Ding
authors: Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and Comparative Medicine Center, Peking Union Medical College, Beijing, China.
authors: Chuan
authors: C
authors: Qin
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Center for Influenza Research and Early-Warning, Chinese Academy of Sciences, Beijing, China.
authors: Xin
authors: X
authors: Ye
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Office of Director-General, Chinese Center for Disease Control and Prevention, Beijing, China.
authors: George F
authors: GF
authors: Gao
authors: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China liuwj@im.ac.cn.
authors: Wenjun
authors: W
authors: Liu
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism
chemical_list: C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D028901:alpha Karyopherins