Novel phosphoprotein-interacting region in Nipah virus nucleocapsid protein and its involvement in viral replication.
The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.
Omi-Furutani
Mio
M
Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Yoneda
Misako
M
Fujita
Kentaro
K
Ikeda
Fusako
F
Kai
Chieko
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045405:Nipah Virus / Q000235:genetics / Q000502:physiology*; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells; D014779:Virus Replication / Q000502:physiology; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C527701:enhanced cyan fluorescent protein