Mapping the phosphoprotein binding site on Sendai virus NP protein assembled into nucleocapsids.
To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.
Cevik
Bayram
B
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Kaesberg
Jeffrey
J
Smallwood
Sherin
S
Feller
Joyce A
JA
Moyer
Sue A
SA
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019251:Nucleocapsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D029082:Sendai virus / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010750:Phosphoproteins; D012367:RNA, Viral; D014758:Viral Core Proteins