Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.
Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.
Myers
T M
TM
Smallwood
S
S
Moyer
S A
SA
D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D016679:Genome, Viral; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D018108:Respirovirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D014758:Viral Core Proteins