Summary auto-generated
This study identified critical amino acid residues in the Sendai virus nucleocapsid (NP) protein required for viral RNA replication and nucleocapsid assembly. Researchers generated four alanine-substitution mutants targeting conserved residues at positions 362-375 of the NP protein and tested their effects on protein stability, protein-protein interactions, and RNA replication in vitro. NP362 (F362A) could not support RNA replication despite forming extended oligomers lacking RNA. NP364 (L362A, G365A) was unstable and failed to form protein complexes. NP370 (K370A, D371A) maintained normal protein-protein interactions and formed morphologically normal nucleocapsid-like particles containing RNA, but could not replicate viral RNA, suggesting a specific defect in genome RNA recognition or encapsidation. NP373 (conserved charged residues at 373 and 375 altered) exhibited wild-type replication activity. All replication-defective mutants retained the ability to form NP-P complexes essential for encapsidation. Results indicate that specific NP residues function independently in different aspects of viral replication: protein-protein interactions, nucleocapsid assembly, and viral RNA encapsidation.
Key findings
- Phenylalanine at position 362, and lysine and aspartate at positions 370-371 are essential for NP function in RNA replication, while non-conserved residues at 373-375 are dispensable.
- NP362 mutant formed structurally abnormal oligomers lacking RNA, indicating defective protein-protein interactions; NP364 was unstable and could not form protein complexes.
- NP370 maintained normal NP-P and NP-NP interactions and assembled morphologically normal nucleocapsid-like particles containing RNA, but failed to replicate viral RNA, indicating a specific defect in viral genome encapsidation.
- The study demonstrates that different regions of NP protein can be functionally dissected into domains for protein-protein interactions, nucleocapsid assembly, and specific viral RNA encapsidation.
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Abstract
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 NP(0)--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.