Dissection of individual functions of the Sendai virus phosphoprotein in transcription.
The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
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Bowman
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Smallwood
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Moyer
D000818:Animals; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016296:Mutagenesis; D019251:Nucleocapsid / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018108:Respirovirus / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
C055859:P protein, Sendai virus; D010750:Phosphoproteins; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases