Functional significance of alternate phosphorylation in Sendai virus P protein.
Phosphorylation of the negative-sense RNA virus phosphoproteins is highly conserved, implying functional significance. Sendai virus (SV) phosphoprotein (P) is constitutively phosphorylated at S249. Abrogation of the SV P primary phosphorylation causes phosphorylation of P at alternate sites, creating a problem in determining the function of phosphorylation. We have now identified the alternate phosphorylation sites using two-dimensional phosphopeptide analysis of several deletion and point mutants of the P protein. The alternate phosphorylation sites were mutagenized to create P with (S249combo) or without (combo) primary phosphorylation. The combo protein has less than 10% phosphorylation compared with the wild-type P or S249combo. Functional analysis of the mutant proteins using a Sendai virus minigenome replication system showed that the combo P protein was as proficient in supporting minigenome replication as the wild-type P in cell cultures. These studies suggest that like the primary, the alternate phosphorylation of the P protein is also dispensable for virus replication in cell cultures. Interestingly, the ability of the multiple site mutant of P (combo mutant has eight serine residues changed to alanine residues) to support efficient virus RNA synthesis suggests that the P protein has a high flexibility at least in its sequence and perhaps also in structure.
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Department of Immunology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, 60612, USA.
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D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010748:Phosphopeptides / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017354:Point Mutation; D018108:Respirovirus / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
C055859:P protein, Sendai virus; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D014764:Viral Proteins