Functional Significance of Alternate Phosphorylation in Sendai Virus P Protein

Abstract

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.