title: The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro.
abstract: The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of (33)P(i)-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012367:RNA, Viral / Q000096:biosynthesis; D018357:Respiratory Syncytial Virus Infections / Q000821:virology; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016205:Sigmodontinae; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D010750:Phosphoproteins; D012367:RNA, Viral; D014764:Viral Proteins