Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75.
The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.
Arrese
M
M
Centro Nacional de Biología Celular y Retrovirus, Instituto de Salud Carlos III, Madrid, Spain.
Portela
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005561:Formates / Q000494:pharmacology; D009980:Influenza A virus / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D016601:RNA-Binding Proteins; D012694:Serine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000737:chemistry* / Q000378:metabolism
D005561:Formates; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; C030544:formic acid; D012694:Serine