SERINE 3 IS CRITICAL FOR PHOSPHORYLATION AT THE N-TERMINAL END OF THENUCLEOPROTEIN OF INFLUENZA-VIRUS-A VICTORIA/3/75/

The influenza A virus nucleoprotein (NP) is a phosphoprotein that enca psidates the viral genomic RNA. To map the in vivo phosphorylation sit e(s) of this protein, P-32-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chroma tography. The purified protein was then subjected to chemical digestio n with formic acid, which cleaves proteins at Asp-Pro bonds, and the r esulting products were analyzed by sodium dodecyl sulfate-polyacrylami de gel electrophoresis. Two of the phosphorylated products obtained we re identified as fragments corresponding to the N-terminal 88 amino ac ids and to the C-terminal 196 residues of the NP. To identify the phos phate 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 phosp horylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of P-32 label incorporated into NP, whereas the other substitutions did not have a discernible effect on t he phosphorylation level of the protein. In addition, all serine-alter ed proteins were tested for their functionality in an artificial syste m in which expression of a synthetic chloramphenicol acetyltransferase RNA molecule is driven by influenza virus proteins synthesized from c loned 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 acetyltran sferase RNA in vivo. These data are discussed regarding the possible r oles of NP phosphorylation for the viral replicative cycle.