The structure and synthesis of influenza virus phosphoproteins.

The synthesis and phosphorylation of influenza virus nucleoprotein and nonstructural protein were analyzed. The nucleoprotein (NP) was found to be phosphorylated in both infected cells and in isolated virions. The phosphate is in a monoester linkage to a serine residue. Two-dimensional tryptic peptide maps of the 32P-labeled protein, as well as measurements of specific activity, suggests that NP is phosphorylated at one site per molecule. The viral nonstructural (NS 1) protein is also phosphorylated, but on threonine residues. Up to a maximum of two sites per NS 1 molecule could be so modified in infected cells, as demonstrated by two different methods of tryptic peptide analysis and by measurements of the ratio of 32P to 3H-amino-acids incorporated into NS 1 protein species. The NS 1 protein is resolved into four major species of differing isoelectric point in a two-dimensional electrophoretogram. The most acidic species was found to have two phosphorylated sites per molecule, and the next most acidic species contained on the average one phosphate per molecule. Treatment of the phosphorylated species with bacterial alkaline phosphatase demonstrated that the level of phosphorylation is the only identifiable difference between the phosphorylated and unphosphorylated NS 1 species. The distinction between the two unphosphorylated species could not be determined. The distribution of the un-, mono-, and diphosphorylated NS 1 species was characterized at different times after synthesis. These modifications were found to occur very rapidly after translation (30 to 60 s), after transport of the unmodified species from cytoplasm to nucleus of the infected cell. The phosphorylation of NP also takes place rapidly after its synthesis; the site within the cell of the NP phosphorylation has not been unambiguously determined.