Recombinant expression and modification analysis of protein agno-1b encoded by avian polyomavirus BFDV

Among two pairs of agnoproteins encoded in upstream positions in the late mRNAs of avian polyomavirus BFDV, either agno-1a or its splice derivative agno-1b are required for viral propagation. Out of the two proteins both of which consist of multiple electrophoretic subspecies, the smaller and less complex agno-1b has been cDNA-cloned into an influenza-virus /RNA-polymerase I expression system for production of higher amounts of this protein in infected chicken embryo fibroblasts. Fractional modification of agno-1b by phosphorylation at residues serine 51, serine 53, and threonine 73 is demonstrated through dephosphorylation by alkaline phosphatase, mass spectrometry of individual protein species isolated by strong anion exchange chromatography, and single or multiple alanine substitutions of serine or threonine residues in site-directed mutagenesis.