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 m RNAs of avian polyomavirus BFDV, either agno-1a or its splice derivative ag no-1b are required for viral propagation. Out of the two proteins both of w hich consist of multiple electrophoretic subspecies, the smaller and less c omplex agno-1b has been cDNA-cloned into an influenza-virus /RNA-polymerase I expression system for production of higher amounts of this protein in in fected chicken embryo fibroblasts. Fractional modification of agno-1b by ph osphorylation at residues serine 51, serine 53, and threonine 73 is demonst rated through dephosphorylation by alkaline phosphatase, mass spectrometry of individual protein species isolated by strong anion exchange chromatogra phy, and single or multiple alanine substitutions of serine or threonine re sidues in site-directed mutagenesis.