W protein expression by Newcastle disease virus

Differential editing of transcripts from the Newcastle disease virus (NDV) phosphoprotein gene results in mRNAs capable of encoding the phosphoprotein (P), the V protein, and the W protein which share a common N-terminus but specify different C-termini. Whereas the expression and viral incorporation of the P - and V proteins by NDV has been documented, evidence for the existence of a W protein was lacking. To analyze expression of the NDV W protein, two peptides encompassing predicted antigenic sites of the unique C-terminal W protein amino acid sequence of NDV Clone 30 were used for the generation of W-specific rabbit antisera. One of them detected plasmid-expressed W protein and identified W protein after infection by indirect immunofluorescence and Western blot analyses. W protein was absent in cells infected by a newly generated recombinant NDV lacking W protein expression. Furthermore, Western blot and mass spectrometric analyses indicated the incorporation of W protein into viral particles. Confocal microscopic analyses of infected cells revealed nuclear accumulation of W protein that could be attributed to a bipartite nuclear localization sequence (NLS) within its unique C-terminal part. Redistribution of the W protein to the cytoplasm within transfected cells confirmed functionality of the NLS after mutation of its two basic clusters. This finding was additionally corroborated in cells infected with a recombinant virus expressing the mutated W protein.