Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its in tracellular cleavage activation occurs in a variety of cells; therefore, th e viruses cause systemic infections in poultry, To explore the protease res ponsible for the cleavage in the natural host, we examined detailed substra te specificity of the enzyme in chick embryo fibroblasts (CEF) using a pane l of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-l ike proteases such as furin, PC6 and PACE4 which are candidates for F prote in processing enzymes, It was demonstrated in CEF cells that Arg residues a t the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage, On the other hand, f urin and PC6 expressed by vaccinia virus vectors showed cleavage specificit ies against the F protein mutants consistent with that shown by the process ing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type, These results indicate that the proteolytic processing enzym es of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4, The significance of individual contribution of the three amino acid s at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.