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 intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at 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, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes 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 acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.
Fujii
Y
Y
Department of Bacteriology, Hiroshima University School of Medicine, Hiroshima, Japan.
Sakaguchi
T
T
Kiyotani
K
K
Yoshida
T
T
D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008253:Macaca mulatta; D009522:Newcastle disease virus / Q000254:growth & development* / Q000472:pathogenicity; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014760:Viral Fusion Proteins / Q000378:metabolism*; D014774:Virulence; D014775:Virus Activation
D006023:Glycoproteins; D011498:Protein Precursors; D014760:Viral Fusion Proteins; D010450:Endopeptidases; C080654:PCSK6 protein, human; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins