Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site.
Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F1 and F2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P.W. Choppin [1976). Virology, 265-277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60-70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005796:Genes; D005814:Genes, Viral; D009154:Mutation; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000254:growth & development; D010447:Peptide Hydrolases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics*; D014775:Virus Activation
D012333:RNA, Messenger; D014760:Viral Fusion Proteins; D010447:Peptide Hydrolases