Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses

The structural basis for the different proteolytic cleavability of influenza virus hemagglutinin (HA) was investigated with a group of pathogenic and nonpathogenic avian influenza viruses belonging to the antigenic subtype H7 (Hav1). Infected cel lysates or lystates of purified virus particles were subjected to two-dimensional gel electrophoresis. The first dimension, isoelectric focusing,- was done under nonreducing conditions, the second dimension, SDS-PAGE, under reducing conditions. The results obtained permit the following conclusions: The amino acid sequence of the connecting peptide between HA₁ and HA₂ determines proteolytic cleavability by a trypsin-like cellular enzyme. Upon proteolytic cleavage of HA of pathogenic strains, peptides of differing positive charge were eliminated. These HAs have, however, significantly more basic connecting peptides than HAs of nonpathogenic viruses. HAs of nonpathogenic H7 strains appear to have a connecting peptide similar to the human influenza viruses, since treatment of these viruses with trypsin results in a similar small charge shift which probably corresponds to the elimination of one basic amino acid. Thus, the primary structure of the connecting peptide determines biological activation and thereby pathogenicity of these viruses.