Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus.

The virulence of avian influenza A viruses depends on the cleavability of the haemagglutinin (HA) by an intracellular protease at multiple basic amino acids. Although previous studies have demonstrated the importance of these amino acids for processing by the cellular protease, with emphasis on conserved residues near the cleavage site, the minimal requirements for cleavage remain unknown. By expressing site-specific mutants of the HA of a virulent avian influenza virus, A/turkey/Ireland/1378/85 (H5N8), in the simian virus 40 system and testing for their cleavability by an endogenous protease in CV-1 cells, and their fusion activity in a polykaryon formation assay, we were able to show that glycine at the amino terminus of HA2 is not essential for cleavage and that maximal cleavage requires at least five basic residues at the cleavage site, when carbohydrate is nearby. Moreover, we confirmed, that a conserved proline upstream of the cleavage site is not essential for HA cleavage or fusion activity, and that lysine replacement of the carboxyl-terminal arginine of HA1 abolishes cleavability. These findings should help identify the proteases responsible for intracellular cleavage of the HA of virulent avian influenza viruses.