N1 NEURAMINIDASE OF INFLUENZA-VIRUS A/FPV/ROSTOCK/34 HAS HEMADSORBING ACTIVITY

The neuraminidase (NA) gene of influenza virus A/FPV/Restock/34 virus (H7N1) was cloned and expressed in Sf9 cells using a baculovirus vector. The expression product had the expected molecular mass and showed neuraminidase activity. NA expressed in Sf9 cells also showed haemagglutinating activity as indicated by its ability to induce haemadsorption of chicken red blood cells. Haemadsorption depended on the presence of neuraminic acid on the erythrocytes, but was not blocked by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, a specific inhibitor of the enzymatic activity. These observations suggest that the N1 neuraminidase has two distinct reactive sites: a catalytic site and a receptor binding site. This concept was confirmed by site-directed mutagenesis which showed that the haemadsorbing activity of FPV NA was abolished when amino acids located on two surface loops at the putative binding site were exchanged. Substitutions on either of the two loops were sufficient for this effect. The mutated NAs retained full neuraminidase activity. In contrast, a mutated NA lacking neuraminidase activity still had haemadsorbing activity.