Studies on the size, chemical composition, and partial sequence of the neuraminidase (NA) from type A influenza viruses show that the N-terminal region of the NA is not processed and serves to anchor the NA in the viral membrane

Abstract

Intact neuraminidase (NA) molecules were isolated by detergent treatment of influenza virus particles from NA subtypes, N1, N2, N5, and N8. Incomplete NA molecules (“heads”) were isolated by Pronase digestion of virus particles from the N2, N5, and N8 strains. Sedimentation equilibrium analysis of NA heads from A/Tokyo/67 (N2) influenza virus gave a molecular weight for the tetramer of 200,000. Under nonreducing denaturing conditions these tetramers were found to dissociate into two disulphide-linked dimers of molecular weight 100,000. Using a monomer molecular weight of 50,000, amino acid and carbohydrate analyses show that the Pronase-released monomer contains approximately 397 amino acid residues and 31 carbohydrate residues. Only N-acetylglucosamine, mannose, galactose, and fucose were found suggesting that all sugar units are in N-glycosidic linkage to asparagine residues. Comparative peptide mapping and direct N-and C-terminal sequence analysis of intact NA and Pronase-released heads showed that the membrane-associated stalk region of the enzyme comes from the N-terminal region of the molecule. Maps of tryptic peptides from intact NA molecules yielded a peptide which had the same composition as the N-terminal hexapeptide (predicted from the nucleotide sequence of the NA genes), which is common to all these strains. This peptide was not present in the Pronase-released NA heads of the N2, N5, and N8 strains. Amino acid sequence analysis showed that the N-terminal sequence of intact NA was very different from that of Pronase-released heads, some 73–76 amino acids being removed by Pronase digestion. The C-terminal regions of both forms of NA were the same. The sequence data also show that protein synthesis does start at the AUG codon that is equivalent to the first ATG in the cDNA sequence of the NA gene and that the N-terminus of the NA polypeptide is not modified following its synthesis. No initiating methionine or signal peptide is cleaved off the neuraminidase molecule.