BIOSYNTHESIS, INTRACELLULAR-TRANSPORT AND ENZYMATIC-ACTIVITY OF AN AVIAN INFLUENZA-A VIRUS NEURAMINIDASE - ROLE OF UNPAIRED CYSTEINES AND INDIVIDUAL OLIGOSACCHARIDES

Intracellular transport, glycosylation, tetramerization and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were ana lysed in vertebrate cells after expression from a vaccinia virus vecto r. Tetramerization occurred with a halftime of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membra ne occurred with half-times of 2 and 3 h, respectively, suggesting a s tep in NA maturation beyond tetramerization that limits the rate of tr ansport to the medial Golgi. NA transport rates were about fourfold sl ower than those of haemagglutinin (HA), Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the trans port rate of HA influenced by NA, The slow transport kinetics of NA we re also observed in FPV-infected CV-1 cells, As deduced from the codin g sequence, FPV NA has the shortest stalk of all naturally occurring N As described to date and contains only three potential N-glycosylation sites, which are all located in the globular head domain, Elimination of each of the three N-glycosylation sites revealed that the two olig osaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose-rich form, The glycosylation mut ants showed also that oligosaccharides at positions 124 and 213 of FPV NA modulate enzymatic activity, Transport of NA is not influenced by single elimination of any of the three oligosaccharide attachment site s, Mutational analysis of the three Cys residues not involved in intra chain disulfide pairing revealed that Cys-49 in the stalk of the NA mo lecule is responsible for the formation of disulfide-linked dimers. An alysis of cysteine mutants of FPV NA also demonstrated that disulfide- linked dimers are not absolutely necessary for the formation of enzyma tically active tetramers but may stabilize the quaternary structure of NA.