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 analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a half-time of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPV-infected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs 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 oligosaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose-rich form. The glycosylation mutants 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 sites. Mutational analysis of the three Cys residues not involved in intrachain disulfide pairing revealed that Cys-49 in the stalk of the NA molecule is responsible for the formation of disulfide-linked dimers. Analysis of cysteine mutants of FPV NA also demonstrated that disulfide-linked dimers are not absolutely necessary for the formation of enzymatically active tetramers but may stabilize the quaternary structure of NA.
Hausmann
J
J
Institut für Virologie, Philipps-Universität Marburg, Germany.
Kretzschmar
E
E
Garten
W
W
Klenk
H D
HD
D000818:Animals; D001692:Biological Transport; D001717:Birds / Q000821:virology*; D003545:Cysteine; D004789:Enzyme Activation; D006031:Glycosylation; D009980:Influenza A virus / Q000201:enzymology*; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides
D009844:Oligosaccharides; D009439:Neuraminidase; D003545:Cysteine