Summary auto-generated
This study examined the biosynthesis, transport, glycosylation, and enzymatic activity of neuraminidase (NA) from fowl plague virus (FPV), an avian influenza A virus. Using vaccinia virus expression systems and site-directed mutagenesis, researchers tracked NA maturation in vertebrate cells. FPV NA exhibited unusually slow transport kinetics—about four times slower than hemagglutinin (HA)—with tetramer formation occurring rapidly (15 min half-time) but passage through the Golgi and transport to the plasma membrane occurring much more slowly (2-3 hour half-times). FPV NA has the shortest stalk of any naturally occurring influenza NA, containing only three N-glycosylation sites, all in the globular head domain. Two sites contained complex-type sugars while one retained mannose-rich form. Oligosaccharides at positions 124 and 213 modulated enzymatic activity. Mutational analysis revealed that cysteine-49 in the stalk forms intermolecular disulfide-linked dimers that stabilize tetrameric structure but are not absolutely required for function. Coexpression of NA and HA showed no mutual effects on their transport rates, though NA removed terminal sialic acids from HA's oligosaccharides, as expected from NA enzymatic activity.
Key findings
- FPV NA transport to the cell surface is approximately four times slower than HA, with a 100-120 minute half-time to reach the medial Golgi and additional delays before plasma membrane expression
- FPV NA contains the shortest stalk region of any naturally occurring influenza virus NA (19 amino acids) with only three N-glycosylation sites located exclusively in the globular head domain
- Oligosaccharides at positions 124 and 213 modulate enzymatic activity of NA, while single elimination of any glycosylation site does not affect transport competence
- Cysteine-49 in the stalk forms disulfide-linked dimers that stabilize the NA tetramer but are not absolutely required for enzymatic activity
- Rapid tetramer formation occurs within 15 minutes, but this precedes much slower glycan processing and transport to the plasma membrane
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Abstract
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.