Steps in maturation of influenza A virus neuraminidase.
We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus-infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers.
Saito
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T
Department of Virology/Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Taylor
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G
Webster
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RG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D004220:Disulfides / Q000378:metabolism; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D009980:Influenza A virus / Q000201:enzymology*; D007700:Kinetics; D009439:Neuraminidase / Q000276:immunology / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D013696:Temperature
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D009439:Neuraminidase