Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.
We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.
Biomolecular Research Institute, Parkville, Victoria, Australia. anjali@bioresi.com.au
A
A
Sahasrabudhe
L
L
Lawrence
V C
VC
Epa
J N
JN
Varghese
P M
PM
Colman
J L
JL
McKimm-Breschkin
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005557:Formaldehyde / Q000494:pharmacology; D014644:Genetic Variation; D006146:Guanidines; D006923:Hymecromone / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000201:enzymology* / Q000235:genetics*; D017354:Point Mutation; D011714:Pyrans; D012794:Sialic Acids / Q000494:pharmacology; D055672:Static Electricity; D013379:Substrate Specificity; D053243:Zanamivir
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D005557:Formaldehyde; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase; D053243:Zanamivir