Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en.
A variant of the influenza virus NWS/G70C has been generated which has decreased sensitivity in vitro to the neuraminidase-specific inhibitor, 4-guanidino-Neu5Ac2en. The virus is 1000-fold less sensitive to the 4-guanidino-Neu5Ac2en in a plaque assay, but only 10-fold less sensitive to 4-amino-Neu5Ac2en. In an enzyme inhibition assay 250-fold more drug was needed to achieve inhibition comparable to that observed with the parent virus. In contrast to the plaque assay, the virus was fully sensitive to 4-amino-Neu5Ac2en in the enzyme inhibition assay. Kinetic analysis of 4-guanidino-Neu5Ac2en binding demonstrated that the variant no longer exhibited the slow binding characteristic seen with the parent and other influenza viruses and inhibition by Neu5Ac2en was also decreased. However, binding to 4-amino-Neu5Ac2en remained the same as the parent. Sequence analysis of this virus revealed a mutation at a previously conserved site in the enzyme active site of the neuraminidase, Glu 119 to Gly. Crystallographic analysis of the mutant neuraminidase with and without bound inhibitor confirmed this mutation and suggested that the reduced affinity for the 4-guanidino-Neu5Ac2en derives partly from the loss of a stabilizing interaction between the guanidino moiety and the carboxylate at residue 119, and partly from alterations to the solvent structure of the active site.
Blick
T J
TJ
Biomolecular Research Institute, Parkville, Australia.
Tiong
T
T
Sahasrabudhe
A
A
Varghese
J N
JN
Colman
P M
PM
Hart
G J
GJ
Bethell
R C
RC
McKimm-Breschkin
J L
JL
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines; D006386:Hemagglutination Tests; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D011714:Pyrans; D012794:Sialic Acids / Q000494:pharmacology*; D013379:Substrate Specificity; D010948:Viral Plaque Assay; D053243:Zanamivir
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D009439:Neuraminidase; D053243:Zanamivir