Mutations affecting the sensitivity of the influenza virus neuraminidase to 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid.
4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en) specifically inhibits the influenza virus neuraminidase (NA) through interaction of the guanidino group with conserved Glu 119 and Glu 227 residues in the substrate binding pocket of the enzyme. To understand the mechanism by which influenza viruses become resistant to 4-guanidino-Neu5Ac2en, we investigated mutations at amino acid residues 119 and 227 in the influenza virus NA for their effects on this compound and on NA activity. The NA gene was cloned from the NWS-G70c virus, and mutations were introduced at the codon for amino acid residue 119 or 227. All of the 13 mutants containing a change at residue 119 were transported to the cell surface, although their expression levels ranged from 68.2 to 91.3% of wild type. Mutant NAs that retained at least 20% of the wild-type enzymatic activity were tested for their sensitivity to 4-guanidino-Neu5Ac2en and found to be sevenfold less sensitive to this compound than was the wild-type NA. By contrast, only 6 of 13 mutants defined by modifications at residue 227 were transported to the cell surface, and those NAs lacked substantial enzymatic activity (9% of wild type, at most). These results suggest that only a limited number of resistant viruses arise through mutations at Glu 119 and Glu 227 under selective pressure from 4-guanidino-Neu5Ac2en and that the development of compounds which interact with 227 Glu more strongly than does 4-guanidino-Neu5Ac2en may reduce the likelihood of drug-resistant viruses still further.
Goto
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Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Bethell
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RC
Kawaoka
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Y
D000998:Antiviral Agents / Q000494:pharmacology*; D002461:Cell Line, Transformed; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006146:Guanidines; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D011714:Pyrans; D012794:Sialic Acids / Q000494:pharmacology*; D053243:Zanamivir
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D018698:Glutamic Acid; D009439:Neuraminidase; D053243:Zanamivir