Lv. Gubareva et al., CATALYTIC AND FRAMEWORK MUTATIONS IN THE NEURAMINIDASE ACTIVE-SITE OFINFLUENZA-VIRUSES THAT ARE RESISTANT TO 4-GUANIDINO-NEU5AC2EN, Journal of virology, 71(5), 1997, pp. 3385-3390
Here we report the isolation of influenza virus A/turkey/Minnesota/833
/80 (H4N2) with a mutation at the catalytic residue of the neuraminida
se (NA) active site, rendering it resistant to the novel NA inhibitor
4-guanidino-Neu5Ac2en (GG167), The resistance of the mutant stems from
replacement of one of three invariant arginines (Arg 292-->Lys) that
are conserved among all viral and bacterial NAs and participate in the
conformational change of sialic acid moiety necessary for substrate c
atalysis, The Lys292 mutant was selected in vitro after 15 passages at
increasing concentrations of GG167 (from 0.1 to 1,000 mu M), conditio
ns that earlier gave rise to GG167-resistant mutants with a substituti
on at the framework residue Glu119, Both types of mutants showed simil
ar degrees of resistance in plaque reduction assays, but the Lys292 mu
tant was more sensitive to the inhibitor in NA inhibition tests than w
ere mutants bearing a substitution at framework residue 119 (Asp, Ala,
or Gly), Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en a
nd Neu5Ac2en) varied among mutants resistant to GG167, being Lowest fo
r Lys292 and highest for Asp119, All GG167-resistant mutants demonstra
ted markedly reduced NA activity, only 3 to 50% of the parental level,
depending on the particular amino acid substitution, The catalytic mu
tant (Lys292) showed a significant change in pH optimum of NA activity
, from 5.9 to 5.3. All of the mutant NAs were less stable than the par
ental enzyme at low pH, Despite their impaired NA activity, the GG167-
resistant mutants grew as well as parental virus in Madin-Darby canine
kidney cells or in embryonated chicken eggs, However, the infectivity
in mice was 500-fold lower for Lys292 than for the parental virus, Th
ese findings demonstrate that amino acid substitution in the NA active
site at the catalytic or framework residues, followed by multiple pas
sages in vitro, in the presence of increasing concentrations of the NA
inhibitor GG167, generates GG167-resistant viruses with reduced NA ac
tivity and decreased infectivity in animals.