S. Bantia et al., GENERATION AND CHARACTERIZATION OF A MUTANT OF INFLUENZA-A VIRUS SELECTED WITH THE NEURAMINIDASE INHIBITOR BCX-140, Antimicrobial agents and chemotherapy, 42(4), 1998, pp. 801-807
Influenza neuraminidase (NA) plays an important role in viral replicat
ion, and characterization of viruses resistant to NA inhibitors will h
elp elucidate the role of active-site residues. This information will
assist in designing better inhibitors targeted to essential active-sit
e residues that cannot generate drug-resistant mutations. In the prese
nt study,ve used the benzoic acid-based inhibitor BCX-140 to select an
d characterize resistant viruses. BCX-140 binds to the NA active site
in an orientation that is opposite that of a sialic acid-based compoun
d, anidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thu
s, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA th
e guanidino group binds to Glu-119. We passaged influenza A/Singapore/
1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-
140, and virus resistant to this inhibitor was selected after six pass
ages. The NA of this mutant was still sensitive to inhibition by BCX-1
40. However, the mutant virus was resistant to BCX-140 in plaque and 3
-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assa
ys. Sequence analysis of hemagglutinin (HA) and NA genes revealed chan
ges in both, although none were in the active site of the NA. Dependin
g on the method of selection of the resistant virus, two types of chan
ges associated with the sialic acid binding site were seen in the HA.
One is a change in HAI of Ala-133 to Thr, a residue close to the bindi
ng site, while the other change was Arg-132 of HA1 to Gin, which in HA
I of serotype H3 is a sialic acid contact (Asn-137). Binding studies r
evealed that both types of resistant viruses had reduced receptor bind
ing affinity compared to that of the wild type. Thus, resistance to BC
X-140 IO was generated by modifying the HA. NA active-site residue 276
may be essential for activity, and thus, it cannot be changed to gene
rate resistance. However, drug-induced changes in the HA can result in
a virus that is less dependent on NA activity for growth in cells and
, hence, resistant to NA inhibitors.