Generation and Characterization of a Mutant of Influenza A Virus Selected with the Neuraminidase Inhibitor BCX-140

Abstract

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acidbased compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the 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 passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate 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.

Characterization of 2 Influenza A(H3N2) Clinical Isolates with Reduced Susceptibility to Neuraminidase Inhibitors Due to Mutations in the Hemagglutinin Gene

“…Growth inhibition of influenza virus isolates in MDCK cells was performed using a colorimetric method based on the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by viable cells, as described elsewhere [20]. The percentage of cell survival was calculated using the for-…”

Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities

“…These studies are generally related to altering the activity of HA or NA and observing the relative infectivity or compensatory mutations that occur after passage. Studies involving the inhibition of NA with inhibitors have shown that escape mutants can be generated with substitutions in HA alone (7–9), and sensitivity to these inhibitors is determined by both HA and NA (3). These substitutions are hypothesized to reduce HA binding, allowing efficient release of virus with reduced NA activity.…”

Difluorosialic acids, potent novel influenza virus neuraminidase inhibitors, induce fewer drug resistance-associated neuraminidase mutations than does oseltamivir

“…This is achieved by reducing the affinity of HA to the sialic acid, therefore the progeny virus particles rely less on NA activity when leaving the cell surface (Bantia et al, 1998;Blick et al, 1998;Ginting et al, 2012;McKimm-Breschkin et al, 1996;Molla et al, 2002). Indeed, sequence analyses revealed Fig.…”