Neuraminidase inhibitor drug susceptibility differs between influenza N1 and N2 neuraminidase following mutagenesis of two conserved residues

Abstract

Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity. The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA. This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.