Mechanisms of reduced binding of influenza B wild type and mutant neuraminidases to NA inhibitors

Session VII – Jennifer McKimm-Breschkin

Mechanisms of reduced binding of influenza B wild type and mutant neuraminidases to NA inhibitors
Aaron J. Oakley, Susan Barrett, Thomas S. Peat, Victor Streltsov, Janet Newman, Lynne Waddington and Jennifer L. McKimm-Breschkin
CSIRO Molecular and Health Technologies, Parkville, Australia, 3052

Abstract:
Neuraminidase (NA) inhibitors are specific drugs for the treatment and prophylaxis of both influenza A and B.  While both oseltamivir (Tamiflu™) and zanamivir (Relenza™) have been in clinical use for several years, a third inhibitor peramivir is still in clinical trials.  While each inhibitor has unique modifications compared to the initial inhibitor, DANA, peramivir has modifications common to each of zanamivir and oseltamivir.  Hence while viruses may be resistant to only one of either oseltamivir or zanamivir, they are often also cross-resistant to peramivir.  The clinical effectiveness of oseltamivir against influenza B infection in children is less than against influenza A.  In addition the influenza B NA binds oseltamivir with reduced efficiency in enzyme inhibition assays in vitro.  While emergence of resistance is rare in influenza B viruses, we have recently identified a virus, B/Perth/211/2001, with a spontaneous mutation, D197E in the NA, which confers cross resistance to all NA inhibitors. Viruses with a D197N mutation have previously been isolated from patients treated with oseltamivir. However, unlike many other mutations conferring NA inhibitor resistance D197 is not conserved in all influenza strains.  In order to understand the basis for this resistance, we have expressed both the wild type B/Perth D197 and mutant E197 full length NAs using the baculovirus expression system.  The recombinant NAs have the same wild type and resistant properties as the virus associated NAs.  We have developed a purification method for production of the NA heads, and we have determined the crystal structures of the native B/Perth wild type D197 and mutant E197 NAs, and these NAs in complex with peramivir. The D to E mutation results in increased disorder of R150, compromising its interaction with the N-acetyl group of the inhibitors. This N-acetyl group is common to the substrate sialic acid and inhibitors zanamivir, oseltamivir and peramivir. This result explains the observed cross resistance to all the NA inhibitors.  Additionally, the structure of B/Perth E197 has been determined in complex with oseltamivir.  We demonstrate that upon binding oseltamivir rotation of E275 in the NA active site, which is required for high affinity binding of oseltamivir, does not occur.  This would correlate with the reduced binding of oseltamivir in influenza B strains.