A single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires intera ction of the viral hemagglutinin-neuraminidase (HN) glycoprotein,vith its s ialic acid receptor. 4 Guanidino 2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4 GU-DANA; zanamivir), a sialic acid transition-state analog designe d to fit the influenza virus neuraminidase catalytic site, possesses antivi ral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DA NA-resistant HPF3 virus variant (ZM1) was generated by serial passage in th e presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morpholo gy and harbored two HN gene mutations resulting in two amino acid alteratio ns, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not r esistant to 4-GU-DANA. Neuraminidase assays revealed a 15 fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0, The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less tha n for both WT and C-0 in the presence of 1 mM 4 GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-re sistant properties. An understanding of ZM1 and other escape variants provi des insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.