Paramyxovirus sensitivity to receptor binding and neuraminidase inhibition

Human parainfluenza virus type 3 (HPIV3) is an enveloped, non-segmented negative strand RNA virus responsible for a significant percentage of annual pediatric lower respiratory disease. Entry of HPIV3 into the host cell cytoplasm requires interaction of the envelope glycoprotein hemagglutinin-neuraminidase (HN) with sialic acid receptors on the cell surface. 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA, zanamivir, Relenza(TM)) was rationally designed as an inhibitor of the influenza neuraminidase (NA) glycoprotein, and is effective against NA at nanomolar concentrations [100]. Previous studies in our lab have identified the efficacy, at micro- and millimolar concentrations, of 4-GU-DANA against the receptor binding and neuraminidase activities of HPIV3 HN [119]. The current study presents the characterization of the first drug resistant paramyxovirus variant, ZM1. Initial characterization revealed that ZM1, generated by passage of WT HPIV3 in the presence of 4-GU-DANA, possesses two HN alterations (T193I and I567V), a fusogenic plaque morphology and enhanced receptor binding and neuraminidase activities that exhibit reduced 4-GU-DANA sensitivity. The altered phenotype of ZM1 is attributable solely to the T193I HN alteration. Further investigation revealed that both enhanced receptor binding avidity and reduced inhibitor binding at the HN active site contribute to the diminished 4-GU-DANA sensitivity of ZM1. Interestingly, Newcastle disease virus (NDV, a related paramyxovirus) harbors an isoleucine at the residue corresponding to HPIV3 HN 193 and possesses a reduced receptor binding sensitivity to 4-GU-DANA, providing evidence of the importance of this residue for both HN function and inhibitor sensitivity in other paramyxoviruses. Comparison of the 4-GU-DANA resistance mechanisms identified in HPIV3 and influenza virus revealed significant and informative differences. Influenza variants with decreased 4-GU-DANA sensitivity often possess decreased affinity for receptor, whereas the ZM1 variant utilizes increased receptor binding avidity to circumvent inhibition. Moreover, the ability of 4-GU-DANA to inhibit the neuraminidase activity of HPIV3 HN and to displace sialic acid receptors bound to HN has implications for the clinical use of this inhibitor and others in its class. In summary, the data presented here provide both a model for resistance of HPIV3 to 4-GU-DANA as well as functional and structural clues to the function of paramyxovirus HN.