A Second Receptor Binding Site on Human Parainﬂuenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the Fusion Mechanism (cid:1)

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuramin-idase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion- triggering functions of HN has not been clear. For human parainﬂuenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site’s receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains pSV2-TAT (AIDS Reference Reagent Program) were added to the plates of Vero cells and incubated at 37°C for 12 h. Luciferase expression occurs only when cells containing Tat fuse with those transfected with luciferase. The cells were then washed, lysed (using luciferase lysis buffer [Promega]), and luciferase activity resulting from fusion of the two cell types was quantiﬁed using luciferase assay substrate (Promega) and a Spectramax M5 (Molecular Devices) luminescence microplate reader. Beta-galactosidase complementation-based fusion assay. We adapted an assay (19) that detects early stages of fusion activation, since the readout does not depend upon downstream transactivation events. We used this assay for experiments in which the greater range of detection of this assay was necessary. The assay is based on alpha complementation of beta-galactosidase; the beta-galac-tosidase protein lacking the N-terminal 85 residues (omega peptide) is expressed from one plasmid, and the N-terminal 85 residues (alpha peptide) is expressed from a second plasmid. Cell fusion leads to complementation, and beta-galac- tosidase is quantitated using the Galacto-Star (Applied Biosystems) chemilumi-nescent reporter gene assay system. Receptor-bearing cells expressing the omega peptide are mixed with HPIV3 HN/F-coexpressing cells that also express the alpha peptide, at various temperatures and at speciﬁc time points. Fusion is stopped by lysing the cells with lysis buffer. G values (relative binding energies with respect to sialic acid bound at site I) as an indication of binding efﬁciency, but the absence of crystallographically bound template for binding at site II leads to a fairly high degree of uncertainty. The modeling approach chose the best scoring pose from the many generated by the GOLD docking program and used that to calculate binding energy. This is subject to an uncertainty as to whether we have obtained the overall best pose. Statistical analysis. The Student-Newman-Keuls multiple-comparison test was applied for statistical analyses, using the InStat 3.0 software (GraphPad Software Inc., San Diego, CA).