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

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 (neuraminidase), 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 both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion

Paramyxovirus Receptor-Binding Molecules: Engagement of One Site on the Hemagglutinin-Neuraminidase Protein Modulates Activity at the Second Site

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three different activities: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. These three discrete properties each affect the ability of HN to promote viral fusion and entry. For human parainfluenza type 3, one bifunctional site on HN can carry out both binding and neuraminidase, and the receptor mimic, zanamivir, impairs viral entry by blocking receptor binding. We report here that for Newcastle disease virus, the HN receptor avidity is increased by zanamivir, due to activation of a second site that has higher receptor avidity. Only certain receptor mimics effectively activate the second site (site II) via occupation of site I; yet without activation of this second site, binding is mediated entirely by site I. Computational modeling designed to complement the experimental approaches suggests that the potential for small molecule receptor mimics to activate site II, upon binding to site I, directly correlates with their predicted strengths of interaction with site I. Taken together, the experimental and computational data show that the molecules with the strongest interactions with site I—zanamivir and BCX 2798—lead to the activation of site II. The finding that site II, once activated, shows higher avidity for receptor than site I, suggests paradigms for further elucidating the regulation of HN′s multiple functions in the viral life cycle

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin- neuraminidase (HN) and the fusion protein (F). The receptor- bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc