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

Abstract

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 fusiontriggering 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.Paramyxoviruses, including the human parainfluenza viruses, possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor cleaving as well as receptor binding activity. HN is also essential for activating the fusion protein (F) to mediate the merger of the viral envelope with the host cell membrane. For the parainfluenza viruses as well as other HN-containing paramyxoviruses, this one molecule carries out three different but critical activities at specific points in the process of viral entry. A clear mechanistic understanding of how these activities are regulated is key for the design of strategies to block viral entry (21). The specific role of HN in triggering F is a key question and has eluded simple explanations (3); we propose that a second binding site on HN exists and serves an important function in the triggering mechanism.Crystallographic studies of the human parainfluenza virus type 3 (HPIV3) HN (17), the Newcastle disease virus (NDV) HN (6,33), and the simian virus 5 HN (32) indicated that a single active/catalytic site on the globular head of the HN molecule can indeed provide both receptor binding and neuraminidase activities. For NDV, experimental evidence for the importance of this site ("site I") to receptor binding includes the finding that mutations in site I alter receptor binding (2, 18). For HPIV3, mutations at the putative receptor binding site also affect receptor binding avidity (22,23,25). However, the effects of the receptor analog zanamivir on activities of the two HN molecules were different: for HPIV3 HN, both receptor binding and neuraminidase activity were blocked by zanamivir, while for NDV HN, receptor binding was partially resistant to the e...

Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery

“…CSC11 affects neither receptor-binding nor neuraminidase activity. We have identified a second functional binding site (site II) that contains both binding and F-triggering activities (29,32). For NDV, our experimental data (32) and the revised crystal structure (21) revealed a second receptor binding site in the HN head, and we showed that this second binding site is activated by engagement of the primary binding site (64).…”

“…For HPIV3, however, the dynamics are different. Although the crystal structure of HPIV3 HN revealed only one binding site (22), we experimentally identified a second receptor binding site that works totally differently from that of NDV; its binding is weak (whereas for NDV activation of site II leads to stronger receptor binding), its binding activity is pH-regulated (26), and it plays a direct role in triggering F (29). To evaluate whether the F-triggering activity of CSC11 might act via an interaction with site II, the structure of CSC11 (and in parallel zanamivir, for comparison) were docked into site II of HN.…”

“…Fig. 6B reveals that CSC11 did not inhibit RBC fusion if exposed to HN/F-coexpressing cells at 4°C, a temperature that does not permit F-activation (29). If, however, CSC11 was exposed to the HN/F-expressing cells at 37°C (a temperature that permits F-activation (29)) and before receptor engagement, then cell fusion was ultimately inhibited.…”

“…F-activation (19,26,29). This assay allowed us to address whether fusion and infectivity is modulated by CSC11 before or after HN engages the target cell receptor.…”

“…␤-Galactosidase Complementation-based Fusion AssayWe previously adapted a fusion assay based on alpha complementation of ␤-galactosidase (29,36); receptor-bearing cells expressing the omega peptide of ␤-galactosidase are mixed with HPIV3 HN/F-coexpressing cells that also express the alpha peptide of ␤-galactosidase, in the presence of various compound concentrations. Cell fusion leads to complementation.…”