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

Abstract

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 HNs multiple functions in the viral life cycle.Paramyxoviruses, including human parainfluenza virus type 3 (HPIV3) and the avian paramyxovirus Newcastle disease virus (NDV), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving as well as receptorbinding 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 both HPIV3 and NDV, this one molecule carries out three different but critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block viral entry (19).We have previously used a small molecule receptor mimic, zanamivir (4-guanidino-neu5Ac2en [DANA]), to probe paramyxovirus active sites and found distinctions between HPIV3 HN and NDV HN that are manifested by differences in the sensitivity of individual HN functions to zanamivir. Zanamivir can reversibly occupy the neuraminidase active site/receptor-binding site of HPIV3 HN (17). The compound was originally developed as an influenza neuraminidase inhibitor and has a sialidase K i (M) of approximately 1 ϫ 10 Ϫ9 for the influenza A neuraminidase (13). The K i for the parainfluenza virus neuraminidases is lower, approximately 8 ϫ 10 Ϫ4 for HPIV2, the virus for which values have been obtained (13).For HPIV3, zanamivir inhibited both the receptor-binding function and the neuraminidase function of HN (12) but had its primary anti-infective effect by blocking receptor binding and thus impairing entry. A mutation at the active site (T193I...

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…The analyses of NDV revealed two sialic acid binding regions, sites I and II, on HN. We previously reported that site II can be activated for receptor binding by small molecules (e.g., zanamivir) that occupy site I (25), and this finding was supported by recent analysis of a series of NDV HN mutants (25)(26)(27).…”

“…The neuraminidase activity was reduced in the presence of zanamivir for all of the expressed viral chimeric proteins, indicating that zanamivir effectively interacts with catalytic site I. Figure 3C and 3D show that receptor binding activity is maintained despite zanamivir's blockade of site I (as evidenced by the inhibition of neuraminidase), indicating that the NDV site II was activated for receptor binding in the chimeric proteins as described for NDV AV HN wt and other G-HN and HN-HN chimeric proteins (25)(26)(27).…”

“…6. In the absence of zanamivir, receptor binding site II in NDV's globular head is not activated (25), so that there is no fusion ( Fig. 12A and C) and the RBCs are released.…”

“…Neuraminidase activity was assessed in the presence or absence of zanamivir, which inhibits NDV's neuraminidase activity (25). For chimeric proteins with mutated stalk domains, neuraminidase activity was decreased compared to NDV AV HN wt, especially for G 1-180 -HN 124-571 (Fig.…”

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

“…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.…”