For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4°C are transferred to 22°C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HNreceptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptorbinding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22°C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HNs F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANAresistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.

In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37°C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HNs competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).Human parainfluenza virus 3 (HPIV3), like other paramyxoviruses, possesses two envelope proteins directly involved in viral entry and cytopathology. Hemagglutinin-neuraminidase (HN), by binding to sialic acid-containing cellular surface receptors, brings the viral envelope in proximity to the plasma membrane. Fusion protein (F) is the active mediator of fusion between the viral and cell membranes, which leads to release of the viral nucleocapsid into the cytoplasm. In order to mediate fusion, F must be present in its cleaved state and then undergo a second activation step to assume its fusion-ready conformation. It has been shown that HN plays an essential role in the fusion process, and one proposed role of HN is to drive the final conformational change in F that renders it fusion active (30). Fusion of the virus with the target cell, which permits synthesis of viral macromolecules and expression of viral envelope proteins on the host cell's surface, is followed by two processes that lead to the spread of infection and HPIV3 cytopathogenesis. One is the assembly and budding of new virions that then infect distant as well as neighboring cells. Second, by virtue of HN and F expressed on its surface, an infected cell can fuse with adjacent uninfected cells, forming syncytia. In the spread of infection via budding, HNЈs receptordestroying neuraminidase activity plays the essential role of preventing progeny virions from remaining aggregated on the infected cell's surface rather than spreading to additional cells (29). For syncytium formation, no direct role has yet been found for HNЈs neuraminidase activity, although the magnitude of the receptor-cleaving activity...

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...

Attachment of human parainfluenza virus type 3 (HPIV3) to the host cell is mediated by the envelope protein hemagglutinin-neuraminidase (HN). HN binds to sialic-acid-containing receptors on the cell surface and also contributes to the process whereby the other surface protein (the fusion protein F) is triggered and mediates fusion of the viral envelope and the cell membrane. The third role of HN in the infection process is receptor cleavage (via neuraminidase action), allowing for the release of progeny virions and the spread of infection to additional cells (for a review, see reference 9).One method for interfering with infection by viruses that make use of sialic-acid-containing receptors for entry is the blockade of receptor binding by the use of sialic acid analogs. Monomeric analogs of sialic acid can inhibit the attachment that is required for fusion and entry, and transition-state analogs of sialic acid, identified on the basis of their ability to inhibit influenza neuraminidase, are also effective inhibitors of HPIV3 binding, entry, and fusion (11). 4-guanidino-Neu5Ac2en (4-GU-DANA, or zanamivir) inhibits not only the neuraminidase activity but also the receptor interaction of HPIV3 HN (6), blocking receptor binding and subsequent fusion. For influenza virus, in which 4-GU-DANA inhibits the neuraminidase (NA) and interferes with viral replication by preventing the release of newly formed virions, resistance is conferred by mutations which decrease the binding of 4-GU-DANA to the NA and/or by mutations in the hemagglutinin (HA), which decrease the affinity for the cellular receptor (12). In contrast, for HPIV3, 4-GU-DANA reduces infectivity instead by inhibiting HN-receptor interaction, and hence HN mutants with increased receptor-binding avidities are among those that can escape 4-GU-DANA's growth-inhibitory effect. In fact, for all of the HPIV3 wild-type (wt) and HN variant viruses that we have studied, decreased sensitivity correlated with an increased avidity for the receptor (16,17).In earlier work, we selected for an HPIV3 HN variant in tissue culture that was less sensitive to 4-GU-DANA's effects on both HN activities. We thereby generated a fusogenic HPIV3 virus variant (called ZM1 in previous publications [16,17]; HN T193I/I567V) that harbors two HN gene mutations that result in amino acid alterations and phenotypic resistance to the effects of 4-GU-DANA on both neuraminidase activity and receptor binding (16,17). One of these mutations (T193I) is responsible for an increase in receptor binding and in neur-* Corresponding author. Mailing address: