Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HNs receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HNs F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HNs F-triggering activity may have promise in vivo.Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry, the viral surface glycoproteins hemagglutinin-neuraminidase (HN) (the receptor-binding molecule) and F (the fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. To understand these mechanisms, elucidate how paramyxoviruses enter cells, and develop strategies to prevent or treat infection, we study human parainfluenza virus (HPIV), an important cause of croup and bronchiolitis in children. Our results have uncovered fundamental roles of the receptor-binding protein in paramyxovirus fusion and principles of coordinated interaction between the glycoproteins during the viral life cycle.To understand how the diverse functions of the viral glycoproteins are regulated during the viral life cycle, we have used viruses bearing variant HN molecules with mutations at the binding/F-triggering site (and/or the primary receptor-binding site) to study how this molecule works to trigger F (2,3,7,10,15,18,20). The correct timing of F activation (triggering) by HN is essential for entry. For infection to occur, triggering must occur only when F is in proximity to the target cell membrane, and we propose that the regulation of F triggering is essential for the survival of the virus. The outcome of infection is determined by the target cell's propertie...

No abstract

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptorengaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo.IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

Background The first step in infection by human parainfluenza viruses (HPIVs) is binding to the surface of respiratory epithelial cells via interaction between viral receptor-binding molecules and sialic acid-containing receptors. DAS181, a recombinant sialidase protein containing the catalytic domain of A. viscosus sialidase, removes cell surface sialic acid, and we proposed that it would inhibit HPIV infection. Methods Depletion of sialic acid receptors by DAS181 was evaluated by lectin binding assays. Anti-HPIV activity in cultured cell lines and in human airway epithelium (HAE) was assessed by the reduction in viral genomes and/or plaque forming units (PFU) upon treatment. In vivo efficacy of intranasally administered DAS181 was assessed using a cotton rat model. Results DAS181-mediated desialylation led to anti-HPIV activity in cell lines and HAE. Intranasal DAS181 in cotton rats, a model for human disease, significantly curtailed infection. Conclusions Enzymatic removal of the sialic acid moiety of HPIV receptors inhibits infection with all tested HPIV strains, both in vitro and in cotton rats. Enzyme-mediated removal of sialic acid receptors represents a novel antiviral strategy for HPIV. The results of this study raise the possibility of a broad spectrum antiviral agent for influenza virus and HPIVs.