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Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutininneuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo. [ABSTRACT FROM AUTHOR]

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptorbound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. How-ever, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect. [ABSTRACT FROM AUTHOR]

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation. [ABSTRACT FROM AUTHOR]