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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. Receptor-bound 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.

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.

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. However, 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.

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses., Author Summary Nipah (NiV) and Hendra (HeV) viruses are two lethal emerging zoonotic paramyxoviruses. In addition to acute infection, these viruses may lead to late-onset disease or relapse of encephalitis years after initial infection, as well as persistent or delayed neurological sequelae. We present a new strategy to prevent and treat NiV/HeV infection that may be broadly applicable for enveloped viral pathogens. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection, and blocking this step can prevent or treat infection, as clinically validated for HIV. For paramyxoviruses, however, peptides that bind the viral fusion protein have been shown to inhibit fusion in vitro, but not in vivo. The new strategy that we present here opens the door to clinically effective paramyxovirus fusion-inhibitory peptides. By targeting fusion-inhibitory peptides to the target membrane using a cholesterol tag, we capture an early stage in the viral fusion-activation process, thus drastically enhancing the efficacy of these peptides at inhibiting viral entry. Importantly, this strategy prevents and treats lethal Nipah virus infection in vivo. Membrane targeting of antiviral peptides thus offers a new approach to development of highly effective peptide fusion antivirals against important human pathogens.