Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 M in vitro (50% inhibitory concentration, 2 M), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine's antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.

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