White arrowheads indicate syncytia. levels that would allow for fusion. Attempts to restore fusion having a reducing agent were unsuccessful, suggesting the launched disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a prefusion conformation-specific antibody prior to cell disruption, indicating that the launched disulfide bonds did not significantly impact GO6983 protein folding. This study is the 1st to statement that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion. IMPORTANCE The paramyxovirus Hendra disease (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further GO6983 studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion (F) protein may be relevant to a broad span of enveloped viruses. In this study, we demonstrate that disulfide bonds launched between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still collapse properly and bind a prefusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and increase our knowledge of the TMD and its part in HeV F stability and fusion promotion. family consists of negative-sense single-stranded RNA viruses enclosed within lipid membranes. Hendra (HeV) and Nipah (NiV) viruses, members of the genus, are highly pathogenic zoonotic viruses within the family (1). Due to the high mortality rates associated with HeV and NiV infections and the lack of a human being vaccine or effective treatment, they have been designated biosafety level 4 pathogens (2). HeV and NiV were recognized in Australia and Malaysia, respectively, in the 1990s following outbreaks of severe encephalitis and respiratory disease in humans (2,C5). Further investigation exposed that fruit bats of the family were the natural reservoir for the viruses, and transmission to other organisms, including pigs and horses, contributed to the zoonotic spread to humans (6,C8). The potential for long term outbreaks of henipavirus infections and for the emergence of related zoonotic viruses warrants further study into the access mechanisms of these pathogens. Membrane fusion is an essential step in access of enveloped viruses that relies on the coordination of specialized proteins at the viral membrane surface. HeV and NiV possess two surface glycoproteins: the attachment protein (G), which allows the computer virus to bind a target cell, and the fusion protein (F), which promotes merger of the viral membrane with the target membrane (9, 10). Both glycoproteins, F and G, are required for paramyxovirus membrane fusion, but it is still unclear how interactions between F and G and receptor binding promote triggering of F (11). The henipaviruses and other members of the family make use of a trimeric class I F EIF2AK2 protein to drive membrane fusion (12,C14). Before the F protein can participate in fusion events, the inactive precursor (F0) must be proteolytically cleaved within the host cell to form a fusion-active disulfide-linked heterodimer (F1+F2) (Fig. 1A). For HeV and NiV, the F protein traffics to the cell surface and is subsequently endocytosed to be cleaved by the protease cathepsin L before being recycled back to the surface (15,C17). Following the cleavage event, the F protein is managed at the surface in a metastable prefusion GO6983 state until it is triggered to undergo the conformational changes needed to promote membrane fusion. These conformational changes from your prefusion to postfusion form involve an essentially irreversible rearrangement of the F protein ectodomain that results in.