Transmembrane Domain Dissociation Is Required for Hendra Virus F Protein Fusogenic Activity.

Hendra virus (HeV) is a zoonotic paramyxovirus that utilizes a trimeric fusion (F) protein within its lipid bilayer to mediate membrane merger with a cell membrane for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the prefusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, and N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced 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 introduced disulfide bonds did not significantly affect protein folding. This study is the first to report that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion. The paramyxovirus Hendra virus (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion (F) protein may be applicable to a broad span of enveloped viruses. In this study, we demonstrate that disulfide bonds introduced between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still fold properly and bind a prefusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and expand our knowledge of the TMD and its role in HeV F stability and fusion promotion.