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

Abstract

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. IMPORTANCE 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.

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

“…S1 ). This protein turnover is similar to turnover rates seen in PIV5 fusion protein, also activated by cellular furin [ 99 ], and slightly slower turnover than Hendra fusion protein, activated by cellular cathepsins [ 100 , 101 ]. Over-expression of cellular proteases that may process S did not affect these protein turnover rates.…”

Viral Membrane Fusion and the Transmembrane Domain

“…Following triggering, the F protein of paramyxoviruses undergoes large conformational changes that include insertion of the FP into the target membrane, followed by the protein refolding back on itself to facilitate formation of the six-helix bundle. While the details of the TMD throughout this process are still being investigated, there is clear evidence for an active role of the TMD and TMD-TMD interactions along the fusion cascade [105,106,[110][111][112][113]. Illustrating the role of the TMD in the fusion process, replacement of the NDV F protein TMD with the TMD of a related viral fusion protein abolished fusion, including hemi-fusion intermediates, despite the chimeric fusion protein being expressed and cleaved at the cell surface [110].…”

“…To further probe specific residues of the TMD that are critical for fusion, several studies performed mutagenesis on paramyxovirus F protein TMDs and analyzed differences in fusion activity [106,108,109,[111][112][113]. Alanine scanning mutagenesis found that β-branched or just branched amino acid residues at the C-terminus of the TMD appear to play an important role in fusion in both PIV5 [106] and Hendra [113].…”

“…Mutating the L-I Zipper motif in the F protein TMD completely suppressed fusion activity in both PIV5 and Hendra, despite there being cleaved protein at the cell surface in both cases [109,111]. Interestingly, introduction of disulfide bonds to prevent TMD-TMD dissociation also disrupts fusion in Hendra [112], as does introduction of disulfide bonds directly N-terminal to the TMD in PIV5 F [114]. These studies suggest that TMD-TMD association and dissociation must be intricately controlled for membrane fusion to occur.…”

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

“…Throughout this trafficking, the F protein remains associated as a trimer [4,5,12,13]. Interactions within the transmembrane (TM) regions of F trimers have been implicated in protein stability and fusion function [11,12,[14][15][16]. Additionally, residues S490 and Y498 have been demonstrated to be involved in the endocytosis of F [11].…”