A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability

Abstract

Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development. IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre-to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.

Viral Membrane Fusion and the Transmembrane Domain

“…The ability of TMD peptides to interfere with fusion protein function was also demonstrated in viral infection, as TMD peptides homologous to the TMD of the PIV5 F protein decreased PIV5 infection when incubated with the virus prior to cellular infection. This inhibition was sequence specific, as pre-incubation with PIV5 F TMD peptides did not decrease the infection of a related virus (HMPV) [174]. A similar antiviral strategy was demonstrated using peptides derived from the MPER of Flavivirus E protein.…”

“…The Env TMD peptides were able to directly associate with WT full-length Env protein, suggesting that the decrease in virus infectivity is due to the peptide disrupting the native TMD-TMD association [82]. Similarly, co-expression of small proteins mimicking the TMD of the Hendra F protein led to de-stabilization of the full-length F protein, consistent with the presence of additional TMDs reducing the trimeric interactions needed for pre-fusion stability [174]. The TMD proteins also disrupted cell-cell fusion when co-expressed with WT full-length Hendra fusion protein.…”

Molecular dynamics simulation and experimental study of the surface-display of SPA protein via Lpp-OmpA system for screening of IgG

“…The e ciency of surface display systems and the correct and e cient protein folding and its stability is highly related to the speci cations of the carrier protein, passenger protein, and fusion method Barrett ., et al 2019). LPP-ompA is an e cient surface display system that has been developed and applied for various applications (Fasehee ., et al 2018;Rigi ., et al 2014 ;Tafakori., et al 2012).…”

Differential Features of Fusion Activation within the Paramyxoviridae

“…Finally, co-expression of the isolated HeV-F TM domain with native, full-length HeV-F resulted in reduced expression of the native HeV-F and disrupted fusion activity, likely because interaction of the two forms leads to either protein misfolding during trafficking or premature triggering at the cell surface [135]. These effects are virus-specific, as the isolated HeV-F TM domain has no deleterious effect on native PIV5-F.…”