Bis(Monoacylglycero)Phosphate Promotes Membrane Fusion Facilitated by the SARS-CoV-2 Fusion Domain.

Membrane fusion is a critical component of the viral lifecycle. For SARS-CoV-2, fusion is facilitated by the spike glycoprotein and can take place via either the plasma membrane or the endocytic pathway. The fusion domain (FD), which is found within the spike glycoprotein, is primarily responsible for the initiation of fusion as it embeds itself within the target cell's membrane. A preference for SARS-CoV-2 to fuse at low pH akin to the environment of the endocytic pathway has already been established; however, the impact of the target cell's lipid composition on the FD has yet to be explored. Here, we have shown that the SARS-CoV-2 FD preferentially initiates fusion at the late endosomal membrane over the plasma membrane, on the basis of lipid composition alone. A positive, fusogenic relationship with anionic lipids from the plasma membrane (POPS: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine) and endosomal membrane (BMP: bis(monoacylglycero)phosphate) was established, with a large preference demonstrated for the latter. When comparing the binding affinity and secondary structure of the FD in the presence of different anionic lipids, little deviation was evident while the charge was maintained. However, it was discovered that BMP had a subtle, negative impact on lipid packing in comparison to that of POPS. Furthermore, an inverse relationship between lipid packing and the fusogenecity of the SARS-CoV-2 FD was witnessed. In conclusion, the SARS-CoV-2 FD preferentially initiates fusion at a membrane resembling that of the late endosomal compartment, predominately due to the presence of BMP and its impact on lipid packing.

A Salt Bridge and Disulfide Bond within the Lassa Virus Fusion Domain Are Required for the Initiation of Membrane Fusion.

Infection with Lassa virus (LASV), an Old-World arenavirus that is endemic to West Africa, causes Lassa fever, a lethal hemorrhagic fever. Delivery of LASV's genetic material into the host cell is an integral component of its lifecycle. This is accomplished via membrane fusion, a process initiated by a hydrophobic sequence known as the fusion domain (FD). The LASV FD (G260-N295) consists of two structurally distinct regions: an N-terminal fusion peptide (FP: G260-T274) and an internal fusion loop (FL: C279-N295) that is connected by a short linker region (P275-Y278). However, the molecular mechanisms behind how the LASV FD initiates fusion remain unclear. Here, we demonstrate that the LASV FD adopts a fusogenic, helical conformation at a pH akin to that of the lysosomal compartment. Additionally, we identified a conserved disulfide bond (C279 and C292) and salt bridge (R282 and E289) within the FL that are pertinent to fusion. We found that the disulfide bond must be present so that the FD can bind to the lipid bilayer and subsequently initiate fusion. Moreover, the salt bridge is essential for the secondary structure of the FD such that it can associate with the lipid bilayer in the proper orientation for full functionality. In conclusion, our findings indicate that the LASV FD preferentially initiates fusion at a pH akin to that of the lysosome through a mechanism that requires a conserved salt bridge and, to a lesser extent, an intact disulfide bond within the internal FL.

Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein Fusion Domain Essential for Membrane Interaction.

The SARS-CoV-2 spike protein is the primary antigenic determinant of the virus and has been studied extensively, yet the process of membrane fusion remains poorly understood. The fusion domain (FD) of viral glycoproteins is well established as facilitating the initiation of membrane fusion. An improved understanding of the structural plasticity associated with these highly conserved regions aids in our knowledge of the molecular mechanisms that drive viral fusion. Within the spike protein, the FD of SARS-CoV-2 exists immediately following S2' cleavage at the N-terminus of the S2 domain. Here we have shown that following the introduction of a membrane at pH 7.4, the FD undergoes a transition from a random coil to a more structurally well-defined postfusion state. Furthermore, we have classified the domain into two distinct regions, a fusion peptide (FP, S816-G838) and a fusion loop (FL, D839-F855). The FP forms a helix-turn-helix motif upon association with a membrane, and the favorable entropy gained during this transition from a random coil is likely the driving force behind membrane insertion. Membrane depth experiments then revealed the FP is found inserted within the membrane below the lipid headgroups, while the interaction of the FL with the membrane is shallower in nature. Thus, we propose a structural model relevant to fusion at the plasma membrane in which the FP inserts itself just below the phospholipid headgroups and the FL lays upon the lipid membrane surface.