Investigation into the distinct structural features of the internal fusion loop within the LASV fusion domain.

Abstract

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 widely accepted to be 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–P275) and an internal fusion loop (FL: G276–N295). Here, we identified a conserved disulfide bond (C279 and C292) and salt bridge (R282 and E289) within the FL that are pertinent to fusion. Through a FRET-based fusion assay, we witnessed a significant reduction in fusion when the disulfide bond was broken. Additionally, we found that fusion is reduced in the presence of high salt concentrations. When single point mutations were made such that each residue was the same charge (i.e., R282E and E289R) to ablate the salt bridge, we observed reduced fusion, which was partially returned when a double mutation was introduced (R282E/E289R). This was corroborated by ITC where the binding affinity to the lipid bilayer was decreased in comparison to wildtype for each salt bridge mutant and without the disulfide bond. While it could be suggested aggregation is causing this loss in function, CD spectroscopy and gel filtration revealed that both the global secondary structure and oligomeric state, respectively, of the FD remains unchanged, even after mutagenesis or disruption of the disulfide bond. In conclusion, our findings suggest that both the salt bridge and disulfide bond within the FL are critical for the proper association of the complete FD with the membrane to elicit efficient fusion.