RESUMEN
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.
RESUMEN
Lassa virus (LASV) is the most prevalent arenavirus afflicting humans and has high potential to become a threat to global public health. The transmembrane domain (TM) of the LASV glycoprotein complex forms critical interactions with the LASV stable signal peptide that are important for the maturation and fusion activity of the virus. A further study of the structure-based molecular mechanisms is required to understand the role of the TM in the lifecycle of LASV in greater detail. However, it is challenging to obtain the TM in high quantity and purity due to its hydrophobic nature which results in solubility issues that makes it prone to aggregation in typical buffer systems. Here, we designed a purification and detergent screen protocol for the highly insoluble TM to enhance the yield and purity for structural studies. Based on the detergents tested, the TM had the highest incorporation in LMPG. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were utilized to confirm the best detergent system for structural studies. Through CD spectroscopy, we were able to characterize the secondary structure of the TM as largely alpha-helical, while NMR spectroscopy showed a well-structured and stable TM in LMPG. From these results, LMPG was determined to be the optimal detergent for further structural studies.
RESUMEN
Lassa virus (LASV), an arenavirus endemic to West Africa, causes Lassa fever-a lethal hemorrhagic fever. Entry of LASV into the host cell is mediated by the glycoprotein complex (GPC), which is the only protein located on the viral surface and comprises three subunits: glycoprotein 1 (GP1), glycoprotein 2 (GP2), and a stable signal peptide (SSP). The LASV GPC is a class one viral fusion protein, akin to those found in viruses such as human immunodeficiency virus (HIV), influenza, Ebola virus (EBOV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are enveloped and utilize membrane fusion to deliver their genetic material to the host cell. Like other class one fusion proteins, LASV-mediated membrane fusion occurs through an orchestrated sequence of conformational changes in its GPC. The receptor-binding subunit, GP1, first engages with a host cell receptor then undergoes a unique receptor switch upon delivery to the late endosome. The acidic pH and change in receptor result in the dissociation of GP1, exposing the fusion subunit, GP2, such that fusion can occur. These events ultimately lead to the formation of a fusion pore so that the LASV genetic material is released into the host cell. Interestingly, the mature GPC retains its SSP as a third subunit-a feature that is unique to arenaviruses. Additionally, the fusion domain contains two separate fusion peptides, instead of a standard singular fusion peptide. Here, we give a comprehensive review of the LASV GPC components and their unusual features.
