Lassa virus (LASV) enters host cells through membrane fusion, a critical step mediated by the viral glycoprotein complex (GPC). Within this complex, glycoprotein 2 (GP2) functions as the primary fusion machinery. The N-terminal fusion domain engages the host membrane (HM) under acidic conditions, while the transmembrane domain (TM) anchors GP2 in the viral membrane (VM). Because LASV acquires its envelope during budding at the plasma membrane, the viral lipid composition reflects that of the host cell, which contains up to 30% cholesterol. Cholesterol regulates membrane fluidity, packing, and protein conformational dynamics, suggesting that it may directly shape GP2-lipid interactions at the TM-VM interface and thereby modulate fusion efficiency. To test this hypothesis, we performed fusion assays using viral and host mimetic membranes of defined compositions of 30:20:10:10:30 (PC:SM:PS:Chol) for VM; 50:20:20:10 (PC:PE:BMP:PS) for HM. The results showed that VM lipid composition had a stronger influence on fusion efficiency compared to HM complexity. We next examined the role of cholesterol directly and observed that fusion increased in cholesterol-rich VM vesicles, with concentrations varying from 0% to 30%. Higher cholesterol content enhanced fusion, emphasizing its importance in LASV fusion. To probe the potential protein-lipid interaction of this effect, we incorporated 19 F-labeled Phe into the TM of GP2 and monitored spectral changes during cholesterol titration into DMPC/DHPC bicelles by NMR. Distinct chemical shift perturbations revealed direct interactions between cholesterol and the TM, indicating that cholesterol modifies the local environment and stabilizes fusion-ready states. Together, these findings show that cholesterol is not only a structural component of the viral membrane but also an active regulator of LASV fusion. By directly engaging the GP2, cholesterol reshapes the local lipid environment and promotes conformations that favor membrane fusion.
Nicholas P. Schifano (Sun,) studied this question.