Mechanistic Studies of Lassa Fever Virus Glycoprotein-Mediated Membrane Fusion

Abstract

Lassa Fever Virus (LASV) GPC glycoprotein binds to α-dystroglycan on the cell surface, allowing virus uptake and transport to acidic late endosomes. GPC then engages the LAMP1 receptor and mediates fusion of viral and endosomal membranes. To overcome difficulties associated with mechanistic studies of virus-endosome fusion, we investigated LASV GPC-function using a cell-cell fusion model. We found that cells expressing LASV GPC fuse to mammalian cells upon exposure to mildly acidic pH 5.5-6.2 and that this fusion is enhanced by ectopic expression of LAMP1. Consistent with the role of LAMP1 as the LASV receptor, avian cells expressing a divergent LAMP1 orthologue did not support GPC-mediated fusion at mildly acidic pH, but ectopic expression of human LAMP1 or exposure to pH below 5.0 rescued fusion. These findings demonstrate that human LAMP1 enhances fusion efficiency and shifts the fusion optimum to less acidic pH, but it is dispensable for GPC function. Consistent with this notion, we show that LASV GPC is irreversibly inactivated when exposed to pH below 5.0 in the absence of target cells. Further mechanistic experiments show that, similar to other viral glycoproteins, LASV GPC-mediated fusion proceeds through a hemifusion intermediate, which can be arrested by exposure to low pH in the cold, and that this intermediate can be converted to full fusion by chlorpromazine treatment. Incorporation of lyso-phosphatidylcholine into the plasma membrane inhibits GPC-induced hemifusion, consistent with fusion proceeding through a highly curved, negative curvature lipid intermediate (stalk). Real-time measurements of transfer of fluorescent cytoplasmic markers shows that LASV GP-mediated fusion pores did not enlarge as readily as pores formed by other viral glycoproteins, likely due to a less optimal composition of the plasma membrane compared to the limiting membrane of late endosomes where LASV fusion occurs.