Functional assessment of the cell entry pathway of Lymphocytic choriomeningitis virus

Abstract

Lymphocytic choriomeningitis virus (LCMV) is the prototypic member of the mammarenaviruses, the majority of which are rodent-borne, zoonotic viruses primarily associated with a particular rodent host. LCMV has been utilized as the model to understand the host immune response against viral infection and its use has been critical in multiple seminal discoveries in the field of immunobiology. Although LCMV is primarily a rodent pathogen, it is known to cause human disease, including fatal meningitis in transplant patients as well as congenital birth defects and spontaneous abortion if acquired during pregnancy. Although the receptor for LCMV was identified in 1998 as glycosylated a- Dystroglycan (aDG), subsequent investigation revealed that LCMV could enter cells in a aDG-independent manner. In these studies, we used a genetic screen to identify additional host factors involved in LCMV entry. We identified the lysosomal mucin CD164, genes involved in N-linked glycosylation, sialylation, the heparan sulfate biosynthesis machinery, as well as the previously known receptor aDG. Through cellular and biochemical approaches, we found that the LCMV glycoprotein (GP) uses a receptor switch strategy. LCMV can utilize aDG and heparan sulfate as cell surface attachment molecules. However, after endocytosis and trafficking to lysosomes, exposure to acidic pH leads to a conformational change in GP that allows for the recognition of the lysosomal protein CD164 in a manner dependent on a sialylated glycan on residue N104 within CD164, an interaction that promotes GP-mediated membrane fusion. There are currently no approved vaccines or therapeutic options to treat LCMV. Research on the epitopes targeted by neutralizing antibodies against Lassa virus, a related mammarenavirus, revealed that these antibodies only bind the pre-fusion GP structure. In these studies, we identified M28 as a potent neutralizing antibody against LCMV in vitro and were able to reconstruct a high-resolution structure of the pre-fusion, trimeric LCMV GP bound by the antibody M28 using cryo-electron microscopy (cryo-EM). This is the first trimeric pre-fusion structure of LCMV GP and the first structurally characterized neutralizing antibody against it. Together, this work expands on our understanding of LCMV entry biology and presents a foundation for both LCMV vaccine design and antibody discovery.