Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Intervention strategies that target the arenavirus envelope glycoprotein complex (GPC) hold promise for combating these lethal infections. In contrast to other class I fusion proteins, GPC contains three subunits: GP1, GP2 and a unique stable signal peptide (SSP) that acts in conjunction with GP2 to sense acidic pH in the endosome and trigger the structural transitions leading to virus-cell membrane fusion. Small-molecule inhibitors target this interaction to block arenavirus entry. A detailed understanding of GPC membrane fusion and its inhibition has been hampered by the lack of structural knowledge of the membrane-anchored GPC trimer. Crystallographic structures of soluble ectodomain fragments of GPC lack SSP and the transmembrane domain of GP2. We have determined a low-resolution structure of the Lassa virus (LASV) GPC on virion particles using cryo- electron microscopy (cryoEM) tomography, but were unable to resolve these missing elements. Remarkable technical advances in single-particle cryoEM now allow the capture of significantly more information than cryotomography and should enable us to generate high-resolution structures. In preliminary studies, we have produced LASV GPC in insect cells and used cryoEM to visualize the trimeric complex, in solution and embedded in lipid nanodiscs (NDs). This proposal entails a collaboration between two experienced research groups with complementary strengths in the biochemical analysis of arenavirus membrane fusion and single- particle cryoEM structure determination, with the goal of obtaining near-atomic resolution structural knowledge of the native LASV GPC complex in a membrane environment. We will pursue the following specific aims: (1) Determine the cryoEM structure of the precursor form of LASV GPC stabilized in lipid nanodiscs. NDs offer an ordered lipid environment to stabilize the structure of membrane proteins beyond the limits of nonionic detergents. We have incorporated cleavage-defective (cd) LASV GPC into NDs and will determine the cryoEM structure of the trimeric precursor. (2) Determine the structure of the mature prefusion form of LASV GPC. Purified wild-type LASV GPC contains a mixture of cleaved and uncleaved molecules. We will take advantage of our recent finding that only the mature complex is susceptible to photoaffinity labeling by fusion inhibitors to selectively enrich for cleaved GPC NDs. (3) Determine the low-pH structures of LASV GPC bound to its LAMP1 co-receptor and in its postfusion state. LASV GP1 undergoes a conformational switch at moderate endosomal pH to bind LAMP1. We have produced soluble LAMP1 and will determine the cryoEM structure of LAMP1-bound GPC NDs. Further acidification will trigger the fusogenic reorganization of the complex. Collectively, the structures will provide an unprecedented view of GPC and the conformational transitions that promote arenavirus membrane fusion and its inhibition. This knowledge will guide the development of novel vaccines and therapeutics against arenaviral hemorrhagic fevers.
Hemorrhagic fever arenaviruses endanger public health and biodefense. Intervention strategies that target the viral envelope glycoprotein (GPC) hold promise for treating these lethal infections. A detailed understanding of GPC membrane fusion and its inhibition by small-molecule fusion inhibitors has been hampered by the lack of structural knowledge of the membrane-anchored GPC trimer. We will take advantage of remarkable technical advances in single-particle cryo-electron microscopy to generate near-atomic resolution structural information on the Lassa virus GPC in a membrane environment. This structural insight will guide the development of novel vaccines and therapeutics.
