Pathogenic Old and New World arenaviruses cause severe hemorrhagic fever in humans. There are currently no approved vaccines or drugs to battle arenavirus infection. Despite intensive studies, arenavirus entry pathways into host cells and the mechanism of arenavirus glycoprotein (GP) mediated virus-cell fusion are not well understood. Old World and New World arenaviruses use distinct cellular receptors and poorly characterized endocytic pathways to enter and infect cells. Based on our preliminary data and published work, we propose a novel overarching hypothesis that Old and New World arenaviruses use common post-uptake pathways to enter a subset of late endosomes that contain a specific lipid cofactor, LBPA, but lack the host restriction factor IFITM3. We found that fusion of diverse arenaviruses is promoted by LBPA and that the Lassa virus bypasses endosomes enriched in IFITM3, thereby escaping restriction. We also found that GP-mediated fusion requires thiol-disulfide interchange within this glycoprotein and obtained evidence that GP mediates arenavirus membrane permeabilization prior to fusion with acidic endosomes, which we hypothesized promotes subsequent virus uncoating. The following four Specific Aims will test the above hypotheses. (1) Delineate the entry pathways of representative Old and New World arenaviruses, using real-time single pseudovirus imaging. (2) Define the role of LBPA in arenavirus fusion and identify the GP lipid-interacting motif. (3) Examine the requirement for GP thiol-disulfide interchange in arenavirus fusion. (4) Elucidate a role of GP-mediated virus membrane permeabilization in uncoating. Successful completion of the proposed experiments will provide important fundamental insights into the mechanism of arenavirus entry/fusion and identify new therapeutic targets for intervention.