biostudies-literatureMann CJHoward Hughes Medical InstituteNIAID NIH HHSFoundation for the National Institutes of HealthFoundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)National Natural Science Foundation of ChinaNIGMS NIH HHSNIH HHSNational Natural Science Foundation of China (National Science Foundation of China)2207-2220https://www.ebi.ac.uk/biostudies/studies/S-EPMC12408356biostudies-literatureUnknown10(9)Of the multiple arenaviruses that cause haemorrhagic fevers in the Americas, all lack reliable therapeutic options, and only one has a vaccine. The arenavirus glycoprotein complex (GPC) binds cellular receptors and mediates pH-dependent fusion of viral and host cell membranes during entry. GPC comprises GP1, GP2 and stable signal peptide (SSP) subunits. SSP remains associated with the mature glycoprotein complex and regulates pH-dependent membrane fusion through an unclear mechanism. We report cryo-EM structures of Junin virus and Machupo virus GPC stabilized in the prefusion conformation using an amino acid substitution in the transmembrane region of SSP at 3.0 Å and 2.9 Å resolution, respectively. Mutational analyses, cell-cell fusion assays and molecular dynamics simulations reveal how Nature microbiologyMolecular organization of the New World arenavirus spike glycoprotein complex.PMC12408356T32AI007245T32 GM144273T32 AI007245DP5 OD023084T32GM144273DP5OD02308482404516Olal DFan XKrammer FMann CJClark LEYang PSmith KNBian YAbraham JfalseMolecular organization of the New World arenavirus spike glycoprotein complex.Of the multiple arenaviruses that cause haemorrhagic fevers in the Americas, all lack reliable therapeutic options, and only one has a vaccine. The arenavirus glycoprotein complex (GPC) binds cellular receptors and mediates pH-dependent fusion of viral and host cell membranes during entry. GPC comprises GP1, GP2 and stable signal peptide (SSP) subunits. SSP remains associated with the mature glycoprotein complex and regulates pH-dependent membrane fusion through an unclear mechanism. We report cryo-EM structures of Junin virus and Machupo virus GPC stabilized in the prefusion conformation using an amino acid substitution in the transmembrane region of SSP at 3.0 Å and 2.9 Å resolution, respectively. Mutational analyses, cell-cell fusion assays and molecular dynamics simulations reveal how 2025-01-01T00:00:00Z2025 Sep2026-05-29T21:50:56.063Z2026-04-08T06:08:55.121ZS-EPMC124083564078144710.1038/s41564-025-02085-6