In line with their importance, bo lcoroteins have been widely studied by us and others, and the x-ray structures of the isolated Gn BIOLOGY AND HEALTH SCIENCES “head” (the N-terminal two-thirds of the ectodomain, Gn) [3] and SYNCHROTRON SOLEIL HIGHLIGHTS 2020 H ectodomain of Gc reported [4]. The structures of the latter showed to feature the characteristic β sheet-cdoain observed in the class II fusion proteins of flaviviruses, alphaviruses, phleboviruses, and rubella PROXIMA-1 & PROXIMA-2A virus, suggesting an evolutionary link between otherwise different viral BEAMLINES families. The organization of the outer lattice has been studied at low resolution by electron microscopy [5]. Despite these advances, the actual organization of the spike and the molecular interactions between Gn Associated publication and Gc have remained elusive. The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control RESULTS Mechanism. Thanks to data collected in Proxima 1 and 2 beamlines, we have obtained A.Serris, R. Stass, E. A. Bignon, the X-ray structures of the metastable pre-fusion Gn/Gc complex ofH N. A., Muena, J. C. Manuguerra, Andes (ANDV) and Maporal (MAPV, closely related to ANDV) viruses to R. K. Jangra, S. Li, K. Chandran, 3.2 and 2.2 Å resolution, respectively. We have also obtained the X-ray N. D. Tischler, J. T. Huiskonen, structure of the Gn “base” (the missing C-terminal third of Gn, Gn), F. A. Rey, P. Guardado-Calvo. B at 1.9 Å resolution and showed that if forms a tight tetramer. We have Cell, 183(2): 442 (2020). fitted these structures into a cryo-electron tomography map of Tula virus (TULV, a non-pathogenic Old world hantavirus) refined to 11.4 References Å resolution. The fitting confirmed that the heterodimeric and homo- [1] C. B. Jonsson etal. , Clin Microbiol tetrameric conformations captured in the crystals correspond to their Rev. 23, 412 (2010). active form in the spikes of infectious particles, and provided a complete [2] P. Guardado-Calvo & F.A. Rey, Adv model for the surface glycoprotein lattice (Fig. 1). This model provided a Virus Res, 98, 83 (2017). framework to understand an accumulating body of data on hantavirus [3] I . Rissanen et al., J Virol, 91(21) biology. For example, it revealed that the conserved N-linked glycans (2017). are buried in the spike, explaining why they remain high-mannose in the [4] P. Guardado-Calvo et al., PLoS released particles, and supporting the hypothesis that the tetrameric Pathog, 12(10): p. e1005813 (2016). spikes form early in the ER and are transported as such to the Golgi [5] S. Li et al., Cell Rep, 15, 959 (2016. apparatus. Moreover, the model paves the way for the development of novel immunogens by providing ways to design stabilized versions Corresponding authors of the heterodimer and the spikes. We provided a proof of principle Felix A. Rey & Pablo Guardado-Calvo that such stabilization is possible by designing disulfide bonds linking Institut Pasteur, Structural Virology both subunits. Also, using data collected in the Proxima beamlines, we Unit, and CNRS UMR 3569 have also obtained the structure of the post-fusion form of Gc from Paris, France ANDV and MAPV at 2.4 and 2.7 Å resolution, respectively. Unlike other rey@pasteur.fr class-II fusion proteins studied so far, the tip of Gc domain II, where a guardado@pasteur.fr critical membrane-interacting region is located, adopts an alternative conformation that reduces surface hydrophobicity, preventing the Caption interaction with membranes in the presence of Gn. Last but not least, FIGURE 1: The structure of the hantavirus spike. the structure of the heterodimer Gn/Gc revealed a strong similarity with Reconstruction of a Tula virus particle at 11 Å resolution. The resulting surface protein lattice is shown in cyan and the fusion machinery E1/E2 of the alphavirus, suggesting a common the viral membrane in gray. The middle and right panels origin and therefore providing unanticipated evolutionary links. show top and side views of a single spike, with one of the fourprotomers colored in red (Gn), orange (Gn) andH B yellow (Gc) and the sugar residues in green. 53