(a) X-ray structure of the crystallographic Hantaan virus Gc dimer (upper panel, PDB: 5LJY, Guardado-Calvo et al., 2016), displayed alongside the crystallographic alphavirus E1 dimer (PDB:2ALA, Roussel et al., 2006) also observed in the contact between spikes on the alphavirus particles (PDB:3J2W, Sun et al., 2013) (lower panel). The two-fold axis is drawn in black. The three domains are labeled; domain I, red; domain II, yellow; domain III, blue. The fusion loops are indicated in orange (FL). Domain III of Hantaan virus Gc appears to have adopted an orientation different than would be expected in the spike, but as it is not part of the dimer interface, it does not affect the contacts made by domain I examined here. (b) Closeup of the crystallographic Gc dimer interface, view slightly titled with respect to the 2-fold axes to show the selected residues for cysteine substitution as well as other residues at the interface, shown in sticks color-coded according to atom type (nitrogen blue, oxygen red, carbon same as the domain color). The Cα of Gly187 and H303 are as green spheres and the Cα-Cα distances across the interface for Gly187-Gly187 and His303-His303 are drawn as green dotted lines. Salt bridges and hydrogen bonds between the carboxyl groups of Glu26 and Asp28 with the guanidinium group of Arg301 are drawn as gray dotted lines. The imidazole rings of His303 interact by π-stacking, indicated with a yellow dotted line. The domain I β-strands B0, H0 and I0 from each protomer are labeled. (c) Expression yields and cell localization of ANDV Gc mutants G187C and H303C representative for two biological replicates. Western blot of fractions obtained from 293FT cells expressing Gn and wild type (WT) or mutant Gc after surface biotinylation using anti-Gc or anti-β-actin antibodies. (d) SDS Page and western blot under reducing and non-reducing conditions of VLPs obtained from supernatants of cells expressing wild type Gn together with wild type, G187C or H303C Gc, representative for four biological replicates. The absence or presence of the reducing agent β-mercaptoethanol is indicated by Red: - and +, respectively.