Fig 1.
GP1LCMV and GP1MORV do not interact with LAMP, although GP1MORV adopts the same global fold as GP1LASV.
(A) Pull-down assay by the indicated GP1-Fc fusion proteins. The presence of LAMP1 is detected by anti-LAMP1 antibody, and Fc levels are shown using anti-Fc for load control. The pull-down assay was independently repeated three times, and a representative image is shown. (B) SPR analysis of the indicated GP1-Fc analytes. Each analyte was injected at 500 nM over immobilized distal domain of LAMP1. (C) A phylogenetic tree based on the sequences of the GPCs from the indicated OW mammarenaviruses. The scale bar represents a substitution rate of 0.4 per site. (D) Graphical representation of the 16 chains, each colored differently, that make the asymmetric unit. The chains are traced as tubes with a radius proportional to B-factor. N-linked glycans are shown as spheres. (E) Cα traces of all 16 chains superimposed. N-linked glycans are shown with lines. The cysteine residues that make the three disulfide bonds (marked with a ‘D’) are shown with spheres and are labeled, and secondary structure elements are numbered. (F) Multiple sequence alignment of GP1s from the indicated OW mammarenaviruses showing the secondary structure elements as observed in the crystal structure of GP1MORV. The numbering of the amino acids is based on the sequence of GPCMORV. Fully conserved residues are highlighted with red background, and partially conserved residues are shown in red. Numbers below the sequences mark the locations and connectivity of the disulfide bridges. The symbol ‘Ψ’ marks the locations of N-linked glycans seen in the crystal structure. We used ESPript [21] (http://espript.ibcp.fr) for generating this graphical representation.
Table 1.
Data collection and refinement statistics.
Fig 2.
Structural comparison between GP1MORV and GP1LASV and the vicinity of the histidine triad.
(A) Superimposition of GP1MORV (blue) and GP1LASV (grey) (PDB: 4ZJF). The histidine triad is indicated as well as β5 & β6. Disulfides are shown with spheres for the sulfur atoms (B) The solvent accessible surfaces of GP1MORV and GP1LASV are presented on the right and left sides, respectively. The proteins are positioned as in panel ‘A’, and the histidine triads are indicated with green arrows. A red arrow marks bulky residues on GP1MORV. The surfaces are colored according to the electrostatic potential in the range of ± 5 kT/e, as calculated by APBS tools at pH 5.0. (C) A close up view of the vicinity of the histidine triad in a superimposition of GP1MORV (blue) and GP1LASV (grey). Non-conserved sites in this region are indicated. (D) Pull-down experiment of LAMP1 by the indicated GP1LASV-Fc mutants. A representative image of three independent repeats. (E). SPR analyses of GP1LASV point mutations. The various mutants in GP1LASV were injected as analytes at 450 nM over immobilized distal domain of LAMP1. WT GP1LASV was injected multiple times at the beginning, middle and end of the injection series to monitor the consistency of the immobilized LAMP1, producing indistinguishable sensograms (only a representative curve is shown). The binding curves were manually inspected and binned into three groups: showing no or very little effect on binding (light-blue background), moderately reduced binding (pink background), and strongly diminished binding (purple background).
Fig 3.
Mapping of the LAMP1 binding site on GP1LASV.
(A) Structural comparison of the loop preceding β5. A yellow dashed line marks an apparent hydrogen bond. (B) Representative images of LAMP1 pull-down experiments by the indicated GP1LASV-Fc mutants, each one selected from three independent repeats. The panel is composed of three intact membranes as designated by the horizontal lines. (C) Superimposition of the β–hairpins of GP1LASV and GP1MORV presented as Cα traces and side-chains only. (D) Ribbon representation of the β–hairpins and their close vicinity in GP1MORV and GP1LASV. (E) Surface representation of GP1LASV. Residues that were mutated and critically affected, weakly affected, or had no affect on LAMP1 binding are colored purple, pink, and blue, respectively, using the same color scheme used in Fig 2E. The histidine triad is red. (F) The crystal structure of GP1LASV (white surface) docked into the EM density of the trimeric spike complex of LASV at pH 5.0 (EMDB: 3292), shown as green mesh at 1.5 σ. The histidine triad is red and all residues identified as important for LAMP1 binding are purple. The crevices between the 3 GP1 subunits are marked with black arrows.
Fig 4.
Grafting the LAMP1 binding site onto MORV.
(A) Sequence alignment of residues 170–219 of GP1 (LASV numbering) from LASV, MORV, and the generated chimera. Red arrows indicate all residues identified as important for LAMP1 binding. (B) Pull-down assay of LAMP1 by the indicated GP1-Fc fusion proteins. (C) SPR analysis of the indicated GP1-Fc fusion proteins as analytes. A 2-fold dilution series of the analytes starting at 450 nM were injected over immobilized distal domain of LAMP1 (orange curves). Bivalent binding models were fitted to data (black curves), and affinity constants (KD) were calculated from the rate constants of the first binding event, when appropriate. The binding curve of 450 nM GP1MORV-Fc is included in the sensogram of GP1Chimera-Fc (blue curve) as a reference. (D) Western blot analysis using Flag-tagged GPCs. The upper molecular weight bands correspond to the full-length unprocessed GPCs. Lower molecular weight bands correspond to the cleaved GP2s. (E) Syncytia formation assays. Bright-field images of HEK293 cells showing formation of syncytia induced by the indicated GPCs. The boundaries of the syncytia are shown with yellow lines as automatically traced by ImageJ [22]. (F) Multiple sequence alignment of GP1s from LASV strains that represent the 4 major lineages. The LAMP1 binding motif is highlighted in red. Fully conserved residues indicated with a red background and chemically similar residues are shown in red font.