Figure 2.

Targeted mutations at two sites in L4 stabilize pre-fusion RABV-G

Wild-type (WT) and mutant RABV-G constructs were expressed on transiently transfected Expi293 cells, and reactivity with site I (RVC20), site II (1112-1), and site III (17C7) IgG was assessed by flow cytometry. Cell-surface expression levels of all constructs are shown in Table S3.

(A) Effect of histidine substitutions. All histidine residues in the RABV-G ectodomain were mutated to alanine and to leucine, with exceptions detailed in methods. Pre-fusion protein stability was measured by calculating median fluorescence intensity (MFI) of the pre-fusion-specific mAb RVC20 (Hellert et al., 2020; De Benedictis et al., 2016) after binding at pH 5.8 as a proportion of that after binding to the same construct at pH 7.4: this proportion was 0.11 for untagged WT RABV-G, and 0.12 for WT RABV-G expressed as a fusion protein. Results are expressed as fold change in this proportion compared with WT protein. Filled and open symbols denote introduction of alanine and leucine, respectively. Black and green symbols denote untagged constructs and those expressed as GFP fusion proteins, respectively. Points represent median and error bars represent range of four technical replicates across two experiments (a transfection with each of two independent DNA preparations on each of 2 days). “Poor exp” denotes constructs with cell-surface expression <33% of the level of WT RABV-G, as assessed by RVC20 binding at pH 7.4 (Table S3).

(B) Effect of potentially helix-breaking substitutions with proline. Residues in L4/L5 regions expected to form helices in post-fusion protein were substituted with proline. Colors, replication (points and error bars), and the definition of poor cell-surface expression are as for (A).

(C) H261A/L and H270P retain site II and site III antigenicity, as evidenced by 1112-1 and 17C7 binding. Untagged constructs were used. MFI is expressed as a proportion of that observed with WT RABV-G with each antibody. “None” denotes MFI on cells transfected with an irrelevant antigen. The replication strategy and meaning of points and error bars were as for (A).

(D and E) H261L and H270P mutations abolish RABV-G-mediated cell-cell fusion. Acid-triggered cell-cell fusion was monitored in a dual-reporter luminescence and fluorescence assay. (D) shows luciferase activity (upper graph) and expression level measured by ELISA (lower graph) for samples from the same experiment. Points each represent median of three or more technical replicates (as described in STAR Methods section) using a single DNA preparation. Line indicates median of biological replicates using independent DNA preparations. ND indicates not detectable. (E) shows GFP activity, imaged by fluorescence microscopy. Scale bars, 100 μm.