Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain

FIG 1. Location of substitutions within the F protein from CNS-adapted virus. (A) Schematic of MeV F with fusion peptide (FP), N-terminal heptad repeat (HRN), C-terminal heptad repeat (HRC), transmembrane (TM), and cytoplasmic (CT) domains indicated. (B) Ribbon diagrams of the prefusion MeV F protein (MeV F; PDB 5YXW [16]). Five substitutions (M337L, L454W, E455G, T461I, and N462K) in the F protein structure are shown.

FIG 2. Infection with wild-type (WT) versus virus bearing the L454W F; the CNS-adapted virus outcompetes the WT virus in organotypic brain cultures (OBC). (A) Schematic representation of OBC preparation from the organ, slicing using a tissue chopper II device, and transfer of slices on insert for further culture and infection. (B and C) OBC from IFNARKO murine brains were infected with 5,000 PFU/slice WT virus bearing EGFP (green fluorescence). (D and E) OBC from IFNARKO murine brains were coinfected with 5,000 PFU/slice of WT virus bearing tdTomato (red fluorescence) and MeV IC323-EGFP-F L454W (green fluorescence) at 5,000 PFU/slice and monitored over 96 h. Photos were taken at 24 h (D) and 96 h (E). Scale bar = 500 μm. (F to H) MeV F-derived fusion inhibitor peptide (HRC4) inhibits the dissemination of MeV bearing L454W F in OBC. OBC from IFNARKO murine brains were infected with MeV IC323-EGFP-F L454W at 5,000 PFU/slice for 4 days. OBC were treated at the indicated concentrations or left untreated (NT control) by adding HRC4 fusion inhibitory peptide 24, 48, and 72 h after initial infection. (F) Schematic of the procedure. (G) Total RNA was harvested from organotypic slices at 4 days…

FIG 3. CNS-adapted MeV variants spread efficiently in human pluripotent stem cell (hiPSC)-derived brain organoids. (A) Two separate sets of 90-day-old human brain organoids (derived from two hiPSCs, FA10 and FA11) were infected with recombinant MeV viruses (with either EGFP or tdTomato fluorescent protein) bearing the indicated MeV fusion (F) proteins. For each virus, 3 separate wells each containing 2 to 4 organoids were infected (5,000 PFU/well). The brain organoids were monitored over time, and the fluorescence shown here reflects the infection after 10 days. Bar = 1,000 μm. (B) Viral titer of the inoculum used for infection was assessed on Vero CD150 cells (PFU/ml, log). (C) Total RNA was harvested from the human brain organoids at 10 days postinfection, and the level of MeV N gene expression was quantified by RT-qPCR. (D) RNA-Seq analysis of WT versus L454W F-bearing virus infection in brain organoids (data from three separate experiments). Seven replicates of uninfected and MeV-infected brain organoids were transcriptionally profiled (n = 6 uninfected, n = 5 WT, n = 2 L454W F-bearing virus). (E) RPM values for MeV for each sample are depicted in the same order as for the heatmap…

FIG 4. Fusion activity and thermal stability of MeV fusion (F) proteins bearing the indicated mutations. (A) Cell-to-cell fusion between HEK293T cells coexpressing the indicated MeV F proteins + MeV WT hemagglutinin (H) and HEK293T cells (no known measles receptor) was assessed by a B-gal complementation assay. The values on the y axis are expressed as relative luminescence unit (RLU) averages (with standard error, SE) of results from three independent experiments. ****, P < 0.0001 (2-way analysis of variance [ANOVA]). (B) HEK293T cells were transfected with MeV F protein bearing the indicated mutations and incubated at 37°C for 24 h and then raised to 55°C for the indicated times (x axis). The values on the y axis represent the percentages of prefusion conformation specific antibody binding to the indicated F proteins (compared to the WT F protein at time zero). The values are the average of three independent experiments. ****, P < 0.0001 (2-way ANOVA). (C) Fusion mediated by cells coexpressing MeV H and F proteins bearing the indicated F mutation in the presence of nectin 4, CD150, or no receptor compared to WT F (100%). HEK293T cells were transfected with MeV F protein bearing the…