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

Abstract

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

Keywords: central nervous system infection; host-pathogen interaction; viral evolution; viral fusion.

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 IFNAR^KO murine brains were infected with 5,000 PFU/slice WT virus bearing EGFP (green fluorescence). (D and E) OBC from IFNAR^KO 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 IFNAR^KO 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 postinfection (dpi), and the level of MeV N gene expression was quantified by RT-qPCR. Results are expressed as means ± standard deviations of cultures from 5 different mice (**, P < 0.01; ***, P < 0.001 [Mann-Whitney-U test]). (H) Green fluorescence related to infection was observed 4 dpi by epifluorescence microscopy in OBC treated at the indicated concentrations (scale bar = 500 μm). (I) Ex vivo virus bearing the L454W F infection in fully immunocompetent OBC. OBC from C57BL/6 murine brains were infected with L454W F-bearing virus (using both viral preparations, one with the additional E455G in F and the one with G506E in F) at 1,000 PFU/slice for 7 days. Pictures were taken at 4 days after infection as indicated. (L) L454W-bearing virus growth in WT and IFNAR^KO OBC. OBC from WT or IFNAR^KO murine brains were infected with 1,000 PFU/slice MeV IC323-EGFP-F L454W for 7 days. Total RNA was harvested from OBC at 4 days postinfection, and the level of MeV N gene expression was quantified by RT-qPCR. Results are expressed as means ± standard deviations in cultures from at least 5 different mice (*, P < 0.05; ***, P < 0.001 [Mann-Whitney-U test]). Pictures where reconstituted using the Stitching plugin with ImageJ software.

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 in panel D. Raw counts were normalized across all samples, and differential expression analysis was performed. The 50 genes with the lowest adjusted P value between MeV IC323-EGFP-F L454W and uninfected are depicted in the heatmap, colored by log₂ fold change of each sample relative to the mean normalized counts for each gene.

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 indicated mutations and incubated at 37°C for 24 h and then raised to 55°C. The time (minutes) at 55°C that decreases the fraction of prefusion epitope to 50% (TS₅₀) and to 10% (TS₁₀) is indicated compared to the WT F at time zero (100%). Data are averages from three experiments +/− SE. (D) OBC from IFNAR^KO murine brains were coinfected with 5,000 PFU/slice of WT virus bearing tdTomato (red fluorescence) and MeV-IC323-EGFP-F L454W/E455G (green fluorescence) and monitored over 96 h. Photos were taken at 96 h. Pictures where reconstituted using the Stitching plugin with ImageJ software. Scale bar = 500 μm. (E) Two separate sets of 90-day-old human brain organoids (derived from two hiPSCs, FA10 and FA11) were infected with recombinant MeV viruses bearing the L4545W/E455G F. Three separate wells containing 2 to 4 organoids were infected (5 000 PFU/well). The brain organoids were monitored over time, and the fluorescence shown here reflects infection after 10 days. Bar = 1,000 μm. (F) 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. (G) Viral titer of the viral inoculum used for infection was assessed on Vero CD150 cells (PFU/ml). Photos show the extent of infection after 2 days (PFU/well are indicated). Pictures were reconstituted using the Stitching plugin with ImageJ software.