Collective fusion activity determines neurotropism of an en bloc transmitted enveloped virus

Abstract

Measles virus (MeV), which is usually non-neurotropic, sometimes persists in the brain and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection, serving as a model for persistent viral infections. The persisting MeVs have hyperfusogenic mutant fusion (F) proteins that likely enable cell-cell fusion at synapses and "en bloc transmission" between neurons. We here show that during persistence, F protein fusogenicity is generally enhanced by cumulative mutations, yet mutations paradoxically reducing the fusogenicity may be selected alongside the wild-type (non-neurotropic) MeV genome. A mutant F protein having SSPE-derived substitutions exhibits lower fusogenicity than the hyperfusogenic F protein containing some of those substitutions, but by the wild-type F protein coexpression, the fusogenicity of the former F protein is enhanced, while that of the latter is nearly abolished. These findings advance the understanding of the long-term process of MeV neuropathogenicity and provide critical insight into the genotype-phenotype relationships of en bloc transmitted viruses.

Fig. 1.. Influence on the F fusogenicity of cumulative mutations and the WT F protein coexpression.

(A) The MeV isolates from patients with SSPE who have the T461I substitution and other hyperfusogenic mutations in the F protein. Their strain names, accession numbers (GenBank) and references, other hyperfusogenic mutations, and references for the mutations are shown. (B and C) The WT H protein, MeV F [WT F, F(T461I), F(N462S/N465S), F(T461I/N462S/N465S), F(G264E), F(G264E/T461I), F(F375S/N465K), or F(F375S/T461I/N465K)], CADM1, and EGFP were expressed in 293FT cells without (B) or with (C) the WT F protein. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bars, 500 μm. For type A and type B, refer to the main text and fig. S8. (D and E) The WT H protein, MeV F [F(T461I), F(N462S/N465S), F(T461I/N462S/N465S), F(G264E), F(G264E/T461I), F(F375S/N465K), or F(F375S/T461I/N465K)], and CADM1 were expressed in mixed 293FT/DSP1 and 293FT/DSP2 cells without or with the WT F protein. The ratio of each mutant F protein to the WT F protein was 1:1 (D) or 4:1, 1:1, or 1:4 (E). The Renilla luciferase activity in the transfected cells was analyzed 24 hours after transfection. RLU, relative light units. Each data point represents one biological replicate (N = 3). Error bars indicate SDs. Significance of the difference in the luciferase activity (as compared with that obtained by the expression of the corresponding mutant F protein only) was analyzed by two-way ANOVA: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. The fusogenicities of MeV F proteins at a low temperature.

(A and B) These figures depict the possible relationship between the stability of the F protein prefusion form and its fusion activity. The cases of ordinary hyperfusogenic F proteins (A) and unstable nonfusogenic proteins (B) are shown, respectively. (C) The WT H protein, MeV F [WT F, F(T461I), or F(F375S/T461I/N465K)], CADM1, and EGFP were expressed in 293FT cells at 37° or 32°C. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm. (D) The WT H protein, MeV F [WT F, F(T461I), or F(F375S/T461I/N465K)], and CADM1 were expressed in mixed 293FT/DSP1 and 293FT/DSP2 cells at 32°C. The Renilla luciferase activity in the transfected cells was analyzed 24 hours after transfection. Each data point represents one biological replicate (N = 3). Error bars indicate SDs.

Fig. 3.. Infection of mouse primary neurons with mixed genome MeVs.

(A) Schematic diagram of the experimental procedures for the isolation of mouse hippocampus primary neurons and infection with mixed genome viruses. Created with BioRender.com. E17, embryonic day 17. (B) Mouse primary neurons were infected with the MV323-mCherry (WT) or one of mixed genome viruses [MV323-mCherry + MV323-Venus-F(T461I) and MV323-mCherry + MV323-Venus-F(F375S/T461I/N465K)] at an MOI of 0.0001, and the cells were observed 4 days post infection (dpi) under a fluorescence microscope. Scale bar, 200 μm.

Fig. 4.. The F fusogenicity of the patient B strain.

(A) The WT H protein, MeV F [WT F, F(T461I), F(G264E/T461I), and Patient-B-F], CADM1, and EGFP were expressed in 293FT cells. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm. (B) The F mutations that the patient B strain has and the fusogenicities of F proteins having certain combinations of the mutations are shown. N-MSI indicates three amino acids (methionine, serine, and isoleucine) added at the N terminus. ΔC19 indicates a 19–amino acid deletion at the C terminus. (C) The WT H protein, MeV F [WT F, F(G264E/T461I), F(I62T/G264E/T461I), or F(G264E/T461I/I446T)], CADM1, and EGFP were expressed without or with the WT F protein in 293FT cells. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm. (D) The WT H protein, MeV F [WT F, F(T461I), F(I62T), or F(I446T)], CADM1, and EGFP were expressed in 293FT cells. The cells were observed 72 hours after transfection under a fluorescence microscope. Scale bar, 500 μm.

Fig. 5.. The F fusogenicity of the OSA-3/Bs/B strain.

(A) The WT H protein, MeV F [WT F, F(T461I), F(F375S/T461I/N465K), and OSA-3/Bs/B-F], CADM1, and EGFP were expressed without or with the WT F protein in 293FT cells at 37° or 32°C. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm. (B) The WT H protein, MeV F [F(T461I), F(F375S/T461I/N465K), and OSA-3/Bs/B-F], and CADM1 were expressed in mixed 293FT/DSP1 and 293FT/DSP2 cells without or with the WT F protein at 37° or 32°C. The Renilla luciferase activity in the transfected cells was analyzed 24 hours after transfection. Each data point represents one biological replicate (N = 3). Error bars indicate SDs. Significance of the difference in the luciferase activity (as compared with that at 37°C) was analyzed by two-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) The F mutations that the OSA-3/Bs/B strain has and the fusogenicities of F proteins having certain combinations of the mutations are shown. ΔC5 indicates a 5–amino acid deletion at the C terminus. (D) The WT H protein, MeV F [WT F, F(F375S/T461I/N465K), F(I62V/F375S/T461I/N465K), F(R70G/F375S/T461I/N465K), or F(F375S/T461I/N465K/I446N)], CADM1, and EGFP were expressed without or with the WT F protein in 293FT cells at 37° or 32°C. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm. (E) The WT H protein, MeV F [WT F, F(T461I), F(I62V), F(R70G), or F(I446N)], CADM1, and EGFP were expressed in 293FT cells. The cells were observed 24 hours after transfection under a fluorescence microscope. Scale bar, 500 μm.