The SARS-CoV-2 NSP4 T492I mutation promotes double-membrane vesicle formation to facilitate transmission

Figures

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  Fig. 1. The SARS-CoV-2 NSP4 T492I mutation promotes DMV formation. A Schematic diagram of mutation sites of NSP3, NSP4 and NSP6 in Omicron BA.1 variant. B Conventional TEM images of DMV in HeLa cells induced by NSP3/4/6 with different mutations. C Statistical analysis of DMV numbers per cell. Mean ​± ​SEM, N ​= ​3, n ​= ​16 (in all figure legends, N indicates the number of independent experiments and n indicates the number of total measurements or observations). D Statistical analysis of DMV diameters in different mutation samples. Mean ​± ​SEM, N ​= ​3, n ​= ​8. E Immunoblotting of NSPs protein expression level in different mutation. F Conventional TEM images of DMV in HEK293T cells induced by NSP3/4. G Statistical analysis of DMV numbers per cell. N ​= ​3, n ​= ​15. All data are shown as mean ​± ​SEM, using unpaired two-tailed t-test, ∗P ​< ​0.05, significant.

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  Fig. 2. Phylogeny of NSP3, NSP4, NSP6 mutations in 3986 SARS-CoV-2 genomes from 2020 to 2024. A A phylogenetic tree of SARS-CoV-2 strains showing the T492I mutation associated VOCs, marked with circle (generated by the GISAID database). B The ratio of NSP3, NSP4, NSP6 mutations from 2020 to 2024 (generated by the covSPECTRUM).

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  Fig. 3. NSP4 T492I mutation does not affect its stability and its interaction with NSP3. A To assess whether the T492I mutation affected the stability of NSP4, HEK293T cells transfected with NSP4(WT)-FLAG or NSP4(T492I)-FLAG were treated with cycloheximide (CHX) to block nascent protein synthesis for the time points 0, 4, 8, 12 ​h, and the stability of NSP4 protein was assessed by immunoblotting. B The fold change in NSP4 expression was calculated by normalization to GAPDH expression. N ​= ​3. Data are presented as mean ​± ​SEM, using repeat measures two-way ANOVA. C–F Co-IP analysis of the effect of T492I mutation on the interaction between NSP3 and NSP4. HEK293T cells co-expressing GFP-NSP3 and NSP4-FLAG (WT or T492I mutation) were lysed, and immunoprecipitates pulled down by FLAG antibody were analyzed by immunoblotting with the indicated antibodies (C). The fold change in immunoprecipitated GFP-NSP3 was calculated by normalization to immunoprecipitated NSP4-FLAG. N ​= ​3 (D). HEK293T cells co-expressing GFP-NSP3 and NSP4-FLAG (WT or T492I mutation) were lysed, and immunoprecipitates pulled down by GFP antibody were analyzed by immunoblotting with the indicated antibodies (E). The…

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  Fig. 4. NSP4 T492I mutation increases its homodimerization and the size of puncta induced by NSP3/4. A Co-IP analysis of the effect of T492I mutation on the homodimerization of NSP4. HEK293T cells co-expressing GFP-NSP4 (WT) and NSP4-FLAG (WT), or GFP-NSP4 (T492I) and NSP4-FLAG (T492I) were lysed, and immunoprecipitates pulled down by FLAG antibody were analyzed by immunoblotting with the indicated antibodies. B The fold change in immunoprecipitated GFP-NSP4 was calculated by normalization to immunoprecipitated NSP4-FLAG. N ​= ​3. All data are shown in mean ​± ​SEM, using paired two-tailed t-test, ∗P ​< ​0.05, significant. C BiFC analysis of the effect of T492I mutation on the homodimerization of NSP4. HEK293T cells transfected with NSP4-nYFP (WT) ​+ ​NSP4-cYFP (WT) or NSP4-nYFP (T492I) ​+ ​NSP4-cYFP (T492I) were analyzed by flow cytometry. Non-transfected HEK293T cells served as a negative control. D Quantitative analysis of the YFP-positive rate. N ​= ​3 (D). All data are shown in mean ​± ​SEM, using paired two-tailed t-test, ∗P ​< ​0.05, significant. E Representative confocal images of the puncta structure induced by GFP-NSP3 and NSP4-mCherry in HeLa cells. F Quantitative analysis of…

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  Fig. 5. NSP4 T492I mutation increases ER membrane curvature and the DMV density per fluorescent puncta. A TEM images of ER containing DMV regions in NSP3/4/6 samples. B Statistical graph of the total ER membrane length and distribution curve of ER membrane curvature for NSP3/4/6 WT and T492I mutation samples. C TEM images of ER in HEK293T cells only expressing WT or T492I mutant NSP4. D Statistical graph of the total ER membrane length and distribution curve of ER membrane curvature for NSP4 WT and T492I mutation samples. E–H Cryo-CLEM image of HeLa cells co-expressing GFP-NSP3 and NSP4 (WT or T492I)-mCherry. White box indicates the area for cryo-ET data collection (WT for E, F and F1; T492I for G, H and H1). Averaged tomographic slice and segmentation of the area depicted in figure E, showing a small number of DMVs induce by NSP3 and NSP4 (WT) (F and F1). Averaged tomographic slice and segmentation of the area depicted in figure G, showing a large number of DMVs induced by NSP3 and NSP4 (T492I) (H and H1). Pink represents DMV, yellow represents ER, purple represents single membrane vesicle (SMV), and green represents microtubules. I, J Averaged slices of a tomogram showing a single DMV…

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  Supplementary Figure S1. Proteomic analysis of host factors interaction with T492I mutant NSP4. A Correlation plot of NSP4 (T492I/WT) and NSP3-T2A-NSP4 (T492I/WT) interaction proteome expressed as fold changes of peptide abundance. B List of upregulated interaction proteins in both NSP4 (T492I) to NSP4 (WT) and NSP3-T2A-NSP4 (T492I) to NSP3-T2A-NSP4 (WT). C List of downregulated interaction proteins in both NSP4 (T492I) to NSP4 (WT) and NSP3-T2A-NSP4 (T492I) to NSP3-T2A-NSP4 (WT). D Confocal microscopy analysis of colocalization of candidate proteins with NSP3 and NSP4. Trace outline is used for line-scan analysis of the relative fluorescence intensity of host factor proteins with NSP3 and NSP4. Scale bar, 20 μm. The selected candidate proteins are the host factors in red in Supplementary Fig. S1B and S1C.

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  Supplementary Figure S2. Measurements of ER membrane curvature. A Curvature of the ER membrane was manually traced using ImageJ. B, C The number and total length of ER membranes were calculated in HEK293T cells co-expressing NSP3/4/6 or solely NSP4.

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  Supplementary Figure S3. Cryo-CLIEM analysis of DMVs induced by NSP3/4. A Cryo-fluorescence light microscopy (cryo-fLM) of GFP-NSP3 and NSP4-mCherry colocalization fluorescence on EM grid. A1 FIB image of the corresponding region in Supplementary Fig. S3A. B Cryo-fLM image of lamella (∼300 ​nm) via FIB direction. B1 FIB image of final lamella (∼150 ​nm). C Cryo-fLM image of lamella (∼150 ​nm) via SEM direction. C1 SEM image of the final lamella. D Cryo-EM image of the lamella. E Merge image of the fluorescence and Fig.S3D. F Tomographic slice of the fluorescent area. Scale bars: 2 ​μm (D and E), 100 ​nm (F).