Fig. 3: Mechanism of CHIKV nsP1 curvature sensing. | Nature Communications

Fig. 3: Mechanism of CHIKV nsP1 curvature sensing.

From: Saddle curvature association of nsP1 facilitates the replication complex assembly of Chikungunya virus in cells

Fig. 3

a Model of nsP1 dodecamer identifying putative membrane binding domains: membrane-association (MA) loops (a.a. 220–233 and 407–427) with a palmitoylation site (a.a. 417–419), one amphipathic helix (AH; a.a. 244–263), and a disordered tail (a.a. 474–535). b Mutations of nsP1 used in this study. c Confocal images of cells on nanobars expressing eGFP-tagged nsP1-WT, nsP1-W258A, nsP1-CCC_3A, nsP1-WCCC_4A. n = 3 repeats. Average images and 3D surface plots are shown below (n = 291, 763, 533, 424 bars). d The end-to-center ratio of PM, nsP1-WT, nsP1-W258A, nsP1-CCC_3A, nsP1-WCCC_4A on nanobars (n = 1027, 1250, 1255, 756). Mean ± SD. e Confocal FRAP images of nsP1-WT, nsP1-W258A, nsP1-CCC_3A in 120 s. n = 3 repeats. f Normalized fluorescence intensity plot of nsP1-WT, nsP1-W258A, nsP1-CCC_3A within the nanobar area from FRAP measurement (2 s interval). Mean ± SD. g Confocal images of SLB (magenta) and 1 μM eGFP-tagged mammalian-expressed nsP1-WT (eGFP-nsP1-WT-(m)) or MA-loop-mutant (eGFP-nsP1-MA-loop-mut-(m)) (green) on gradient nanobars. n = 3 repeats. h Relative protein density of nsP1-WT-(m) (n = 36) and nsP1-MA-loop-mut-(m) (n = 72). Mean ± SD. i Average images and 3D surface plots of nsP1-WT-(m) (n = 75 bars) and nsP1-MA-loop-mut-(m) (n = 93 bars) on 300 nm-wide nanobars. j The end-to-center ratio of nsP1-WT-(m) (n = 145) and nsP1-MA loop mut-(m) (n = 198) on 300 nm-wide nanobars. Mean ± SD. k Normalized nanobar-end density of nsP1-WT-(m) and nsP1-MA loop mut-(m) over 70 bars. Mean ± SEM. l Schematic of P1234, its MA-loop-mutant (P1234-MA-mut) and RNA reporter (polI-Fluc-Gluc) constructs. m RNA reporter expression level directed by genomic (firefly luciferase) and subgenomic (Gaussia luciferase) promoters after co-transfection with P1234 or P1234-MA-mut. Mean ± SD. n Confocal images of cells expressing eGFP-tagged P1E234 on nanobars. n = 3 repeats. Average images and 3D surface plots are shown below (n = 1103 bars). o The end-to-center ratio of PM, nsP1-WT, P1E234 on nanobars (n = 666, 626, 1491). Mean ± SD. Statistical analysis: two-tailed unpaired t test. Scale bars: 2 μm. Arb. units = arbitrary units. Source data are provided as a Source Data file.

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