Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: a Schematic of a spherule on the plasma membrane (PM). The neck displays saddle curvature: positive (+) in the x-z plane and negative (-) in the x-y plane. Two types of nanostructures were designed to recapitulate the 3D saddle curvature into 2D. b Illustration and SEM image of the nanostructures: both 600 nm in height and 2 μm in length. The nanobar is 250 nm wide (pink arrow indicates positive curvature), and the nanocross is 350 nm wide (blue arrow indicates negative curvature). c U2OS cells cultured on nanobars with CellMask-stained PM (magenta) and overexpressed eGFP-nsP1 (green). The cell boundary shown in brightfield. n = 3 repeats. d Average images and 3D surface plots of PM ( n = 489 bars) and eGFP-nsP1 ( n = 223 bars) distribution around nanobars. e The end-to-center ratio of PM ( n = 995) and eGFP-nsP1 ( n = 1005) on nanobars. Mean ± SD [standard deviation]. f Cells cultured on nanocrosses with CellMask-stained PM (magenta) and overexpressed eGFP-nsP1 (green). Yellow arrows indicate negatively curved areas. n = 3 repeats. g Average images of PM and eGFP-nsP1 distribution around nanocrosses ( n = 33 crosses). h Kymograph plots of eGFP-nsP1 on two adjacent nanobars. i Time-lapse images of eGFP-nsP1 clusters on (yellow arrows) and off (pink arrows) nanobars over 270 s. j Mean square displacement (MSD) of eGFP-nsP1 clusters on ( n = 14) and off ( n = 12) the nanobars in 110 s. Mean ± SEM [standard error of the mean]. k The trajectory of nsP1 clusters on ( n = 14) and off ( n = 12) bars over 110 s. l Confocal images of fluorescence recovery after photobleaching (FRAP) test for PM (magenta) and eGFP-nsP1 (green) on nanobar over 120 s. Yellow dashed circles indicate the bleached area. n = 3 repeats. m Normalized fluorescence intensity plot of PM and eGFP-nsP1 within nanobar area from FRAP measurement (2 s interval). Mean ± SD. Statistical analysis: two-tailed unpaired t test. Scale bars: 2 μm. Source data are provided as a Source Data file.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Clinical Proteomics, Membrane, Cell Culture, Staining, Standard Deviation, Fluorescence, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: a Illustration of SLB wrapping around nanobars with increasing widths. Curvature-sensing proteins preferentially bind to highly curved ends of smaller nanobars. b Confocal images showing uniform SLB (magenta) on 300 nm-wide nanobars, with 5.4 μM bacterial expressed nsP1 (eGFP-nsP1-(b); green) bound to the bilayer. n = 3 repeats. c Average images and 3D surface plots of the SLB and eGFP-nsP1-(b) on 300 nm-wide nanobars ( n = 58 bars). d The end-to-center ratio of the SLB and eGFP-nsP1-(b) on 300 nm-wide nanobars ( n = 164). Mean ± SD. e SEM image of nanobar arrays with widths from 100 nm to 600 nm (two columns, one size). The lower panel shows bars with 200, 400, and 600 nm widths. n = 3 repeats. f Average images and 3D surface plots of eGFP-nsP1-(b) on 100–600 nm nanobars over 80 bars. g Normalized nanobar-end density of eGFP-nsP1-(b) on 100–600 nm nanobars over 80 bars. Mean ± SEM. h Confocal FRAP images of SLB (magenta) and eGFP-nsP1-(b) (green) on 200 nm nanobars over 90 s. Yellow dashed circles indicate the bleached area. n = 3 repeats. i Normalized fluorescence intensity plot of SLB and nsP1 within nanobar area from FRAP measurement (2 s interval). Mean ± SD. j Confocal images of SLB (magenta) and 1 μM Alexa Fluor 488-labeled nsP1 (nsP1-(488)-(b); green) on gradient nanobars with 0% POPS, 30% POPS, or 50% POPS lipid compositions. n = 3 repeats. k Relative nsP1 protein density across different lipid compositions over 80 bars. Mean ± SD. l Normalized nanobar-end density of nsP1-(488)-(b) on 100–600 nm nanobars coated with 30% and 50% POPS SLBs over 80 bars. Mean ± SEM. Statistical analysis: two-tailed unpaired t test. Scale bars: 2 μm. Arb. units = arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Fluorescence, Labeling, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: 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.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Membrane, Binding Assay, Expressing, Fluorescence, Mutagenesis, Construct, Luciferase, Cotransfection, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: a Snapshots of all-atom MD simulation showing the interaction of a single nsP1 protein with a planar membrane at initial (left) and equilibrated (right) states. Protein atoms are colored by residue type (acidic: red; basic: blue; hydrophobic: white; and polar: green). Lipid atoms are shown as carbon (C; cyan), oxygen (O; red), phosphorus (P; green), nitrogen (N; blue), and hydrogen (H; white). b Simulation snapshots showing the temporal evolution of nsP1 dodecamer binding to a planar membrane. The top panels show side cutaway views of nsP1 dodecamer-membrane interaction at different time points; the bottom panels highlight the number of nsP1 ring subunits inserted into the membrane. Lipid head beads (PO4, NC3, and CNO) were colored in tan, glycerol linkages (GL1 and GL2) in pink, lipid tail beads in cyan, and only PO4 beads were shown in the top views. Protein beads in the top panels were colored as in ( a ). The inserted protein beads shown in the bottom panels were rendered in such a way that each color refers to a particular subunit of the dodecamer structure. A zoomed-in box shows the insertion of nsP1 MA loop 2 into the membrane. c Top view of the planar membrane-bound nsP1 dodecamer (left) and the corresponding color map for mean membrane curvature (right). d , e Simulation snapshots showing the temporal evolution of the interaction of the nsP1 dodecamer with a flat supported membrane ( d ) and a supported membrane with a radius of curvature of 100 nm ( e ). f Comparison in the area per lipid between planar ( n = 100) and curved ( n = 191452) membranes. Mean ± SD. g Comparison in the total interaction energy of the nsP1 dodecamer with planar ( n = 981) and curved ( n = 501) membranes. Mean ± SD. h Comparison in the maximum insertion depth of nsP1 subunits (averaged over all inserted sub-units) into planar and curved membranes ( n = 100). Mean ± SD. Statistical analysis: two-tailed unpaired t test. Source data are provided as a Source Data file.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Membrane, Residue, Binding Assay, Comparison, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: a SEM image of gradient nanorings with decreasing inner ring diameters, ranging from 1500 nm to 700 nm. n = 3 repeats. b Schematic of a nanoring illustrating three distinct positive curvatures on PM: (i) the sidewall of the outer ring; (ii) the top rim of the outer ring; and (iii) the top rim of the inner ring. c Confocal images of CHIKV-infected cells cultured on a flat surface or a nanoring array. Replication sites were indicated by dsRNA staining (magenta). n = 3 repeats. d Confocal images of nsP3-mCherry tagged CHIKV-infected cells cultured on the nanoring array. The mCherry fluorescent tag was inserted at the hypervariable region of nsP3 (as shown in cyan), which is known to tolerate tags and does not impede virus replication and infection. The cells were stained for nsP1 (yellow) and dsRNA (magenta). n = 3 repeats. e Comparison of the dsRNA density in the inner ring of the nanoring area and on the nearby flat surface ( n = 60 rings). f Average images of the brightfield, PM intensity, and dsRNA intensity on different-sized nanorings (over 30 rings for each size). g The dsRNA intensity per unit inner rim area on different-sized nanorings ( n = 27, 31, 44, 60, 39). Mean ± SD. h The dsRNA intensity per unit inner rim perimeter on different-sized nanorings ( n = 27, 31, 44, 60, 39). 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.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Infection, Cell Culture, Staining, Virus, Comparison, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-025-59402-0

Figure Lengend Snippet: a Schematic of expansion microscopy (ExM) for higher-resolution imaging. b Post-ExM images of a CHIKV-infected cell cultured on fluorescent gelatin-coated nanorings (cyan). The cell was stained for nsP1 (yellow) and dsRNA (magenta). A representative replication site is indicated by a white arrow. n = 3 repeats. c Zoomed-in x-y image (left) and resliced z -axis image along the white dashed line (right) show one nanoring with uniform gelatin coating (cyan). The CHIKV-infected cell exhibits dsRNA signals concentrated in the center of the inner ring (magenta), while nsP1 is located at the periphery of the dsRNA puncta near the nanoring rim (yellow). n = 3 repeats. d Schematic of a nanoring showing that the incomplete PM adhesion to the nanoring sidewall can create space at the bottom of the outer ring, allowing CHIKV replication in this region. e Post-ExM images of a CHIKV-infected cell cultured on fluorescent gelatin-coated nanorings (cyan). Incomplete PM adhesion, indicated by dark voids in the gelatin coating around the nanoring (example highlighted by a yellow ring), allows for viral replication within these spaces. The cell was stained for nsP1 (yellow) and dsRNA (magenta). n = 3 repeats. f Zoomed-in x-y image (left) and resliced z -axis image along the white dashed line (right) show replication occurring at the bottom of the outer ring. n = 3 repeats. g Illustration showing two distinct curvatures generated during spherule formation: 1) a saddle curvature at the neck of the spherule with increasing positive curvature along the x-z plane (pink), sensed by nsP1 via hydrophobic insertion of the MA loops, and a fixed negative curvature along the x-y plane (red) fixed by the size of the nsP1 dodecamer ring; and 2) a progressively increasing positive curvature along the crown of the spherule (blue). Scale bars: 2 μm. Source data are provided as a Source Data file.

Article Snippet: The lipid used to study the POPS lipid charge effect on nsP1 curvature sensing consisted of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti, #850457), 0.5 mol% Texas-Red-PE with increasing mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), Avanti, #840034) from 0 mol%, 30 mol% to 50 mol%.

Techniques: Microscopy, Imaging, Infection, Cell Culture, Staining, Generated