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. 2022 Oct 19;11:e83042. doi: 10.7554/eLife.83042

Figure 3. NsP1 binds to membranes containing monovalent anionic lipids and recruits nsP2 in a concentration-dependent manner.

(A–C) Copelletation of nsP1 with multilamellar vesicles (MLVs) with varying percentages of the anionic phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), phosphatidylinositol (PI) (A), PI(4)P (B), and PI(4,5)P2 (C) in a background of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 20% cholesterol. Representative example gels shown in Figure 3—figure supplement 2. The percentage of protein associated with membranes was quantitated from gels and plotted. Each plot represents the mean ± SD of three independent replicates. (D) Confocal imaging of nsP1- ATTO488 (green) binding to giant unilamellar vesicles (GUVs) (red) with POPC, or POPC including 5 mol% PI(4,5)P2, or 20% POPS. Scale bar, 20 µm. (E) Quantification of nsP1-bound GUVs from three experiment series. Data represent the percentage of nsP1-binding GUVs calculated from total number of GUVs observed for each experiment series plotted against the respective GUVs types. (F) Co-pelletation assay of nsP2 and nsP1 with POPS-containing MLVs. NsP2 and MLV concentrations were kept constant, while the nsP1 concentration was varied. Analysis of supernatant (S) and pellet (P) fractions by SDS-PAGE. (G) Quantification of pelleted nsP2 with nsP1. The experiment shown in (F) was repeated two times. The pellet intensity at each nsP1 concentration was normalized to the total nsP2 intensity and plotted (mean ± SD) against the nsP1 concentration. (H) Schematic of the findings from A to G, in the context of the neck complex. The non-structural proteins nsP3 and nsP4 were not included in these experiments but are displayed for completion as possible components of the neck complex.

Figure 3—source data 1. Contains the uncropped version of the gel image shown in Figure 3F.

Figure 3.

Figure 3—figure supplement 1. The purified chikungunya virus (CHIKV) nsPs are homogeneous and monomeric.

Figure 3—figure supplement 1.

(A) Size-exclusion chromatography (SEC) purification profile of CHIKV nsP1. The elution fraction at 14 ml represents the homogeneous and monodisperse fraction of CHIKV nsP1, which was confirmed by Coomassie-stained 10% SDS-PAGE (B) and mass photometry (C). Mass photometry shows that the protein has a molecular mass of 62 kDa, which is within the error range (±5%) from the expected mass of CHIKV nsP1 (60 kDa). (D) SEC purification profile of CHIKV nsP2. The narrow elution peak represents the homogeneous and monodisperse fraction of CHIKV nsP2, which was confirmed by Coomassie-stained 10% SDS-PAGE (E).
Figure 3—figure supplement 1—source data 1. Contains the uncropped version of the gel images shown in Figure 3—figure supplement 1.
Figure 3—figure supplement 2. Representative gels related to Figure 3.

Figure 3—figure supplement 2.

(A–C). Copelletation of nsP1 with multilamellar vesicles with varying percentages of the anionic phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) (A), PI(4)P (B), and PI(4,5)P2 (C) in a background of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 20% cholesterol. The supernatant and pellet were run on 10% SDS-PAGE. After destaining the Coomassie stained gel, image was acquired with a Chemidoc Imaging System (Bio-Rad), and the relative intensity of bands were quantified using ImageLab software (Bio-Rad) and plotted as shown in Figure 3A-C.
Figure 3—figure supplement 2—source data 1. Contains the uncropped version of the gel images shown in Figure 3—figure supplement 2.