chikungunya virus glycoprotein  (Native Antigen Inc)

Journal: iScience

Article Title: In silico reconstruction of a salmonid alphavirus virion reveals distinctive molecular features implicated in virulence

doi: 10.1016/j.isci.2026.115070

Figure Lengend Snippet: Aquatic and terrestrial alphavirus phylogeny and E2 envelope protein alignment (A) Phylogenetic tree of alphavirus structural protein sequences. The amino acid sequence of 17 representative terrestrial and aquatic alphaviruses were aligned and a maximum-likelihood (ML) phylogenetic tree was generated (LG [G + I] substitution model). Numbers at nodes indicate percent bootstrap support from 1,000 replicates. Tree drawn to scale with branch lengths measured in number of substitutions per site (scale bar). For each alphavirus, the known host range and vectors are indicated by animal diagrams. Experimentally determined virion structures are depicted for the following terrestrial alphaviruses: VEEV (PDB: 3j0c ); EEEV (PDB: 6odf ); SINV (PDB: 6imm ); WEEV (PDB: 8dec ); RRV (PDB: 6vyv ); MAYV (PDB: 7ko8 ); CHIKV (PDB: 3j2w ). (B) Amino acid sequence alignment of the N-term region of alphavirus E2. Protein sequences of the 17 aquatic and terrestrial alphaviruses in (A) were aligned and the region corresponding to E2 N-term is shown. The alignment reveals an amino acid insert present only in fish alphavirus E2 proteins (Indel, top). Salmonid alphavirus (SAV) is unique in harboring an additional stretch of three residues herein referred to as 7-8-9 triplet based on SAV E2 amino acid numbering.

Article Snippet: Crystal structure of the immature envelope glycoprotein complex of Chikungunya virus , Protein DataBank (PDB) , 3n40.

Techniques: Sequencing, Generated

Journal: iScience

Article Title: In silico reconstruction of a salmonid alphavirus virion reveals distinctive molecular features implicated in virulence

doi: 10.1016/j.isci.2026.115070

Figure Lengend Snippet: In vitro biochemical analysis and in silico modeling of SAV E3E2 furin cleavage and comparison with CHIKV (A) SAV E3 C-term furin cleavage site (FCS) and E2 N-term. Position of the FCS, amino acid composition, triplet 7-8-9 and E2 N-term insert α-helix are shown. The residues involved in the E2 N-term α-helix predicted by AlphaFold3, RoseTTAFold, and PsiPred are depicted as helical cartoons. (B) Biochemical analysis of cleavage status of p62 in infected cells and purified SAV virions. Western blot analysis was performed on lysates of mock-infected (lane 1) or SAV2-infected BF-2 cells at an m.o.i. of 1 (lane 2) or 5 (lane 3) or on sucrose cushion-purified SAV2 virions (lane 4). Western blot analyses performed using anti-E2 (17H23) or anti-E1 (78K5) mAbs. GAPDH detection was used as loading control for cell lysates. (C) Structural modeling of SAV p62 (E3E2) cleavage by furin. SAV E1 ecto , E2 ecto , and E3 were modeled as complexes using AlphaFold 3. Top corresponds to uncleaved p62 (E3E2), middle corresponds to E3 cleaved from E2 with E3 remaining non-covalently linked, and bottom corresponds to E1 and E2 ectodomains after E3 release. Arrows indicate location of E2 N-term ɑ-helix and 7-8-9 triplet. The models are colored using AlphaFold pLDDT confidence scoring. (D) Structural alignment of CHIKV and SAV uncleaved p62. The uncleaved model of SAV p62 (pink) was structurally aligned with the crystal structure of CHIKV p62 (gold, PDB: 3n40 ) using MatchMaker in ChimeraX. For panels C and D, structure labeling, representation, and scale bars were visualized using ChimeraX.

Article Snippet: Crystal structure of the immature envelope glycoprotein complex of Chikungunya virus , Protein DataBank (PDB) , 3n40.

Techniques: In Vitro, In Silico, Comparison, Infection, Purification, Western Blot, Control, Labeling

Journal: iScience

Article Title: In silico reconstruction of a salmonid alphavirus virion reveals distinctive molecular features implicated in virulence

doi: 10.1016/j.isci.2026.115070

Figure Lengend Snippet: Structural and evolutionary analyses of aquatic and terrestrial alphavirus proteins (A) E2-E1-Cp subunit of SAV predicted by AlphaFold 3. (B) Detailed view of E2 ectodomain with N-term insert helix and 7-8-9 triplet highlighted (arrows). (C) E2-E1-Cp subunit of VEEV (PDB: 3j0c ) and CHIKV (PDB: 8fcg ) terrestrial alphaviruses and E2-E1-Cp subunit of mammalian aquatic alphaviruses SESV and AHPV predicted by AlphaFold 3. (D) E2-E1-Cp subunit of WHAV, CAV, WCFAV, and WSFAV fish alphaviruses predicted by AlphaFold 3. For Cp, only the ordered C-term region of the respective proteins was modeled. The known host range and vectors are indicated by animal diagrams. For (C and D), a focused view of the E2 N-term of each model is shown in each box. Structure labeling, representation, and scale bars were visualized using ChimeraX. (E) Comparative analyses between alphavirus proteins using structure-based protein alignment and phylogenetic analysis. FoldMason was used to perform multiple protein structure alignments (MSTA) of alphavirus E1 and E2 ectodomains and Cp protein (C-term domain). In each structural alignment, the blue (E1), green (E2), and red (Cp) protein corresponds to SAV while the superimposed proteins in gold are from the other species analyzed. The structure-based protein sequence alignments were used to build Maximum-Likelihood phylogenetic trees using WAG+G+I substitution model for E1 and WAG+G for E2 and Cp. Numbers at nodes indicate percent bootstrap support from 1,000 replicates. Trees drawn to scale with branch lengths measured in number of substitutions per site (scale bars).

Article Snippet: Crystal structure of the immature envelope glycoprotein complex of Chikungunya virus , Protein DataBank (PDB) , 3n40.

Techniques: Labeling, Sequencing

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Immunoblot analysis of total lysates and supernatants harvested from CHIKV-infected Vero and VeroΔTIM/AXL cells. Cells were infected with CHIKV-GFP at 0.5 (1x) or 5 (10x) MOI. Total cell lysates and purified supernatants were probed against CHIKV E1 or vinculin as a control. (B) Diagram of CHIKV-GFP-E2-NLuc virus genome used for release efficiency assays. Nano luciferase (NLuc) was inserted at the N-terminus of E2. Created in BioRender.com (C) Vero cells were infected with either CHIKV-GFP or CHIKV-GFP-E2-NLuc. Supernatants were purified through ultracentrifugation and analyzed using a stain-free gel. (D) Multi-step replication curve of CHIKV and CHIKV-GFP-E2-NLuc in Vero cells (0.01 MOI) was harvested at each indicated time point. (E) Ratio between TCID50U/mL and Relative Luminescence Units (RLU) from samples harvested in the multi-step replication curve of cells infected with CHIKV-GFP-E2-NLuc. Data represents the mean ±SEM from at least three independent trials.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Western Blot, Infection, Purification, Control, Virus, Luciferase, Staining

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Release efficiency assay of CHIKV-GFP-E2-NLuc in Vero, VeroΔTIM, VeroΔAXL, and VeroΔTIM/AXL cells (MOI 0.5, harvested at 18 hours) (A) and corresponding levels of luminescence present in the total cell lysates (TCL) and supernatants (sup) (B) . Release efficiency assay with VeroΔTIM/AXL cells infected with ten times more CHIKV-GFP-E2-NLuc than Vero cells (C) and corresponding luminescence levels (D). Release efficiency assay of VSV-GFP-M-NLuc in Vero, VeroΔTIM, VeroΔAXL, and VeroΔTIM/AXL cells (MOI of 1, harvested at 8hrs) (E) and corresponding luminescence levels (F). Release efficiency assay of CHIKV VLPs in Vero and VeroΔTIM/AXL cells transfected with a plasmid encoding CHIKV’s structural cassette tagged with nano-luciferase (G) and corresponding luminescence levels (H). Data represent the mean ±SEM from at least three independent trials. For each release assay, data was normalized to the parental cell line to determine the relative release efficiency. Unpaired parametric Student’s t-test with unequal variance (Welch’s correction) was performed to determine statistical significance in comparison to the parental cell line. *, p < .05.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Release Assay, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Experimental design for fluorescent liposome competition during release of CHIKV-infected Vero and VeroΔTIM/AXL cells. Created in BioRender.com (B) Increasing concentrations of fluorescent PC:PE:PS liposomes were added to CHIKV-E2-NLuc infected Vero or VeroΔTIM/AXL cells 6 hpi and release efficiency was calculated 18hrs post-infection. Data was normalized to the no-liposome control of each cell line. Vero (C) and VeroΔTIM/AXL (D) cells were transfected with a plasmid encoding hTIM-1 and infected with CHIKV-E2-NLuc 24 hours following transfection. Release efficiency was calculated 18hrs post-infection. (E) 293T cells were transfected with plasmids encoding the indicated surface receptors and infected with CHIKV-E2-NLuc 24 hours following transfection. Release efficiency was calculated 18hrs post-infection. Data was normalized to the GFP-transfected control. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test with unequal variance (Welch’s correction) was performed to determine statistical significance in comparison to the parental cell line. *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Liposomes, Control, Transfection, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Entry efficiency of CHIKV-GFP in HAP1 and HAP1ΔCDC50 cells. (B) Multi-cycle replication curve of CHIKV in HAP1 and HAP1ΔCDC50 cells (MOI 0.01). (C) Release efficiency of CHIKV-GFP-E2-NLuc in HAP1 and HAP1ΔCDC50. Data was normalized to the release efficiency of the parental cell line to determine the relative release efficiency. (D) Surface biotinylation analysis of uninfected HAP1 and HAP1ΔCDC50 cells. Total lysates and surface proteins were probed using a Tyro3 antibody or Actin antibody as a loading control. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test was performed to determine statistical significance in comparison to the parental cell line at each indicated timepoint. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Entry efficiency of CHIKV-GFP in VeroS and VeroSΔCDC50 cells. (B) CHIKV-GFP spread in VeroS and VeroSΔCDC50 cells (MOI 0.1). (C) Multi-cycle replication curve CHIKV in VeroS and VeroSΔCDC50 cells (MOI 0.01). Supernatants were harvested and titrated at the indicated time points. (D) Release efficiency assay of CHIKV-GFP-E2-NLuc when five times more virus as added to VeroSΔCDC50 than Vero cells to equalize cell lysate luminescence levels. (E) VeroSΔCDC50 cells were transfected with a plasmid encoding CDC50a and infected with CHIKV-E2-NLuc, release efficiency was assessed 18 hours post-infection. Surface receptors of VeroS (green) and VeroSΔCDC50a (blue) were assessed via staining using (F) a TIM-1 antibody or (G) binding of DioC 18 (3) fluorescent PC:PE:PS liposomes and analyzed through flow cytometry. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test was performed to determine statistical significance in comparison to the parental cell line at each indicated timepoint. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Virus, Transfection, Plasmid Preparation, Infection, Staining, Binding Assay, Liposomes, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Representative histograms of binding of fluorescent liposomes in Vero and VeroΔTIM/AXL cells as a measure for phospholipid binding receptors. (B) Fold binding of fluorescent PC:PE:PS liposomes in mammalian and mosquito cell lines. To remove differences in fluorescent background levels among cell lines, fold binding was determined by calculating the ratio of DioC 18 (3) mean fluorescent intensity (MFI) over no-liposome control for each cell line. The dotted line represents the threshold where DioC 18 (3) MFI was equivalent to no-liposome background levels indicating no binding occurred. (C) The release efficiency of CHIKV-GFP-E2-NLuc in a panel of mammalian and mosquito cell lines. (D) Correlation analysis between liposome binding and CHIKV release efficiency. The size of circles represents the degree of liposome binding and colors indicate levels of release efficiency. Data represents the mean ±SEM from at least three independent trials.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Binding Assay, Liposomes, Control

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Levels of TIM-1 in the surface of uninfected or CHIKV-infected VeroS cells were assessed via receptor staining using a TIM-1 antibody (A) or binding of fluorescently labeled liposomes (B) and analyzed through flow cytometry. (C) VeroS cells were infected with either CHIKV-GFP (top, MOI 0.5) or LCMV-GFP (bottom, MOI 1) resulting in similar levels of infection. (D) The total protein present in total cell lysates (TCL) and biotinylated surface proteins (SB) of uninfected, CHIKV, and LCMV-infected VeroS cells were compared using a stain-free gel. (E) Immunoblot analysis of samples shown in panel D.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Staining, Binding Assay, Labeling, Liposomes, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Total cell lysate and surface biotinylated proteins of uninfected, CHIKV or LCMV-infected VeroS cells were quantified using densitometry analysis.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Infection

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) CHIKV-GFP infected VeroS cells were infected at different time points, and all were harvested at the same time for surface biotinylation analysis. Infection was maintained for 0, 3, 6, 9 or 12 hours. Samples were probed using TIM-1, E1, or transferrin (Trfn) antibodies. (B) VeroS cells were infected with CHIKV at different time points, and subjected to fluorescent liposome binding simultaneously. Infection was maintained for 0, 3, 6, 9, 12, 15, or 18 hours and analyzed through flow cytometry. (C) Quantification of fluorescent PC:PE:PS liposomes. (D) Cells were transfected with plasmids encoding CHIKV proteins for 48 hours and analyzed through binding of fluorescently labeled liposomes using flow cytometry. Liposome binding was compared to control cells to determine the relative liposome binding levels. Data represents the mean ±SEM from at least three independent trials. An ordinary one-way ANOVA with multiple comparisons was used to evaluate statistical differences in comparison to control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ****, p < .0001.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Binding Assay, Flow Cytometry, Liposomes, Transfection, Labeling, Control, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Stain-free gel analysis of (A) total cell lysates or (B) surface biotinylation proteins infected with CHIKV for different periods. (C) Exogenous expression of CHIKV non-structural proteins tagged with FLAG tag was analyzed through SDS-PAGE using an antibody against FLAG or transferrin as loading control. nsP1 was quickly detected in the cell lysates of transfection cells but longer exposure (right) was needed for detection of nsP2, nsP3, and nsP4. (D) Exogenous expression of CHIKV structural proteins was analyzed using an antibody against CHIKV E1 or transferrin as a loading control.

Article Snippet: Cell lysates and purified supernatants were separated on an SDS-PAGE and analyzed through immunoblotting against vinculin as a loading control (1:2,000, MGA465GA, BioRad) or CHIKV E1 glycoprotein (1:1,000, MAB97792, R&D systems).

Techniques: Staining, Infection, Expressing, FLAG-tag, SDS Page, Control, Transfection