chikv nsp2  (GeneTex)

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: a-f . Scatter plots of metabolite changes for CHIKV infection performed at the indicated multiplicity of infections (MOI). Each dot represents an average fold change (FC) of five independent biological replicates performed at multiple technical replicates (n = 5 biological replicates). Colored dots (red and pink) denote log 2 FC ≅ 1 or –1 for the respective cell lines. Infection dose, a and b : MOI of 0.2, c and d : MOI of 2, e and f : MOI of 20, cell lines: a , c , e : Vero E6 and b , d , f : Huh7.5.1.

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Infection

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: a . Methionine, transsulfuration, and polyamine pathways. Abbreviations of enzymes: AHCY, S -adenosylhomocysteine hydrolase; AMD1, Adenosylmethionine decarboxylase 1; CBS, Cystathionine β-synthase; CTH, Cystathionine ɣ-lyase; GCLC, Glutamate-cysteine ligase; GSS, Glutathione synthase; Mat2a, Methionine adenosyltransferase 2a; MTAP, Methylthioadenosine phosphorylase; MTR, Methionine synthase. Abbreviations of metabolites: MTA, 5ʹ-methylthioadenosine; SAM, S -adenosylmethionine; and SAH, S -adenosylhomocysteine. ** and ↑ denote the CHIKV-induced upregulated enzyme (Mat2a) and metabolite (MTA). Right : Chemical structures of major Met cycle metabolites – Met, MTA, SAM, SAH, and homocysteine. Blue indicates structural similarity of MTA, SAM, and SAH. b and c . qPCR profiles of Met salvage cycle enzymes during CHIKV WT infection at MOI of 2 for 8 hpi under complete medium ( b ) and Met-Cys limiting conditions for Mat2a expression ( c ). Experiments were conducted four times ( b ): Student’s t -test, Mat2a vs reference, t = 3.181, df = 3, * – p = 0.05, and twice (n = 2 independent replicates) ( c ): CHIKV MOI 2 vs reference, t = 27.29, df = 2, *** – p = 0.0013, No infection, t = 36.51, df = 2, *** – p = 0.0007, CHIKV MOI 2 vs No infection, t = 4.372, df = 2, * – p = 0.0485, each in three technical replicates. d . Left: Schematic of isotopic incorporation tracking of methionine during CHIKV infection (8 hpi; MOI of 2) using L-Met ( 13 C-methyl) into MTA. Right : Mass spectra of MTA from CHIKV-infected cell cultures grown with either L-Met (top) or with L-Met ( 13 C-methyl) (bottom). e – f . CHIKV replication regulation by Met-Cys availability. Infected cells were cultured in Met-Cys free or complete medium for indicated hpi and CHIKV infection intensity quantified by plaque assay (e – f). In parallel, cell viability was assessed by CellTiter-Glo ® assay at 48 h (e, f). t -test, CHIKV yield 24 hpi: Complete medium vs 0 µM Met, 200 µM Cys, t = 2.694, p = 0.0243, 100 µM Met, 200 Cys, t =2.995, p = 0.0092, 200 µM Met, 200 µM Cys, t = 2.131, p = 0.05, 200 µM Met, 0 Cys, t = 2.357, p = 0.0437. 48 hpi, Complete vs 0 Met, 200 µM Cys, t = 4.339, p = 0.0005, ns – not significant. g . Infection rescue assay assessed by supplementing the indicated sulfur-containing intermediate metabolites into Met-Cys deprived infected cells. Representative bright field microscopy images of infected cells taken 48 hpi. Scale bar, 300 µm. h . MTA favors CHIKV replication in a Met-Cys free medium. CHIKV infection was conducted with a wild-type virus at MOI of 0.5 for 72 hpi. Viral RNA was extracted from supernatants and absolute genome copies quantified by qPCR using standard CHIKV E1 gene fragment. Student ’s t -test, Met-Cys free + SAM, t = 10.38, df = 22, p < 0.0001, Met-Cys free + MTA, t = 6.682, df = 22, p < 0.0001, Met-Cys free + MTA vs Met-Cys free + SAM, t = 8.485, df = 22, p < 0.0001. i . Comparative CRISPR deletion knockout effects of Met transporter genes and Met-Cys pathways on CHIKV infection (MOI of 2; 48 hpi). Experiments were conducted for two independent replicates in 96 technical replicates. Student ’s t -test, Mat2a KO vs Reference, t = 123, df = 1, p = 0.0052, AHCY KO, t = 145, p = 0.0044, CBS KO, t = 35, p = 0.0182, GCLC KO, t = 53, p = 0.0120, METTL16 KO, t = 20.20, p = 0.0315, KIAA1966 KO, t = 29.50, p = 0.0216, MTR1 KO, t = 21, p = 0.0303, SLC43A2 KO, t = 24, p = 0.0262, ns – not significant.

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Infection, Expressing, Cell Culture, Plaque Assay, Glo Assay, Rescue Assay, Microscopy, Virus, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: a. Expression levels of tRNA modifying enzymes during CHIKV WT infection at MOI of 2 at 8 hpi under complete medium and Met-Cys depleted conditions, determined by qPCR. Experiments were conducted twice (n = 2 independent replicates) in three technical replicates. Student ’s t -test: Complete medium, Kiaa1456 v reference, t = 13.28, df = 1, p = 0.0478. Met-Cys free medium: Ctu2 , t = 41.86, p = 0.0152, Nfs1 , t = 76.5, p = 0.0083, Alkbh8 , t = 13.49, p = 0.0471, Mocs3 , t = 14.21, p = 0.0447, ns – not significant. b . Left : Comparative CRISPR deletion effects of tRNA modifying genes on CHIKV infection (MOI of 2; 48 hpi). Experiments were conducted for two independent replicates (96 technical replicates). Student ’s t -test, MOCS3 KO, t = 24, df = 1, p = 0.0265, Urm1 KO, t = 31.50, df = 1, p = 0.0202, ELP3 KO, t = 95, df = 1, p = 0.0067, NFS1 KO, t = 13.29, df = 1, p = 0.0478, Ctu2 KO, t = 28.50, df = 1, p = 0.0223, ALKBH8 KO, t = 149, df = 1, p = 0.0043, Ctu1 KO, t = 15.67, df = 1, p = 0.0406, ns – not significant. Right : ALKBH8 KO cell viability tested at 48 h. Student ’s t -test, t = 5.480, df = 4, * – p = 0.0276. c . Reaction scheme of ALKBH8-dependent U 34 -tRNA modifications. Blue and pink atoms indicate the specific methylation and thiolation sites. d . Comparative effects of exogenous supplementation of mcm 5 U and mcm 5 s 2 U to ALKBH8 KO cells on CHIKV infectivity for 48 and 72 hpi (MOI of 1; n = 5). Student ’s t -test, 48 hpi, ALKBH8 KO + 20 µM mcm 5 U, t = 4.227, df = 4, p = 0.0134, ALKBH8 KO + 50 µM mcm 5 U, t = 5.6154, df = 4, p = 0.005, ALKBH8 KO + 20 µM mcm 5 s 2 U, t = 4.0833, df = 4, p = 0.0151, ALKBH8 KO + 50 µM mcm 5 s 2 U, t = 2.9074, df = 4, p = 0.0438. 72 hpi, ALKBH8 KO + 20 µM mcm 5 s 2 U, t = 2.7271, df = 4, p = 0.05, ALKBH8 KO + 50 µM mcm 5 s 2 U, t = 2.8105, df = 4, p = 0.0482, ns – not significant. e . Quantification of mcm 5 U and mcm 5 s 2 U tRNA modifications by LC-MS. Two-tailed Student ’s t- test, mcm 5 U: Complete medium (WT) vs Met-Cys free (WT), t = 13.12, df = 3, ** – p = 0.0048, Complete medium (WT) vs Complete medium (ALKBH8 KO), t = 8.812, df = 3, ** – p = 0.0083. mcm 5 s 2 U: Complete medium (WT) vs Met-Cys free (WT), t = 2.079, df = 3, ns – p = 0.1259, Complete medium (WT) vs Complete medium (ALKBH8 KO), t = 8.695, df = 3, ** – p = 0.0013.

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Expressing, Infection, CRISPR, Methylation, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: a . Met-Cys time-of-deprivation assay at MOI of 1 for a single replication cycle. Infection was initiated with complete culture medium and Met-Cys free medium was added at the indicated time and viral infection intensity quantified at 24 hpi. **** – p < 0.0001, ** – < 0.05, and ns, not significant ( Student’s t -test). b . Relative m 6 A RNA methylation analysis using EpiQuik m 6 A RNA methylation quantification fluorescence kit. Unpaired two-tailed Student ’s t -test; NTC vs Infection reference, t = 2.137, df = 4, ns – p = 0.0994, Met-Cys free vs Infection reference, t = 5.467, df = 4, ** – p = 0.0054, Met-Cys free + MTA vs Infection reference, t = 6.951, df = 4, ** – p = 0.0024. c . Analysis of CHIKV RNA transcription priming by MTA. Total RNA samples were collected from MTA-supplemented Met-Cys free + CHIKV-infected cells in presence of 10 µM Actinomycin D (ActD) or its absence (complete medium) at the indicated time points post cell entry. CHIKV RNA copies were quantified by qRT-PCR analysis of E1 gene. Experiments were performed in triplicates for three replicates (n = 3).

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Infection, Methylation, Fluorescence, Two Tailed Test, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: a. Chemical structures of AHCY inhibitors; 3-deazaneplanocin A HCl (DzNep) and adenosine dialdehyde (Adox) exerting CHIKV antiviral activities at indicated potencies. b. Dose-response curves of antiviral effects exerted on CHIKV Gluc MOI of 0.01 by DzNep and Adox for 72 hpi (n = 6 independent replicates). c . Representative images of bright field microscopy of infected cells (MOI of 0.01) on treatment with DzNep and Adox taken at 72 hpi. Scale bar, 300 µm. d . Immunofluorescence image of treatment effects of DzNep, Adox, and Met-Cys free medium on CHIKV infection for 8 hpi at MOI of 2. Infection intensity was probed using CHIKV nsP2 antibody under GFP background. e . Time-of-addition assay for DzNep and Adox for 8 hpi, with reference to ribavirin. Treatment was started following virus cell entry and quantification performed at 24 hpi. Assay conducted in triplicates at MOI of 1 for two independent replicates. Student’ s t -test, **** – p < 0.0001, ** – 4 hpi: Ribavirin, t = 5.6493, p = 0.007, *** – 6 hpi: t = 11.377, p = 0.0002, ** – 8 hpi: DzNep, t = 5.5941, p = 0.0091, Adox, t = 6.0724, p = 0.0064. f . Cycloleucine antiviral activity at 30 mM against a non-treated control. Unpaired Student ’s t -test, t = 25.99, df = 8, ****- p < 0.0001. g . Met-Cys deprivation and DzNep/Adox treatments restores CHIKV-induced reactive oxidative stress (ROS) levels. Cells were infected in complete medium, when the inhibitors were added. NTC, non-infected treatment control. Student’ s t -test, CHIKV WT vs NTC, t = 9.494, df = 16, ****- p < 0.0001, Met-Cys free vs NTC, t = 0.8610, df = 16, p = 0.4017, DzNep vs NTC, t = 0.0296, df = 16, p = 0.9767, Adox vs NTC, t = 0.0531, df = 16, p = 0.9582. ns – not significant.

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Microscopy, Infection, Immunofluorescence, Virus, Activity Assay, Control

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet: CHIKV entry into host cell increases demand for metabolite supply from the methionine pathway, reprogramming the Met salvage pathway into increased production of 5ʹ-MTA, and upregulation of Mat2a. The reprogramming is tightly linked to the availability of sulfur amino acids that maintain downstream transsulfuration pathway to fine-tune CHIKV-modulated ALKBH8 activity. MTA can enhance CHIKV replication in absence of sulfur. Sulfur-dependent processes are targetable to thwart viral infection using the AHCY inhibitors; DzNep and Adox that mimic sulfur deprivation effects. Graphic created on Biorender.com.

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Activity Assay, Infection

Journal: bioRxiv

Article Title: Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

doi: 10.1101/2025.07.11.664323

Figure Lengend Snippet:

Article Snippet: Following washing off of formaldehyde with 1× PBS, the cells were permeabilized for 15 min with 0.1% Triton X-100 (in 1× PBS) and blocked with 2% BSA added for 1 h. The cells were subsequently incubated overnight at 4°C with CHIKV nsP2 antibody (Thermo Fisher #PA5-143493; dilution 1:500).

Techniques: Infection

Journal: bioRxiv

Article Title: Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

doi: 10.1101/2025.01.09.632058

Figure Lengend Snippet: A, Schematic of chikungunya virus (CHIKV) replicon used in this study. B, Inhibition of host factors known to be required for CHIKV replication by CRISPR knockout (KO) in the stable Huh7.5.1-Cas9 CHIKV replicon cell line. CHIKV replication levels were quantified by changes in % eGFP expression upon CRISPR KO of host genes established to be involved in CHIKV replication (open symbols) or genes unrelated to CHIKV replication (gray), with wild-type (WT) CHIKV replicon cells assayed in parallel (black). Experiments were performed in technical triplicates and values represent mean % eGFP ± SD normalized to % eGFP at the start of the experiment. C, Enrichment plot from a CRISPR KO screen with the CHIKV replicon cell line for host factors involved in CHIKV replication. The y-axis represents the significance of enrichment calculated by MAGeCK RRA statistical analysis of genes that were enriched in the selected (eGFP-low) population vs control (unselected) cell population; the x-axis corresponds to genes. D, Independent validation of replicon screen phenotypes: arrayed CRISPR KO in the CHIKV replicon cell line targeting a panel of candidate genes identified in the screen. The fraction of eGFP + cells 18 d after cells were treated with individual targeting guide RNA gRNAs or (non-targeting control gRNA, NT) was measured to quantify CHIKV replicon activity. Values represent mean % eGFP ± SD normalized to WT control from 3 biological replicates. E, Quantification of KO impact on live CHIKV infections. Parental (WT) Huh7.5.1 and independently generated KO cell populations were exposed with CHIKV LR-2006 OPY1 (MOI 0.1) and harvested at 7, 18, and 25 h after exposure. Values for each timepoint represent the mean ± SD of normalized CHIKV expression levels relative to the normalized levels detected in the control cell lines transduced with non-targeting gRNA. Gene names are abbreviated according to the human standard ( https://www.genenames.org/ ).

Article Snippet: CHIKV nsP2 (Genetex, GTX135188), CHIKV nsP3 (Genetex, GTX135189), CHIKV nsP4 (Thermo Fisher Scientific, PA5-117443), beta tubulin (Cell Signaling, 15115) NP (IBT Bioservices, 0301-012), VP35 (Kerafast, Kf Ab02366-1.1), VP30 (GeneTex, GTX134035), 2A (Novus, NBP2-59627),EHMT1 (Abcam, ab241306), EHMT2 (Thermo Fisher Scientific, MA5-14880), USP7 (Thermo Fisher Scientific, PA5-34911), and beta tubulin (Thermo Fisher Scientific, MA5-16308).

Techniques: Virus, Inhibition, CRISPR, Knock-Out, Expressing, Control, Biomarker Discovery, Activity Assay, Generated, Transduction

Journal: bioRxiv

Article Title: Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

doi: 10.1101/2025.01.09.632058

Figure Lengend Snippet: Graphical summary of the host factor dependencies within the cell recovered for DENV-2, CHIKV, and EBOV (this study) as well as hepatitis E virus (HEV) . Created in https://BioRender.com .

Article Snippet: CHIKV nsP2 (Genetex, GTX135188), CHIKV nsP3 (Genetex, GTX135189), CHIKV nsP4 (Thermo Fisher Scientific, PA5-117443), beta tubulin (Cell Signaling, 15115) NP (IBT Bioservices, 0301-012), VP35 (Kerafast, Kf Ab02366-1.1), VP30 (GeneTex, GTX134035), 2A (Novus, NBP2-59627),EHMT1 (Abcam, ab241306), EHMT2 (Thermo Fisher Scientific, MA5-14880), USP7 (Thermo Fisher Scientific, PA5-34911), and beta tubulin (Thermo Fisher Scientific, MA5-16308).

Techniques: Virus

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) BHK-21 or ERMS cells were seeded in a 96-well plate and infected with CHIKV-GFP at 0.1 or 5 MOI for 20 or 32 h, respectively and treated with DMSO (vehicle control) or the test compounds. The top panels show the CHIKV replication inhibition as per cent GFP reduction using different compounds at 10 μM concentration. The bottom panels show the percentage of cell viability for each compound. (B) BHK-21 or EMRS cells were seeded in a 96-well plate and infected with CHIKV-GFP at 0.1 or 5 MOI for 20 or 32 h, respectively and treated with the indicated concentrations of different compounds. The bar graphs show the CHIKV replication inhibition as per cent GFP reduction for the selected compounds at lower concentrations of 0.1, 0.5, and 1.0 μM. (C) BHK-21 cells were infected with CHIKV-GFP (0.1 MOI) and treated with different concentrations of WFA. The dose-response curve demonstrating the per cent GFP and per cent cell viability at different WFA concentrations at 24 h pi is shown.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Control, Inhibition, Concentration Assay

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) ERMS cells were infected with CHIKV at 1 MOI. At 0 h pi, the cell culture medium was supplemented with WFA (1 μM) or the vehicle DMSO (control). The total RNA was isolated at different times pi to quantify the intracellular viral RNA levels by qRT-PCR. The relative levels of the CHIKV RNA are shown in the left panel, where the CHIKV RNA level at 6 h pi in the control was taken as 1. The culture supernatant from the CHIKV-infected cells was collected, and the virus titers determined by the plaque assay are shown in the right panel. (B) ERMS cells infected with CHIKV (1 MOI) were treated with different concentrations of WFA. The cells were processed at 6 h pi for the intracellular viral RNA quantitation by qRT-PCR and cell viability by the MTT assay. The line graph demonstrating the relative viral RNA levels and per cent cell viability at the indicated WFA concentrations is shown. The viral RNA levels and per cent cell viability were normalized to the respective vehicle-only controls. (C) HeLa, Huh7, or C2C12 cells were infected with CHIKV (MOI 1) in the presence of WFA or DMSO (vehicle control), and the total RNA was isolated at 6 h pi. The relative CHIKV RNA levels determined by qRT-PCR are presented where the level of CHIKV RNA in the control cells was taken as 1. The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Cell Culture, Control, Isolation, Quantitative RT-PCR, Virus, Plaque Assay, Quantitation Assay, MTT Assay

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: C57BL/6 mice of 12 weeks of age were mock-infected or infected sub-cutaneous with 10 4 PFU of CHIKV and treated with vehicle alone or WFA (5 mg/kg) given intra-peritoneal twice a day. The first dose of WFA was delivered 4 h pi. The mice in all 4 groups were followed for 2 weeks, and the paw edema was measured daily using a digital plethysmometer. (A) A line graph demonstrating the mouse paw edema on different d pi is presented. The Boneforreni post hoc test, followed by a two-sided independent t-test, was used to calculate the p values: * p <0.0332, ** p <0.0021, *** p <0.0002, **** p <0.0001. (B) The representative images showing the footpad swelling observed in different treatment groups at 7 d pi are presented. (C) The CHIKV load in the mouse serum at 1 and 2 d pi, as determined by the plaque assay, is shown. The student’s t-test was used to calculate the p values: * p <0.05, ** p <0.01, *** p <0.001, ns = not significant.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Plaque Assay

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: ERMS cells were infected with CHIKV (1 MOI) and incubated with 1 μM WFA at different time points after the infection. The control CHIKV-infected cells were incubated with DMSO. The cells were harvested at 6 h pi, and the total RNA was extracted. The qRT-PCR was used to determine the CHIKV RNA levels. The relative viral RNA levels are shown. The CHIKV RNA level at 6 h pi in the control cells was taken as 1. The viral RNA level in the control (DMSO-treated) cells was compared with those treated with WFA at different time points. The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001, ns = not significant.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Incubation, Control, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) ERMS cells were incubated with 1 μM WFA or DMSO (as the vehicle control) for 2 h at 37°C. The cells were then incubated for 1 h with CHIKV (MOI 1) on ice to allow the virus attachment but not its uptake. The cells were washed with ice-cold PBS, and the total RNA was extracted. The qRT-PCR was used to determine the level of CHIKV RNA. The relative levels of CHIKV RNA are plotted where CHIKV RNA in the control cells was taken as 1. (B) ERMS cells were incubated with 1 μM WFA or DMSO (as the vehicle control) for 2 h at 37°C. The cells were washed with PBS and then incubated for 1 h with CHIKV (MOI 1) on ice to allow the virus attachment. The cells were incubated for 1 or 2 h at 37°C for viral uptake and then treated with trypsin to remove uninternalized particles. The cells were then washed with PBS, and the total RNA was extracted. The qRT-PCR was used to determine the level of CHIKV RNA. The relative levels of CHIKV RNA are shown where CHIKV RNA in the control cells was taken as 1. (C) ERMS cells were co-incubated with 1 μM WFA and CHIKV (MOI 1) for 1 h at 4°C and incubated for 1h at 37°C for viral uptake. The control cells were incubated with DMSO and CHIKV. The cells were treated with trypsin to remove the uninternalized virion particles. The cells were then washed with PBS, and the total RNA was extracted. The qRT-PCR was used to determine the level of CHIKV RNA. The relative levels of CHIKV RNA are shown where CHIKV RNA in the control cells was taken as 1. (D) For the virucidal assay, CHIKV and WFA (1 or 2 μM) were incubated at 37°C for 2 h. The control had the virus incubated with DMSO. The viral infectivity was determined by the plaque assay. (E) For the first round of infection, ERMS cells were incubated with CHIKV (MOI 0.1) for 1 h. Following this, the cells were incubated with the culture medium supplemented with WFA (1 μM) or the vehicle DMSO (control). The culture supernatant was collected at 12 h pi for determining the virus titers and cells were harvested for the viral RNA quantitation (left panel). For the second round of infection, ERMS cells were infected (MOI 0.1) with the virus collected at 12 h pi from the first round. The culture supernatant and the cells were harvested at different times pi to determine the CHIKV RNA levels (right panel). The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001, ns = not significant.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Incubation, Control, Virus, Quantitative RT-PCR, Infection, Plaque Assay, Quantitation Assay

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) ERMS cells were infected with CHIKV (1 MOI) and incubated with 1 μM WFA. The control CHIKV-infected cells were incubated with DMSO. The cells were harvested at different time intervals, and total RNA was extracted. The qRT-PCR was used to determine the CHIKV RNA levels. The relative viral RNA levels are shown. The CHIKV RNA level at 1 h pi in the control cells was taken as 1. (B) ERMS cells were infected with CHIKV (MOI 1) and incubated with 1 μM WFA. The control CHIKV-infected cells were incubated with DMSO. The cells were harvested at different time intervals, and total RNA was extracted. The qRT-PCR was used to determine the CHIKV RNA levels. The relative viral RNA levels are shown in the left panel. The CHIKV RNA level at 1 h pi in the control cells was taken as 1. The CHIKV plus- and minus-sense RNA copy numbers were determined using a standard curve and qRT-PCR, and shown in the middle and right panels, respectively. (C) ERMS cells were transfected with 200 ng of CHIKV RNA, and 6 h later, cells were treated with WFA (1 μM) or DMSO (control). The cells and culture supernatants were harvested 24 h post-transfection (pt) to determine the intracellular CHIKV RNA levels by qRT-PCR (left panel) and the extracellular virus titer by the plaque assay (right panel). The relative RNA levels are plotted where the virus RNA level in the control cells at 6 h pt was taken as 1. (D) ERMS cells were mock infected or infected with CHIKV (1 MOI). The virus-infected cells were incubated with 1 μM WFA. The control CHIKV-infected cells were incubated with DMSO. The cells were harvested at different time points and the cell lysates were western blotted with nsP4 antibody. GAPDH was used as the loading control. The relative band intensity compared to the GAPDH band was measured using the ImageJ software and indicated over the band. The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001, ns = not significant.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Incubation, Control, Quantitative RT-PCR, Transfection, Virus, Plaque Assay, Western Blot, Software

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) The CHIKV nsP2 protein is rendered in the cartoon representation and coloured in pink, while WFA is shown in the liquorice representation and coloured atom-wise as C: cyan, and O: red. The binding of WFA at Site 1 and Site 2 is shown. (B-C) The insets show the WFA atomic fitting in the respective pocket; the protein is rendered in the surface view in the pink colour and WFA is shown in the vdW representation. (D) The SiteMap analysis on the CHIKV nsP2 protease. (E-F) The RMSD plots of the WFA-CHIKV complex and WFA at Site 1 and Site 2 from the MD analysis are shown. (G) The interaction map of WFA with the Site 1 residues lining within 4.0 Å is shown. The residues are shown in the liquorice representation and coloured atom-wise as C: white, N: blue, and O: red. The black dotted lines with yellow background represent the hydrogen bonds. The protein is shown in the Quick surf representation.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Binding Assay

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) Microscale thermophoresis was used to study the binding of the labelled CHIKV nsP2 protein (1 μM) with different concentrations of WFA or Wn. The bar graphs display the fluorescence change (ΔF) obtained on nsP2 binding with different concentrations of WFA or Wn in reference to the unbound protein state in the buffer. The left panel shows the nsP2 binding with different WFA concentrations. The right panel shows nsP2 binding with WFA and Wn at the indicated concentration. The student’s t-test was used to calculate the p value; *** p <0.001. (B) To examine the binding and determine the equilibrium dissociation constant between CHIKV nsP2, its protease and helicase domains, and WFA, 1 μM labelled protein was titrated with different concentrations of WFA ranging from 250 to 0.03 μM. (C) To study the effect of the nsP2 mutation on WFA binding 1 μM labelled protein was titrated with different concentrations of WFA. For the binding affinity analysis, ligand-dependent changes in temperature-related intensity change (TRIC) https://nanotempertech.com/nanopedia/tric/ are plotted as F norm values against the WFA concentrations in a dose-response curve. The F norm values are plotted as parts per thousand (‰).

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Microscale Thermophoresis, Binding Assay, Fluorescence, Concentration Assay, Mutagenesis

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: A FRET-based protease assay was used to study the nsP2 protease activity. (A) The real-time profile of the proteolytic assay is presented using 1 μM nsP2 protein with different concentrations of the fluorogenic peptide substrate (Sub-3/4). The fluorescence was monitored every 40 sec. (B) The real-time profile of the proteolytic assay is presented using 1 μM each of the nsP2 protein, or its protease and helicase domains with 25 μM fluorogenic peptide substrate Sub-3/4. The fluorescence was monitored every 40 sec. (C) The real-time profile of the proteolytic assay was obtained using different concentrations of WFA or Wn with 1 μM nsP2 and 25 μM peptide substrate Sub-3/4. A proteolytic assay with 1 μM CHIKV nsP2 helicase domain and 25 μM peptide substrate was also performed as the control. The fluorescence was monitored every 30 sec. (D) The real-time fluorescence profile of CHIKV nsP2 (1 μM) protease assay with different concentrations of WFA and 25 μM fluorogenic peptide substrates (Sub-1/2, Sub-2/3). The fluorescence was recorded every 30 sec. For every assay, the background fluorescence of the substrate peptide was monitored without the enzyme and shown as control (substrate).

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Protease Assay, Activity Assay, Fluorescence, Control

Journal: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) ERMS, HeLa or Huh7 cells were infected with CHIKV at 1 MOI. At 0 h pi, cells were incubated with WFA (1 μM) or the vehicle DMSO (control) in the presence or absence of NAC (3 mM) for 6 h. The total RNA isolated from ERMS cells was used to quantify the intracellular viral RNA levels by qRT-PCR (left panel). The culture supernatant from the CHIKV-infected ERMS cells was collected, and the virus titers were determined by the plaque assay (middle panel). The total RNA isolated from HeLa and Huh7 cells was used to quantify the intracellular viral RNA levels by qRT-PCR (right panel). (B) ERMS cells were infected with CHIKV at 1 MOI. At 0 h pi, cells were incubated with WFA (1 μM) or the vehicle DMSO (control) in the presence or absence of GSH-MEE (3 mM) for 6 h. The total RNA isolated from ERMS cells was used to quantify the intracellular viral RNA levels by qRT-PCR (left panel). The culture supernatant from the CHIKV-infected ERMS cells was collected, and the virus titers were determined by the plaque assay (right panel). (C) The real-time fluorescence profile of CHIKV nsP2 (1 μM) protease assay with 5 μM WFA and 25 μM peptide substrate in the presence or absence of NAC at the indicated concentration was recorded every 30 sec. In the control (substrate) assay, the background fluorescence of the substrate peptide was monitored without the enzyme. The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001, ns = not significant.

Article Snippet: The CHIKV nsP2 cDNA was codon-optimized and synthesised commercially (GenScript).

Techniques: Infection, Incubation, Control, Isolation, Quantitative RT-PCR, Virus, Plaque Assay, Fluorescence, Protease Assay, Concentration Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Intron-derived small RNAs for silencing viral RNAs in mosquito cells

doi: 10.1371/journal.pntd.0010548

Figure Lengend Snippet: (A) Schematic of the CHIKV genome and the location of the 268 nt nsP2 target region (CHI) and each of the 10 sites targeted by small RNAs. (B) Schematic of small RNA expressing plasmids. (C) Strategy for testing small RNA mediated suppression of a CHIKV split replication system (CHIKVRep, replication complex nsP1-4 and viral reporter), chimeric firefly luciferase reporter (CHILuc) and synthetic reporter with the target sequence in the 3’ UTR (LucCHI). Small RNAs containing sequence mismatches (nt 2 and 11) or fully complementary sequences were assessed for their ability to target viral sequences in mosquito cell lines. AAA: polyadenylation signal, Hr5-IE1: Autographa californica nuclear polyhedrosis virus homologous region 5 enhancer-immediate-early gene 1 promoter, Luc: luciferase, mB: Drosophila melanogaster pre- miRNA-1 miRNA backbone, nt: nucleotide, ORF: open reading frame, PUb intron: Ae . aegypti polyubiquitin gene intron, SG: CHIKV subgenomic promoter, SV40: simian virus 40 polyadenylation signal, UTR: untranslated regions, ZsY: ZsYellow fluorescent protein.

Article Snippet: The D . melanogaster pre- miRNA1 stem loop sequence validated by Haley et al. [ ] was modified to target a 268 nt conserved region of the nsP2 viral protease coding region of CHIKV (CHI, ).

Techniques: Expressing, Luciferase, Sequencing, Virus

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Based on PAHO and WHO (PAHO/WHO, 2020) . ZIKV infections colored in blue, DENV in orange and CHIKV in green. A: Confirmed ZIKV, DENV and CHIKV cases in the Americas from 2015 to 2019. B: Confirmed ZIKV, DENV and CHIKV cases in Brazil from 2015 to 2019. C: Confirmed ZIKV, DENV and CHIKV cases in Brazil 2016. D: Confirmed ZIKV, DENV and CHIKV cases in Brazil 2017.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques:

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: The tested compound concentration was 20 µM. HST inhibit ZIKV NS2B/NS3 pro activity about 40% and CHIKV nsP2 pro about 90%. The inhibition of CHIKV protease activity by HSD was higher than 60%. Contrary, HSD shows not relevant inhibition against ZIKV NS2B/NS3 pro . Data shown are the means ± SD from three independent measurements (n = 3). Asterisks mean that the data differs from the control (0 µM inhibitor) significantly at p < 0.01 (**) and p< 0.001 (***), level according to ANOVA and Tukey’s test.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Concentration Assay, Activity Assay, Inhibition, Control

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Chemical structures of HST and HSD (structural formula) and CHIKV nsP2 pro 3D experimental model (PDB code: 3TRK) are shown. CHIKV nsP2 protease domain is presented in pink and the methyltransferase domain in cyan. Normalized activity and inhibition of the virus protease and Lineweaver-Burk plots to determine the inhibition mode is presented: [S] is the substrate concentration; v is the initial reaction rate. The data shown are the means ± SD from three independent measurements (n = 3). A and C: Normalized activity and inhibition of CHIKV nsP2 pro under HST and HSD influence, respectively. B and D: Lineweaver-Burk plot for HST and HSD inhibition of CHIKV nsP2 pro , respectively.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Activity Assay, Inhibition, Virus, Concentration Assay

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Results summarize HST and HSD inhibition experiments of ZIKV NS2B/NS3 pro , and CHIKV nsP2 pro .

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Results summary of the fluorescence spectroscopy experiments of ZIKV NS2B/NS3 pro and CHIKV nsP2 pro with HST and HSD.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Fluorescence, Spectroscopy

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: A: Decomposition of the binding energy of CHIKV nsP2 pro -HST complex. Arrows label the protease domain (gray) and methyltransferase domain (green). The residues involved in the interaction between protease and HST are labeled. B: Decomposition of the binding energy of CHIKV nsP2 pro -HSD complex. The residues involved in the interaction between protease and HSD are labeled. C: 3D structure of CHIKV nsP2 pro (PDB entry: 3TRK), the protease domain is colored in gray and methyltransferase domain in green. Amino acids highlighted that are involved in the interaction with HST (yellow) based on MD simulations. The H-bond between Lys89 and HST is highlighted. D: 3D structure of CHIKV nsP2 pro , amino acids highlighted are involved in the interaction with HSD (yellow) based on MD simulations. The H-bonds between Tyr77, Met240, Val75 and Glu48 with HSD are highlighted. E: Structural overlay of the representative CHIKV nsP2 pro -HST complex structures of two independent MD runs (RMSD: 1.259 Å). Run1: nsP2 pro (gray), HST (yellow) and run2: nsP2 (brown), HST (orange). The binding region of HST is highlighted, the involved amino acids and HST position differs between run1 and run2. In run2 Pro47, Leu51, Leu63 and Ph91 are not interacting by hydrophobic interactions with HST. However, Lys89 forms in run2 like in run1 a hydrogen bond to HST, the distances between donor and acceptor differs slightly between MD run1 and run2 (3.4 to 3.5 Å). F: Structural overlay of the representative CHIKV nsP2 pro -HSD complex structures of two independent MD runs (RMSD: 1.733 Å). Run1: nsP2 pro (gray), methyltransferase domain (green), HSD (yellow) and run2: nsP2 (brown), methyltransferase domain (cyan), HST (orange). The binding region of HST is highlighted, the involved amino acids and HST position differs slightly between run1 and run2. The main difference concerns Met240, which forms in run1 a hydrogen bond, in run2 this interaction is not more formed, the distance increased from 3.0 to 5.5 Å. Because of this observation, Met240 will be not further considered. The hydrogen bonds formed by Glu48, Val75 and Tyr77 remain unaffected.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Binding Assay, Labeling

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Residues involved in forming H-bonds and hydrophobic contacts between the proteins and the ligands.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Residue

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Atoms involved in the H-Bond interaction between flavivirus and alphavirus proteases and ligands.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: