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: 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