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: bioRxiv

Article Title: Arbovirus persistence in mosquitoes is characterized by translation repression of viral RNAs

doi: 10.64898/2026.02.26.708146

Figure Lengend Snippet: (A) Schematic representation of CHIKV RNA genome organization and viral protein expression strategy. (B) CHIKV titers over time in U4.4 and C6/36, determined by plaque assay. (C) Kinetics of CHIKV gRNA and sgRNA levels in U4.4 and C6/36, quantified by RT-qPCR. (D-E) Expression of CHIKV nsP1 and capsid ( C ) proteins in U4.4 cells (D) and C6/36 (E) over the course of infection. Green and blue colors correspond to U4.4 and C6/36 cells, respectively. All infections were performed at an MOI of 4 and samples were collected at the indicated time post infection. Data points represent the mean ± SEM of 3 independent replicates. Viral RNA levels were quantified using a standard curve generated from in vitro -transcribed CHIKV RNA. Protein expression levels correspond to band intensities normalized to tubulin and to the 1 dpi value within each blot. Dotted lines indicate separated lanes in the same membrane.

Article Snippet: Antibodies used in western blotting were: CHIKV nsP1 (1:1500), CHIKV Capsid (1:1500), CHIKV nsP2 (1:5000) and ZIKV NS3 (1:2000), kindly provided by Andres Merits; Tubulin (1:1000; Merck, T6199-100UL), Rpb1 (1:1000; Active Motif, 39497) and P-eIF2α (1:1000; Cell Signaling, 9721S).

Techniques: Expressing, Plaque Assay, Quantitative RT-PCR, Infection, Generated, In Vitro, Membrane

Journal: bioRxiv

Article Title: Arbovirus persistence in mosquitoes is characterized by translation repression of viral RNAs

doi: 10.64898/2026.02.26.708146

Figure Lengend Snippet: (A-B) Representative polysome profile analyses of U4.4 cells (A) and of C6/36 cells (B) at the indicated time points after CHIKV infection. (C-D) Polysome-to-monosome (P/M) ratios reflecting global mRNA translation in U4.4 and C6/36 cells over the course of infection calculated from the area under-the-curve (AUC) of polysome profiles and normalized to non-infected controls and to 1 dpi. (E) Levels of phosphorylated eIF2α in U4.4 and C6/36 cells during CHIKV infection assessed by western blotting. Protein expression levels were normalized to tubulin and to the non-infected value within each blot. Dotted lines indicate separated lanes in the same membrane. (F) Polysome-to-monosome (P/M) ratios of viral CHIKV gRNA and sgRNA in U4.4 and C6/36 cells over the course of infection determined by RT-qPCR of RNA extracted from polysome fractions. Values are expressed relative to 1 dpi. Bars represent the mean ± SEM of 3 independent replicates.

Article Snippet: Antibodies used in western blotting were: CHIKV nsP1 (1:1500), CHIKV Capsid (1:1500), CHIKV nsP2 (1:5000) and ZIKV NS3 (1:2000), kindly provided by Andres Merits; Tubulin (1:1000; Merck, T6199-100UL), Rpb1 (1:1000; Active Motif, 39497) and P-eIF2α (1:1000; Cell Signaling, 9721S).

Techniques: Infection, Western Blot, Expressing, Membrane, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Arbovirus persistence in mosquitoes is characterized by translation repression of viral RNAs

doi: 10.64898/2026.02.26.708146

Figure Lengend Snippet: (A) Western blot analysis of Rpb1 protein levels in U4.4, C6/36, and HEK293T cells non-infected and infected with CHIKV. Protein expression levels were normalized to tubulin and to the non-infected value within each blot. (B) Immunofluorescence analyses of nsP2 subcellular localization in CHIKV-infected C6/36 cells 1 dpi. Cells were labeled with anti-CHIKV nsP2 antibody (green), J2-dsRNA (red), and nuclei were stained with DAPI (blue). Scale bars correspond to 10 μm. (C) LC–MS/MS quantification of mcm⁵U and mcm⁵s²U tRNA modifications in CHIKV-infected U4.4 and C6/36 cells. Values are expressed relative to those in non-infected. Bars represent the mean ± SEM of 3 independent replicates.

Article Snippet: Antibodies used in western blotting were: CHIKV nsP1 (1:1500), CHIKV Capsid (1:1500), CHIKV nsP2 (1:5000) and ZIKV NS3 (1:2000), kindly provided by Andres Merits; Tubulin (1:1000; Merck, T6199-100UL), Rpb1 (1:1000; Active Motif, 39497) and P-eIF2α (1:1000; Cell Signaling, 9721S).

Techniques: Western Blot, Infection, Expressing, Immunofluorescence, Labeling, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Arbovirus persistence in mosquitoes is characterized by translation repression of viral RNAs

doi: 10.64898/2026.02.26.708146

Figure Lengend Snippet: (A) Scheme of ZIKV RNA genome and its protein synthesis strategy. (B) ZIKV titers over time, determined by plaque assay. (C) Infection kinetics of ZIKV RNA levels determined by qPCR. Data points represent the mean ± SEM of 3 independent replicates. Viral RNA levels were quantified using a standard curve of an in vitro generated ZIKV RNA. (D) NS3 protein expression levels assessed by western blotting. Protein expression levels correspond to the intensity quantification of each band normalized to tubulin levels and to the 4 dpi value within each blot. (E) Levels of phosphorylated eIF2α protein levels during U4.4 ZIKV-infection, assessed by western blotting. Protein expression levels correspond to the intensity quantification of each band normalized to tubulin and to the non-infected value within each blot. Infection was performed at an MOI of 1 and samples were collected at the indicated time post infection.

Article Snippet: Antibodies used in western blotting were: CHIKV nsP1 (1:1500), CHIKV Capsid (1:1500), CHIKV nsP2 (1:5000) and ZIKV NS3 (1:2000), kindly provided by Andres Merits; Tubulin (1:1000; Merck, T6199-100UL), Rpb1 (1:1000; Active Motif, 39497) and P-eIF2α (1:1000; Cell Signaling, 9721S).

Techniques: Plaque Assay, Infection, In Vitro, Generated, Expressing, Western Blot

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, Graphical overview of the steps to generate a stable Ebola virus (EBOV) replicon cell line. PiggyBac transposition was applied to generate a “4cis” cell line, in which four EBOV proteins essential for viral replication and transcription (NP, VP35, VP30 and L protein) are stably expressed after integration and subsequent selection in 100 ug/mL hygromycin media. Next, an in vitro transcribed EBOV minigenome RNA (in negative-sense orientation), which encodes the 5ʹ and 3ʹ viral UTRs flanking a reporter-selection (Zeocin-eGFP fusion) cassette, is transfected into the 4cis cell line. Stable population of >90% eGFP + Huh7.5.1-Cas9 EBOV replicon cells were selected and expanded in Zeocin media. B, Validation of the responsiveness of the replicon cell line by CRISPR knockout (KO) of viral L protein and known host factors involved in EBOV entry (Niemann-Pick disease, type C1 [NPC1]) or replication and transcription (dynein light chain LC8-type 1 [DYNLL1]). C, Enrichment plot showing host factor genes recovered from the screen. Enrichment scores are represented as -log10 of the RRA scores obtained from MAGeCK analysis. D, Validation of host factors identified from the screen. Independent KOs of the hits identified in the screen were generated in the EBOV replicon cell line in an arrayed format and flow cytometry was used to measure the eGFP signal over the course of 20 d. The fraction of eGFP + cells on Day 20 of the time course is plotted as mean + SD (three independent technical replicates). E, Quantification of the levels of different types of EBOV minigenome RNA in the replicon cells harboring different host factor knockouts. Total RNA was isolated from replicon cells with different host factor knockouts and used as input for strand-specific RT-qPCR assay. Plotted values are fold expression changes when normalized to the cells expressing non-targeting gRNA (three independent technical replicates). F, Validation of phenotypes in transient EBOV replicon assays in independently generated KOs in parental cell lines. Effect of host factor knock outs on EBOV replication and transcription in a transient replicon assay with a minigenome encoding a secreted nano-luciferase (SecNLuc). Host factor knockout cell lines and controls were co-transfected with an EBOV 4cis plasmid encoding EBOV NP, VP35, VP30, and L protein, and an EBOV minigenome luciferase reporter plasmid. The SecNLuc signal was measured 48 h post-transfection. Data plotted here represent the mean + SD (three independent biological replicates). 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, Stable Transfection, Selection, In Vitro, Transfection, CRISPR, Knock-Out, Generated, Flow Cytometry, Isolation, Quantitative RT-PCR, Expressing, Luciferase, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling

doi: 10.1371/journal.ppat.1011179

Figure Lengend Snippet: ( A ) MRC-5 cells were transfected with 0.1, 1, or 10 ng/ml 5’pppRNA or a control RNA 1 hour prior to infection with CHIKV-LS3-GFP (MOI 0.1) and non-transfected cells (NTF) were included as control. Protein lysates were harvested 24 h p.i. and STAT1 and (p)eEF2 (T56) were detected by WB analysis. ( B ) MRC-5 cells were transfected with 50, 500 or 5000 ng/ml poly I:C or treated with 200 U/ml IFNβ and harvested at 24 h p.t. (p)eEF2 (T56) and ISG15 were detected by WB. Western blots are representative of two independent replicates.

Article Snippet: Primary antibodies used were rabbit antisera against CHIKV nsP1, CHIKV nsP2 helicase, CHIKV nsP2 protease, CHIKV nsP3 and CHIKV nsP4 [ ], CHIKV capsid protein, SFV capsid protein (all kind gifts from prof. Andres Merits, University of Tartu, Estonia), rabbit polyclonal against eEF2 #2332, mouse monoclonal against STAT-1 (9H2) #9176, mouse monoclonal against Rpb1 CTD (48H) #2629 (all three Cell Signaling Technology), mouse monoclonal against β-actin #A5316; mouse monoclonal FLAG M2 #F1804 (both Sigma), goat polyclonal against cyclophilin B (C15) #sc-20361, mouse monoclonal against ISG15 (F-9) # sc-166755 (both Santa Cruz), rabbit polyclonal against Renilla Luciferase #GTX125851 (GeneTex) and an in-house raised rabbit polyclonal serum a-GFP.

Techniques: Transfection, Control, Infection, Western Blot

Journal: Viruses

Article Title: Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner

doi: 10.3390/v14061327

Figure Lengend Snippet: Schematic overview of used plasmids and verification of nsP2 expression in transfected C6/36 cells. ( A ). Trans-replicase plasmid for expression of alphavirus ns-polyprotein. Ubi—full-length Aedes aegypti polyubiquitin promoter; UL—transcribed leader of polyubiquitin gene containing naturally occurring intron; SV40Ter—SV40 late polyadenylation region. ( B ) Constructs expressing template RNAs for trans-replicases. AlbPolI—truncated (−250 to −1) promoter for Aedes albopictus RNA polymerase I; AlbTer—tentative terminator for Aedes albopictus RNA polymerase I. The 5′ and 3′ UTRs and SG promoter are from CHIKV, SINV, SFV, RRV, MAYV, EILV, VEEV or EEEV; nsP1N—region encoding for the N-terminal region of nsP1; HDV RZ—antisense strand ribozyme of hepatitis delta virus. ( C ). Constructs expressing nsP2 of CHIKV or SINV. nsP1*—region encoding for 10 C-terminal amino acid residues of nsP1; DmHSP70Ter—transcription terminator of Drosophila melanogaster hsp70 gene. ( A – C ). The vector backbones are not shown; drawings are not in scale. ( D ). C6/36 cells were transfected with pPubi-CHIKV-nsP2 (left) or pPubi-SINV-nsP2 (right). Cells were harvested at 12, 18, 24, 36 or 48 hpt and lysed in 1× Laemmli buffer. Proteins were separated using SDS-PAGE in 10% gels and transferred to PVDF membranes. nsP2 proteins were detected using anti-CHIKV and anti-SINV nsP2 antibodies, and β-actin was detected as the loading control. ( E ) C6/36 cells were transfected with (left panel) pPubi-CHIKV-nsP2 (WT), pPubi-CHIKV-nsP2 CA , pPubi-CHIKV-nsP2 EV , pPubi-CHIKV-nsP2 YA+EV , pPubi-CHIKV-nsP2 ALT/ERR , pPubi-CHIKV-nsP2 KR/DD ; (right panel) pPubi-SINV-nsP2 (WT), pPubi-SINV-nsP2 CA , pPubi-SINV-nsP2 ND , pPubi-SINV-nsP2 ND+PQ , pPubi-SINV-nsP2 PQ or pPubi-SINV-nsP2 KR/DD . Cells were harvested at 48 hpt and analyzed as described for ( D ).

Article Snippet: Cells were harvested at 12, 18, 24, 36 and 48 h post transfection (hpt), lysed with 1× Laemmli buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 100 mM dithiothreitol, and 0.1% bromophenol blue) and boiled for 10 min. Proteins were separated by SDS-PAGE in 10% gels, transferred to polyvinylidene difluoride (PVDF) membrane followed by staining with primary anti-CHIKV nsP2 (in-house, raised against recombinant protein corresponding to residues 1–470 of nsP2 of CHIKV) or anti-SINV nsP2 (in-house, raised against full-length recombinant nsP2 of SINV) antibodies and antibody against β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Virus, SDS Page, Control