Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A, E) HEK293T, (B, F) A549, (C, G) HFF1, or (D, H) HeLa cells treated with either 0.1% BSA (carrier control) or 500 U/mL of interferon-ß for 0, 6, 12, 24, or 48 hours. Cells were harvested for mRNA analysis (A – D) using RT-qPCR or protein analysis (E–H) using western blot. Panel E includes one HEK293T sample transfected with DDX60 wild type run on the same gel as 24- and 48-hour time points to show relative DDX60 protein levels in interferon-ß treated versus transfected cells. All western blots for 0-, 6-, and 12-hour time points were run on the same gel but are separated by cell line for visualization purposes. All western blots for the 24- and 48-hour time points were run on the same gel but are separated by cell line for visualization purposes. Data is representative of at least 2 biological replicates per time point from experiments performed on different days.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Quantitative RT-PCR, Western Blot, Transfection

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) Schematic of DDX60 protein with putative functional domains. Helicase ATP binding type I domain (amino acids 785 - 921) and C-terminal helicase domain (amino acids 1291-1331) are shown as larger boxes in linear DDX60 schematic. Amino acids are numbered below. Putative functional motifs (I, II, III, and VI) are annotated. The function of amino acids in bold as well as N and C-terminal regions were interrogated in antiviral assays. ( B ) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification by densitometry are shown below. (C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and challenged with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. (D) Effect of DDX60 on replication of bicistronic or monocistronic reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. For (C) and (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least 3 biological replicates from experiments performed on separate days; ns = not significant, *p < 0.05, ****p < 0.0001 using ANOVA and Dunnett’s multiple comparison test against Fluc.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Functional Assay, Binding Assay, Expressing, Transfection, Western Blot, Mutagenesis, Plasmid Preparation, Luciferase, Infection

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5’ cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5’ cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5’ cap-driven Rluc units given either: equal translation of 5’ cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5’ cap-driven Rluc (bottom). (B) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5’ cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Raw data is shown in . Data shows mean ± SD for at least 3 biological replicates from experiments performed on separate days; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA and Dunnett’s multiple comparison test against GFP.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Plasmid Preparation, Luciferase, Transfection, Negative Control, Positive Control, Mutagenesis, Lysis, In Vitro, Construct

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) HEK293T cells transfected with GFP (negative control), DDX60 wt, or different DDX60 mutants described in and subsequently transfected with in vitro transcribed 5’ cap- or different IRES-driven Fluc mRNA constructs. Mean ± SD luciferase units from lysed cells are plotted. Dotted line shows mean luciferase units from Fluc reporters transfected into GFP transfected cells. Conditions below dotted line show inhibition of Fluc production and conditions above the dotted line show enhancement of Fluc production. Data is an average of 3 biological replicates from experiments performed on separate days. (B) HEK293T cells transfected as in . Luciferase units after cell lysis is plotted. Data shows mean ± SD for 3 technical replicates from one representative biological replicate for data shown in .

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Transfection, Negative Control, In Vitro, Construct, Luciferase, Inhibition, Lysis

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5’ cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 24 hours later. ( A ) Abundance of luciferase reporter mRNAs assayed using RT-qPCR. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. Mean ± SD luciferase units are plotted for at least 3 biological replicates from experiments performed on separate days; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Transfection, Negative Control, In Vitro, Construct, Luciferase, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: HEK293T cells stably expressing a type I IFNsensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Cells were then treated with either PBS (negative control), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (A) or western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (B) . Mean ± SD relative luciferase units (RLU) are plotted in (A). Data is representative of at least 3 biological replicates from experiments performed on separate days.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Negative Control, Positive Control, Luciferase, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) RNA-protein binding assays using biotin labeled RNA probes and streptavidin coated beads. DDX60 wt expressing HEK293T cell lysates were combined with either biotin labeled or unlabeled (negative control) IRES RNA sequences (IRESs from poliovirus, EV71, EMCV, or HCV) or 5’ capped Fluc RNA sequence (negative control). After allowing for protein and RNA binding, streptavidin coated beads were used to pull down RNA. After washing, RNA bound proteins were visualized using western blot. Input or DDX60 overexpression (O.E) lanes show DDX60 and other interrogated RNA binding proteins before incubation of cell lysates with interrogated RNAs. Comparisons for enrichment of RNA binding is made by comparing unlabeled and biotin labeled lanes. Visually equivalent band intensities between unlabeled and biotin labeled lanes signify non-specific binding between an RNA and interrogated protein. Lane labeled beads represents amount of protein that binds to streptavidin coated beads in the absence of any interrogated RNA. RNA binding proteins interrogated include Polypyrimidine Tract Binding Protein 1 (PTPB1) (positive control for binding type I and type II IRESs specifically), Far upstream element binding protein 1 (FBP1) (binds EV71 IRES), and DDX60. Data is representative of at least 2 biological replicates from experiments performed on separate days. (B) Protein-RNA binding assays using immunoprecipitated DDX60 and RT-qPCR for Fluc mRNAs. HEK293T cells transfected with DDX60 wt or left untransfected were transfected with either 5’ cap- or EMCV IRES-driven Fluc mRNA. Cell lysates were then subjected to immunoprecipitation using either DDX60 targeting antibody or isotype control IgG. Bound mRNA was detected using RT-qPCR against Fluc. Efficiency of immunoprecipitation is visualized using western blot against DDX60. Bound mRNA is quantified as a percent of input RNA post-immunoprecipitation with either IgG or anti-DDX60 antibody in either DDX60 expressing or untransfected cells. Data is representative of 2 separate immunoprecipitations.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Protein Binding, Labeling, Expressing, Negative Control, Sequencing, RNA Binding Assay, Western Blot, Over Expression, Incubation, Binding Assay, Positive Control, Immunoprecipitation, Quantitative RT-PCR, Transfection

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) Polysome profile of total RNA upon DDX60 overexpression. Untransfected or DDX60 wt expressing HEK293T cells were treated with 100 μg/mL of cycloheximide for 15 minutes to arrest polysomes before being trypsinized and frozen down. All harvested cells per condition were then subjected to polysome profiling by lysing cells in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. The centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance. Data is an average of two replicate plate of cells per condition. (B) Polysome profile of total RNA post transfection of DDX60 and Fluc mRNA reporters. DDX60 wt or DDX60 E890A (negative control) expressing HEK293T cells transfected with in vitro transcribed 5’ cap (left) or EMCV IRES (right) driven Fluc mRNA constructs. Duplicate samples of DDX60 E890A and 5’ cap- or EMCV IRES-driven Fluc mRNA transfected cells were treated with 200 μM puromycin as positive controls for decrease in polysomes. Cells were then treated with 100 μg/mL of cycloheximide for 15 minutes to arrest polysomes before being trypsinized and frozen down. All harvested cells per condition were then subjected to polysome profiling by lysing cells in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. The centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance. Data is representative of 2 biological replicates from experiments conducted on separate days.

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Over Expression, Expressing, Transfection, Negative Control, In Vitro, Construct

Journal: bioRxiv

Article Title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

doi: 10.1101/2022.02.08.479557

Figure Lengend Snippet: (A) Schematic of polysome profiling strategy. HEK293T cells were transfected with DDX60 wt or DDX60 E890A (negative control). 48 hours post transfection, cells were transfected with in vitro transcribed 5’ cap- or EMCV IRES-driven Fluc mRNA constructs for 24 hours. Duplicate samples were treated with 200 μM puromycin for 20 minutes as positive controls for decrease in polysomes. Cells were treated with 100 μg/mL of cycloheximide for 15 minutes to arrest polysomes and subjected to polysome profiling by ultracentrifugation through 15%-50% sucrose gradients. Amount of Fluc reporter mRNA from polysome fractions was determined by RT-qPCR. (B) Effect of DDX60 on polysome occupancy of 5’ cap (left) or EMCV IRES (right) driven Fluc mRNA. Shown is the mean percent of Fluc mRNA in each of the fractions from 2 biological replicates from experiments conducted on separate days. Full polysome profiles are shown in .

Article Snippet: HEK293T (CRL-3216), A549 (CCL-185), HFF1 (SCRC-1041), and H1 clone of HeLa (CRL-1958) cells were purchased from ATCC.

Techniques: Transfection, Negative Control, In Vitro, Construct, Quantitative RT-PCR