DDX60 selectively reduces translation off viral type II internal ribosome entry sites

Figures

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  Figure 1. Functional mapping of DDX60 antiviral domains and interrogation of anti‐HCV activity 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 1,291–1,331) are shown as larger boxes in linear DDX60 schematic. Amino acids are numbered below. Putative functional motifs (I, II, III, and VI) and mutations made are annotated. The amino acids in bold as well as N‐ and C‐terminal regions were interrogated in antiviral assays.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 relative to GAPDH from one representative blot are shown below.HCV antiviral assays with DDX60 wt or mutant panel. Huh‐7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc and negative control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV‐Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein…

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  Figure EV1. DDX60 mRNA and protein expression upon interferon treatment (A–H)(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 h. 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‐h time points to show relative DDX60 protein levels in interferon‐ß treated versus transfected cells. All western blots for 0‐, 6‐, and 12‐h 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‐h time points were run on the same gel but are separated by cell line for visualization purposes. Data information: Individual replicates and mean of mRNA fold‐change compared to unstimulated cells. For data points with at least n = 3 replicates *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, non‐significant (ns) using Mann–Whitney ranked test against carrier control values of same time point. For data points with n = 2 replicates statistics were not determined (nd).…

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  Figure 2. In cell reporter assay for reporters translationally driven by different internal ribosome entry sites Plasmid‐based dual luciferase bicistronic reporter assays. HEK293T cells cotransfected 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 (center). 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 (center), or greater translation of 5′ cap‐driven Rluc (bottom).RNA‐based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control),…

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  Figure EV2. Monocistronic RNA reporter assays for different internal ribosome entry sites and DDX60 mutant panel HEK293T cells transfected as in Fig 2B and luciferase intensity measured from cell lysates. Mean ± SD for n = 3 technical replicates from one representative biological replicate for data shown in Fig 2B; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ANOVA and Dunnett's multiple comparison test against GFP.HEK293T cells transfected with GFP (negative control), DDX60 wt, or different DDX60 mutants described in Fig 1 and subsequently transfected with in vitro transcribed 5′ cap‐ or different IRES‐driven Fluc mRNA constructs and luciferase intensity measured from cell lysates. Mean ± SD of n = 3 biological replicates; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ANOVA and Dunnett's multiple comparison test against GFP.

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  Figure 3. Viral replication assays for a panel of IRES‐containing viruses A–E(A, B, E) Multicycle infection assays with type I and type II IRES‐containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) poliovirus, (B) encephalomyocarditis virus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV‐IRES‐PV) at MOI 0.001. Supernatants were collected 24‐h postinfection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV‐IRES‐PV) and titers determined via plaque assay on HeLa cells. (C, D) Single‐cycle infection assays with type I and type II IRES‐containing viruses. BHK‐J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) bovine enterovirus‐1, or (D) foot and mouth disease virus at MOI 1. Supernatants were collected 5‐h postinfection (hpi) and titers determined via plaque assay on BHK‐21 clone 13 cells. Data information: Data show mean ± SEM percent infectious titers relative to Fluc from at least n = 3 biological replicates; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001,…

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  Figure EV3. Growth kinetics of different internal ribosome entry site containing viruses in the presence of DDX60 AGrowth kinetics of poliovirus, encephalomyocarditis virus (EMCV), or a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV. HeLa cells were infected with the three viruses at MOI 0.001 and supernatants from infected cultures were collected 0, 1, 8, 24, 32 or 48 hpi. Virus in supernatants was then titrated on HeLa cells. Mean of n = 2 biological replicates.BAssessment of exogenous DDX60 expression in HeLa cells. HeLa cells were transduced with lentivirus carrying either DDX60 wt, DDX60 E890A, IRF1, or Firefly luciferase (Fluc) together with a puromycin resistance gene and selected with puromycin. Cell lysates were analyzed using western blot for DDX60, Fluc, IRF1, and GAPDH (loading control) protein products.C–ETime course of antiviral assays with panel of IRES‐containing viruses. HeLa cells stably expressing Fluc (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A were infected with (C) poliovirus, (D) EMCV, or (E) a chimeric poliovirus at a MOI of 0.001. Supernatants from infected cultures were collected at either 0, 1, 8, 24, 32, or 48…

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  Figure 4. mRNA abundance for reporters translationally driven by different internal ribosome entry sites, and interferon‐stimulated response element activity in the presence of DDX60 A, BHEK293T 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 16 h later. (A) Abundance of luciferase reporter mRNAs assayed using RT–qPCR. Data are representative of at least three biological replicates from experiments performed on separate days. ns, not significant using one sample t‐test comparing mean fold changes to theoretical mean of 1. (B) IRES‐ or cap‐driven translation from the same samples assayed in parallel using luciferase assay.C, DHEK293T cells stably expressing a type I IFN sensitive 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. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells…

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  Figure EV4. Probing for a physical interaction between DDX60 and different IRES sequences 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 nonspecific 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 (PTBP1) (positive…

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  Figure EV5. Global translation levels under DDX60 overexpression 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 min 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 (254 nm). Data are an average of two replicate plate of cells per condition.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…

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  Figure 5. Polysome profile of type II IRES Fluc mRNA in the presence of DDX60 Schematic of polysome profiling strategy. HEK293T cells were transfected with DDX60 wt or DDX60 E890A (negative control). 48‐h post‐transfection, cells were transfected with in vitro transcribed 5′ cap‐ or EMCV IRES‐driven Fluc mRNA constructs. 16‐h post‐transfection, duplicate samples were treated with 200 μM puromycin for 20 min as positive controls for decrease in polysomes. Cells were treated with 100 μg/ml of cycloheximide for 15 min 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.Effect of DDX60 on 5′ cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes. Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n = 3 biological replicates. Full polysome profiles are shown in Fig EV5.

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  Figure 6. Polysome profile of wild type (type I IRES) poliovirus versus chimeric (type II IRES) poliovirus in the presence of DDX60 Schematic of polysome profiling strategy. HeLa cells stably expressing wild type DDX60 were infected with parental poliovirus (type I IRES) or chimeric poliovirus (type II IRES). 6 hpi cells were treated with 100 μg/ml of cycloheximide for 15 min to arrest polysomes and subjected to polysome profiling by ultracentrifugation through 15–50% sucrose gradients. Amount of poliovirus VP4 mRNA from polysome fractions was determined by RT‐qPCR.Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15–50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60‐transfected, EMCV‐IRES‐PV‐infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity…