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• W4306732182 abstract "Article18 October 2022Open Access Source DataTransparent process DDX60 selectively reduces translation off viral type II internal ribosome entry sites Mohammad Sadic Mohammad Sadic orcid.org/0000-0002-4728-8908 NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Methodology, Writing - original draft Search for more papers by this author William M Schneider William M Schneider The Rockefeller University, New York, NY, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Olga Katsara Olga Katsara NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Gisselle N Medina Gisselle N Medina Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA National Bio and Agro-Defense Facility (NBAF), ARS, USDA, Manhattan, KS, USA Contribution: Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Ashley Fisher Ashley Fisher NYU Grossman School of Medicine, New York, NY, USA Contribution: Formal analysis, ​Investigation, Writing - review & editing Search for more papers by this author Aishwarya Mogulothu Aishwarya Mogulothu Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT, USA Contribution: Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Yingpu Yu Yingpu Yu The Rockefeller University, New York, NY, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Meigang Gu Meigang Gu The Rockefeller University, New York, NY, USA Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Teresa de los Santos Teresa de los Santos orcid.org/0000-0003-3075-4316 Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA Contribution: Conceptualization, Supervision, Funding acquisition, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Robert J Schneider Robert J Schneider orcid.org/0000-0001-5807-5564 NYU Grossman School of Medicine, New York, NY, USA Contribution: Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Meike Dittmann Corresponding Author Meike Dittmann [email protected] orcid.org/0000-0002-1741-7916 NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft, Project administration Search for more papers by this author Mohammad Sadic Mohammad Sadic orcid.org/0000-0002-4728-8908 NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Methodology, Writing - original draft Search for more papers by this author William M Schneider William M Schneider The Rockefeller University, New York, NY, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Olga Katsara Olga Katsara NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Gisselle N Medina Gisselle N Medina Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA National Bio and Agro-Defense Facility (NBAF), ARS, USDA, Manhattan, KS, USA Contribution: Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Ashley Fisher Ashley Fisher NYU Grossman School of Medicine, New York, NY, USA Contribution: Formal analysis, ​Investigation, Writing - review & editing Search for more papers by this author Aishwarya Mogulothu Aishwarya Mogulothu Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT, USA Contribution: Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Yingpu Yu Yingpu Yu The Rockefeller University, New York, NY, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Meigang Gu Meigang Gu The Rockefeller University, New York, NY, USA Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Teresa de los Santos Teresa de los Santos orcid.org/0000-0003-3075-4316 Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA Contribution: Conceptualization, Supervision, Funding acquisition, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Robert J Schneider Robert J Schneider orcid.org/0000-0001-5807-5564 NYU Grossman School of Medicine, New York, NY, USA Contribution: Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Meike Dittmann Corresponding Author Meike Dittmann [email protected] orcid.org/0000-0002-1741-7916 NYU Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft, Project administration Search for more papers by this author Author Information Mohammad Sadic1, William M Schneider2, Olga Katsara1, Gisselle N Medina3,4, Ashley Fisher1, Aishwarya Mogulothu3,5, Yingpu Yu2, Meigang Gu2, Teresa Santos3, Robert J Schneider1 and Meike Dittmann *,1 1NYU Grossman School of Medicine, New York, NY, USA 2The Rockefeller University, New York, NY, USA 3Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA 4National Bio and Agro-Defense Facility (NBAF), ARS, USDA, Manhattan, KS, USA 5Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT, USA *Corresponding author. Tel: +1 (646) 501 4642; E-mail: [email protected] EMBO Reports (2022)23:e55218https://doi.org/10.15252/embr.202255218 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5′ cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5′ capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements. Synopsis Translational shutdown is an effective countermeasure to viral infections, albeit at the cost of the host cell's protein synthesis. DDX60 selectively reduces translation off viral type II internal ribosome entry sites, leaving cap-mediated translation intact. DDX60 is expressed upon interferon-beta stimulation in primary cells and cell lines. DDX60 selectively decreases translation from type II IRESs of EMCV and FMDV, but not other IRES types or 5′ caps. DDX60 reduces replication of type II IRES-containing viruses. DDX60 modulates the amount of translating ribosomes on viral type II IRES mRNAs, but not 5′-capped mRNA. Introduction During viral infection, competition ensues between viruses and their host cells to control the protein synthesis machinery. To initiate mRNA translation in eukaryotes, a covalent m7GpppG 5′ cap structure on host messenger RNAs (mRNAs) enables the recruitment of a translation initiation factor complex that recruits the 40S ribosome subunit (Jackson et al, 2010; Merrick & Pavitt, 2018). The cap structure is recognized by eukaryotic initiation factor (eIF) protein eIF4E, which forms a complex with the scaffold protein eIF4G. Interaction between eIF4G and eIF3 then assembles a 43S preinitiation complex consisting of a 40S ribosomal subunit bound to eIF3, eIF1, eIF1A, and a ternary complex of GTP bound eIF2 and initiator Met-tRNAiMet, among other factors. This ribosomal complex scans the mRNA in the 5′ to 3′ direction. During scanning, eIF4G-bound RNA helicase eIF4A and activator protein eIF4B unwind RNA secondary structures in the mRNA until the start codon is identified. Subsequently, eIF1, eIF1A, and eIF5 assist in positioning the 40S ribosomal subunit such that the initiator Met-tRNAiMet is at the peptidyl (P)-site of the 40S ribosomal subunit. eIF5 then promotes GTP hydrolysis by eIF2, releasing eIF2 and eIF5 for subsequent cycles of translation initiation. Lastly, the GTPase eIF5B assists in joining the 60S ribosomal subunit to the 40S subunit to form an 80S initiation complex. The poly A-binding protein (PABP) interacts with the 3′-poly(A) tail and eIF4G, further promoting mRNA translation initiation. Viruses evolved diverse mechanisms to compete with and dominate the host protein synthesis machinery, much of it centered on maintaining cap-dependent mRNA translation or bypassing it completely. Some viruses utilize eukaryotic capping enzymes to add a m7Gppp 5′ cap to their mRNAs, while others encode their own viral capping enzymes to add a 5′ cap that functionally mimics a eukaryotic 5′ cap. A number of viruses naturally have uncapped mRNAs but can “snatch” capped 5′ terminal fragments from host mRNAs (Plotch et al, 1981; Decroly et al, 2012), while others covalently link their uncapped mRNA to a 5′ terminal protein that mechanistically acts like a 5′ cap to recruit translation initiation complex proteins (Goodfellow et al, 2005). Others directly recruit ribosomes to the mRNA and bypass the requirement for 5′ cap recognition using structured RNA elements called IRESs (Jang et al, 1988; Pelletier & Sonenberg, 1988; Stern-Ginossar et al, 2019). IRESs assemble the translation initiation apparatus either upstream of or at an initiation codon, independently of a 5′ cap structure (Fraser & Doudna, 2007; Lozano & Martínez-Salas, 2015; Lee et al, 2017; Yamamoto et al, 2017; Martinez-Salas et al, 2018). During recruitment of the translation initiation apparatus, often with structural support from host IRES-transacting factor proteins (ITAFs), IRESs interact with a defined set of eIFs that assist in the recruitment of the 40S ribosomal subunit (Walter et al, 1999; Andreev et al, 2012; Martinez-Salas et al, 2018). Several subtypes of viral IRESs exist, based on their unique RNA structures, differential requirements for eIFs and ITAFs, and start codon recognition mechanisms (Kaminski et al, 1990; Belsham, 1992; Hunt et al, 1993; Ohlmann & Jackson, 1999; Beales et al, 2003; Lozano & Martínez-Salas, 2015; Lee et al, 2017; Yamamoto et al, 2017; Martinez-Salas et al, 2018). Type I IRESs found in picornaviruses such as poliovirus and enterovirus 71 (EV71) employ a ribosomal scanning mechanism for start codon recognition with the assistance of eIFs 1A, 2, 3, 4A, 4B, central domain of 4G, and ITAFs PCBP1/2, PTB, hnRNPA1, and other proteins (Pelletier & Sonenberg, 1988; Thompson & Sarnow, 2003; Sweeney et al, 2014; Martinez-Salas et al, 2018; Stern-Ginossar et al, 2019). Type II IRESs, also found in picornaviruses such as EMCV and FMDV, direct ribosome entry at an AUG in the 3′ end of the IRES, or one located a short distance away with the assistance of the eIFs 2, 3, 4A, 4B, central domain of 4G, the ITAF PTB for EMCV and PTB plus Ebp1/ITAF45 for FMDV with additional assistance from eIFs 1 and 1A for translation initiation at a second downstream AUG only in FMDV (Jang et al, 1988; Belsham, 1992; Pestova et al, 1996a, 1996b; Andreev et al, 2007; Martinez-Salas et al, 2018; Stern-Ginossar et al, 2019). The type III IRES, found uniquely in the picornavirus hepatitis A virus (HAV), requires an intact heterotrimeric complex of eIF4E, eIF4G, and eIF4A (Avanzino et al, 2017). This is in contrast to all other IRES types, which initiate translation independently of eIF4E (Lozano & Martínez-Salas, 2015; Martinez-Salas et al, 2018; Stern-Ginossar et al, 2019). Type IV IRESs, found in some picornaviruses but originally discovered in flaviviruses such as HCV and bovine viral diarrhea virus (BVDV), recruit the 40S ribosomal subunit close to the start codon without the use of eIFs, and subsequently recruit GTP bound eIF2, initiator Met-tRNAiMet and eIF3 to facilitate 60S ribosomal subunit joining (Pestova et al, 1998; Fraser & Doudna, 2007). Type V IRESs, found in different genera of picornaviruses, have a three-dimensional IRES organization resembling a hybrid of type I and type II IRESs and, in some members, have a requirement for the DExH-box protein DHX29 for efficient translation initiation (Yu et al, 2011; Sweeney et al, 2012; Arhab et al, 2020). Finally, IRESs found in dicistroviruses such as cricket paralysis virus (CrPV), require no eIFs or ITAFs for 40S and 60S ribosomal subunit recruitment, and initiate translation at a noncanonical start codon from the A-site of the ribosome (Wilson et al, 2000a, 2000b; Jan & Sarnow, 2002). While viruses must compete for the host's translation machinery, cells respond by enacting different mechanisms to block overall protein synthesis, and in some cases specifically inhibit translation of viral mRNAs. Interferons (IFNs), produced by cells upon viral infection, trigger the expression of a variety of interferon-stimulated genes (ISGs) that have diverse antiviral functions, some of which target translation (Schneider et al, 2014; Hoffmann et al, 2015; Hopfner & Hornung, 2020; Ficarelli et al, 2021; Li & Wu, 2021). Among them is the double-stranded RNA activated protein kinase PKR, which phosphorylates the eIF2 α-subunit to impair GDP to GTP exchange by the eIF2B GTP exchange factor, thus inhibiting global protein synthesis (Stern-Ginossar et al, 2019). Another mechanism involves the activation of oligoadenylate synthase (OAS), which synthesizes short oligoadenylate polymers to stimulate RNase L to indiscriminately degrade ribosomal RNA, as well as viral and certain host mRNAs (Burke et al, 2019). The interferon-induced protein with tetratricopeptide repeats (IFIT) family members and interferon-induced transmembrane protein (IFITM) family members bind specific eIFs to restrict global protein synthesis or recognize structures absent in viral 5′ caps such as 2′O-methylation (Diamond & Farzan, 2013; Schoggins, 2019). Finally, the zinc finger antiviral protein (ZAP) triggers viral RNA degradation and limits interactions between certain eIFs (Schoggins, 2019). While these mechanisms limit translation of viral mRNAs, and in consequence viral replication, they come at the cost of downregulating host protein synthesis. Here, we describe the ISG DExD/H-box helicase 60 (DDX60), an RNA helicase that can inhibit viral type II IRES-driven translation while leaving host 5′ cap-driven mRNA translation intact. The antiviral function of DDX60 was initially discovered in a screen for antiviral ISGs, where it was shown to inhibit a reporter HCV (Schoggins et al, 2011). Later studies probing for the antiviral mechanism of DDX60 generated conflicting data. One group found DDX60 to act as a sentinel for the viral RNA recognition receptor retinoic acid-inducible gene-I (RIG-I) (Miyashita et al, 2011), and to promote degradation of viral RNA independently of RIG-I (Oshiumi et al, 2015). However, another group presented evidence against a role for DDX60 as a sentinel for RIG-I (Goubau et al, 2015), suggesting instead that DDX60 may enact a specific antiviral mechanism for one or a small group of viruses. Here, we aimed to clarify the mechanism for DDX60 antiviral activity. We first show that upon IFN-ß treatment, DDX60 has prolonged and delayed expression dynamics at the mRNA and protein levels, respectively. Through mutagenesis and antiviral assays, we demonstrate that N- and C-terminal regions alongside predicted helicase and ATP binding motifs in DDX60 are important for its antiviral activity. We next use comparative antiviral experiments to show that DDX60 targets type II IRESs found in a group of viruses. We generated in vitro transcribed mRNA reporters to demonstrate that DDX60 specifically inhibits the type II family of IRESs and further show that the type II IRES is sufficient to confer virus inhibition by DDX60. Lastly, we found that DDX60 reduces type II IRES activity by modulating translating ribosome activity both on type II IRES-driven firefly luciferase (Fluc) mRNA and on viral mRNA during viral infection. Importantly, DDX60 shows neither an effect on the overall translation status of the cell nor an effect on the translation of in vitro synthesized 5′ capped Fluc mRNA. Our work suggests that DDX60 acts as an ISG that inhibits type II IRES-mediated mRNA translation and can discriminate between 5′ cap-independent and -dependent translation mechanisms. Studying the anti-IRES mechanism of DDX60 could lead to novel strategies for targeting specific virus translation mechanisms while leaving host translation intact. Results DDX60 displays dynamics of a type I ISG at the mRNA and protein level in multiple cell lines Gene expression of DDX60 at the mRNA level has been shown to be triggered by various stimuli in human cell lines and mouse tissue, including poly(I:C), type I IFN, and virus infections (Miyashita et al, 2011; Goubau et al, 2015). We analyzed the dynamics of DDX60 mRNA in four different human cell lines upon treatment with IFN-ß and compared it with interferon regulatory factor 1 (IRF1), an IFN-stimulated transcription factor with broad antiviral function and known expression dynamics (Schoggins et al, 2011; Forero et al, 2019; Feng et al, 2021). We treated three epithelial cell lines (HEK293T, human embryonic kidney; A549, lung adenocarcinoma; and HeLa, cervical adenocarcinoma) as well as primary human foreskin fibroblasts (HFF) with IFN-ß and analyzed mRNA expression using RT–qPCR. In all cell types tested, both IRF1 and DDX60 expressions increased upon IFN-ß stimulation (Fig EV1A–D). DDX60 mRNA levels reached higher peaks than those of IRF1, most notably in HEK293T cells (Fig EV1A). While IRF1 mRNA levels returned to baseline (0 h values) at 48-h poststimulation, DDX60 mRNA levels remained above baseline in all cell types except primary HFF (Fig EV1C). Overall, our IFN stimulation and mRNA analysis demonstrate that DDX60 displays general characteristics of an ISG. 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 consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Effect of DDX60 on replication of bicistronic or monocistronic infectious 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. Data information: 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 n = 3 biological replicates; ns —not significant, *P < 0.05, ****P < 0.0001, ns, nonsignificant using ANOVA and Dunnett's multiple comparison test against Fluc. Download figure Download PowerPoint Click here to expand this figure. 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). Representative images of Western blots shown. Source data are available online for this figure. Download figure Download PowerPoint We next sought to determine whether protein levels of endogenous DDX60 also change with IFN treatment. We thus analyzed DDX60 protein dynamics in our four IFN-ß treated cell lines by Western blot. Expectedly, DDX60 protein production increased approximately three- to 10-fold upon IFN-ß treatment compared with little to no expression at baseline (Fig EV1E–H). However, compared with DDX60 mRNA levels, DDX60 protein levels showed delayed expression dynamics in all cell lines, peaking at 24 or even 48-h post-IFN-ß treatment (Fig EV1E–H). Together, our findings show that DDX60 is an ISG with very low to undetectable steady-state levels that then peak at both the RNA and the protein levels after IFN treatment in various human cell culture systems. DDX60 decreases replication of a bicistronic reporter HCV carrying an EMCV IRES Previous studies showed that DDX60 inhibits replication of a bicistronic reporter HCV (Schoggins et al, 2011; Oshiumi et al, 2015). To begin determining how DDX60 inhibits HCV, we used InterPro and published literature (Pause & Sonenberg, 1992; Schwer & Meszaros, 2000; Pyle, 2008; Umate et al, 2011; Johnson & Jackson, 2013), to identify putative functional domains and motifs (Fig 1A). We then introduced N-terminal and C-terminal truncations and single point mutations in residues predicted to confer ATP binding/hydrolysis and helicase activity to DDX60. All mutants were ectopically expressed to equal levels as shown by Western blot (Fig 1B). We next used a previously developed virus inhibition assay to assess the antiviral capacity of the different DDX60 constructs (Schoggins et al, 2011). Briefly, we transfected Huh-7 cells with wild-type or mutant DDX60 plasmid containing a red fluorescent protein (RFP) marker to monitor transfection efficiency. Firefly luciferease (Fluc) served as negative control, and IRF1 as positive control. We then infected transfected cells with a yellow fluorescent protein (Ypet)-expressing HCV at a dose yielding approximately 50% infected (Ypet+) cells in Fluc-expressing cells, as previously determined by flow cytometry-based infectivity assays (Jones et al, 2010; Schoggins et al, 2011). The percentage of Ypet-positive (infected) cells within the RFP-positive (transfected) population at 72-h postinfection was assayed by flow cytometry. Wild-type DDX60 reduced the percentage of HCV-infected cells by approximately 30% relative to Fluc-negative control. The predicted ATP binding residues and helicase motif are required for full DDX60 antiviral activity (Fig 1C, K791, E890, and R1328). Interestingly, deletions in either N- and C-terminal extensions were also required for efficient antiviral activity (Fig 1C, Δ1–428, Δ1–556, and Δ1,402–1,712). Although DDX60's extensions are void of characterized function, in other RNA helicases, these extensions allow for protein–protein interactions during RNA substrate recognition (Wang et al, 2005; Thoms et al, 2015; Lingaraju et al, 2019). To characterize the role of essential DDX60 residues and domains in biochemical detail, we next attempted to purify recombinant DDX60 via multiple tagging and protein expression strategies, including yeast and baculovirus systems. However, purification of full-length DDX60 was unsuccessful due to protein aggregation, resulting in low yields. Attempts to solubilize the protein with different salt and glycerol concentrations were unsuccessful. Work by others had characterized purified truncated versions of DDX60 (Miyashita et al, 2011); however, this was not an option for our study, as both N- and C-terminal regions are required for antiviral function (Fig 1C). We therefore speculate but cannot definitively assign DDX60 residues to have specific enzymatic activities. From here on, we use the minimal DEVH helicase motif mutant, DDX60 E890A, as a loss-of-function control in cellular assays. In these initial experiments, we used the same infectious reporter HCV as a screen for ISGs that initially identified DDX60 to be antiviral (Schoggins et al, 2011). This infectious reporter HCV is bicistronic, as translation of the Ypet reporter is driven by the HCV IRES and translation of the HCV polyprotein is subsequently driven by an inserted EMCV IRES (Fig 1D, left schematic) (Jones et al, 2007, 2010; Schoggins et al, 2011). To validate our findings and rule out artifactual observations due to the use of a reporter virus encoding a foreign viral element, we employed our flow cytometry-based virus inhibition assay using a infectious monocistronic reporter HCV where translation is initiated by the endogenous HCV IRES and the Ypet is translated as a part of the HCV polyprotein and subsequently excised due to flanking NS5AB cleavage sites (Fig 1D; Jones et al, 2007; Horwitz et al, 2013). While DDX60 successfully downregulated replication of the infectious bicistronic reporter HCV as observed previously (Fig 1C and D, left panel; Schoggins et al, 2011), DDX60 failed to downregulate the infectious monocistronic reporter HCV (Fig 1D, right). Additionally, the EMCV IRES-driven RFP encoded in our plasmid constructs used in Fig 1B and C showed expression levels that mimicked the expression of our infectious bicistronic Ypet reporter HCV, but 5′ cap-driven proteins such as ß-actin, GAPDH, or our DDX60 transgenes of interest did not (Fig 1B). Flow cytometry revealed that reductions in EMCV IRES-driven RFP was a result of reduced mean fluorescent intensity in RFP-positive cells and not the percentage of RFP-positive cells, thereby still enabling gating on cells that express DDX60. As the main distinguishing feature between the two infectious reporter HCVs is the EMCV IRES, we hypothesized that DDX60's antiviral action may be against the EMCV IRES, and not a component of HCV per se. DDX60 downregulates plasmid- and in vitro transcribed RNA-based reporters translationally driven by type II internal ribosome entry sites To interrogate DDX60's IRES specificity, we next screened for DDX60's ability to inhibit representatives of type I (poliovirus), type II (EMCV and FMDV), or type IV IRESs (EV71); the IRES of CrPV, HAV, and type V picornavirus IRESs were excluded because of low CrPV IRES activity in mammalian cells (Carter et al, 2008) and lack of the tools discussed below to study the HAV and type V IRESs. First, we used a plasmid-based dual luciferase reporter system, where the transfected plasmid is transcribed and the transcript is canonically 5′-capped in the nucleus by the host cell machinery (Pelletier & Sonenberg, 1988; Honda et al, 2000; Jackson, 2013). In the resulting single bicistronic mRNA, translation of the first cistron, Renilla luciferase (Rluc), is initiated by a canonical 5′ cap mechanism, and translation of the second cistron, Fluc, is initiated by an IRES mechanism." @default.
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• W4306732182 title "<scp>DDX60</scp> selectively reduces translation off viral type <scp>II</scp> internal ribosome entry sites" @default.
• W4306732182 cites W118864685 @default.
• W4306732182 cites W144266797 @default.
• W4306732182 cites W146972387 @default.
• W4306732182 cites W1496462301 @default.
• W4306732182 cites W1503339982 @default.
• W4306732182 cites W1525424682 @default.
• W4306732182 cites W1595552461 @default.
• W4306732182 cites W1803296362 @default.
• W4306732182 cites W1807212217 @default.
• W4306732182 cites W1918704078 @default.
• W4306732182 cites W1920555941 @default.
• W4306732182 cites W1938043974 @default.
• W4306732182 cites W1945599304 @default.
• W4306732182 cites W1968676264 @default.
• W4306732182 cites W1969699638 @default.
• W4306732182 cites W1970949525 @default.
• W4306732182 cites W1973243219 @default.
• W4306732182 cites W1976378471 @default.
• W4306732182 cites W1978525119 @default.
• W4306732182 cites W1980892321 @default.
• W4306732182 cites W1981848417 @default.
• W4306732182 cites W1981957524 @default.
• W4306732182 cites W1985638466 @default.
• W4306732182 cites W1986004822 @default.
• W4306732182 cites W1987433368 @default.
• W4306732182 cites W1993844923 @default.
• W4306732182 cites W2000516676 @default.
• W4306732182 cites W2001803866 @default.
• W4306732182 cites W2008410413 @default.
• W4306732182 cites W2010829256 @default.
• W4306732182 cites W2019458198 @default.
• W4306732182 cites W2021812639 @default.
• W4306732182 cites W2022687771 @default.
• W4306732182 cites W2023590131 @default.
• W4306732182 cites W2035792284 @default.
• W4306732182 cites W2036870775 @default.
• W4306732182 cites W2038068681 @default.
• W4306732182 cites W2042077321 @default.
• W4306732182 cites W2042598793 @default.
• W4306732182 cites W2048731469 @default.
• W4306732182 cites W2053003053 @default.
• W4306732182 cites W2057444596 @default.
• W4306732182 cites W2057990302 @default.
• W4306732182 cites W2065533930 @default.
• W4306732182 cites W2069823852 @default.
• W4306732182 cites W2076198632 @default.
• W4306732182 cites W2078646756 @default.
• W4306732182 cites W2079836122 @default.
• W4306732182 cites W2084102697 @default.
• W4306732182 cites W2085297050 @default.
• W4306732182 cites W2085918490 @default.
• W4306732182 cites W2088788596 @default.
• W4306732182 cites W2092019188 @default.
• W4306732182 cites W2094443489 @default.
• W4306732182 cites W2097354001 @default.
• W4306732182 cites W2104573851 @default.
• W4306732182 cites W2104947461 @default.
• W4306732182 cites W2106120955 @default.
• W4306732182 cites W2107277218 @default.
• W4306732182 cites W2109009498 @default.
• W4306732182 cites W2112459919 @default.
• W4306732182 cites W2114084606 @default.
• W4306732182 cites W2122867782 @default.
• W4306732182 cites W2124861729 @default.
• W4306732182 cites W2129195166 @default.
• W4306732182 cites W2133402546 @default.
• W4306732182 cites W2133856510 @default.
• W4306732182 cites W2136563039 @default.
• W4306732182 cites W2138970689 @default.
• W4306732182 cites W2139552435 @default.
• W4306732182 cites W2140197300 @default.
• W4306732182 cites W2142550741 @default.
• W4306732182 cites W2147126871 @default.
• W4306732182 cites W2148824459 @default.
• W4306732182 cites W2150368001 @default.
• W4306732182 cites W2150408070 @default.
• W4306732182 cites W2151216635 @default.
• W4306732182 cites W2151581088 @default.
• W4306732182 cites W2163822189 @default.
• W4306732182 cites W2166288313 @default.
• W4306732182 cites W2192080449 @default.
• W4306732182 cites W2219768227 @default.
• W4306732182 cites W230924981 @default.
• W4306732182 cites W2412727661 @default.

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