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      An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication


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          Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5′ and 3′ untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5′-UTRs lack internal ribosomal entry site function. However, the 5′-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5′-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5′-UTR. The L 5′-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5′-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a “weak” Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10–100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target.

          Author Summary

          Filoviruses (Ebola and Marburg viruses) are emerging zoonotic pathogens that cause lethal hemorrhagic fever in humans and have the potential to be employed as bioterrorism agents. Currently, approved therapeutics to treat filovirus infections are not available and new treatment strategies could be facilitated by improved mechanistic insight into the virus replication cycle. Compared to other related viruses, filovirus messenger RNAs have unusually long 5′ untranslated regions (UTRs) with undefined functions. In the Zaire ebolavirus (EBOV) genome, four of its seven messenger RNAs have 5′-UTRs with a small upstream open reading frame (uORF). We found that a uORF present in the EBOV polymerase (L) 5′-UTR suppresses L protein production and established a reporter assay to demonstrate that this uORF maintains L translation following the induction of an innate immune response; a phenomenon observed with several uORF-containing cellular messenger RNAs. The presence of the uORF is important for optimal virus replication, because a mutant virus lacking the upstream reading frame replicates less efficiently than a wildtype virus, an attenuation which is more pronounced following the induction of cellular stress. These studies define a novel mechanism by which filovirus upstream open reading frames modulate virus protein translation in the face of an innate immune response and highlight their importance in filovirus replication.

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          Most cited references73

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          Coping with stress: eIF2 kinases and translational control.

          In response to environmental stresses, a family of protein kinases phosphorylate eIF2 (eukaryotic initiation factor 2) to alleviate cellular injury or alternatively induce apoptosis. Phosphorylation of eIF2 reduces global translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions. Accompanying this general protein synthesis control, eIF2 phosphorylation induces translation of specific mRNAs, such as that encoding the bZIP (basic leucine zipper) transcriptional regulator ATF4 (activating transcription factor 4). ATF4 also enhances the expression of additional transcription factors, ATF3 and CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 (growth arrest and DNA-damage-inducible protein), that assist in the regulation of genes involved in metabolism, the redox status of the cells and apoptosis. Reduced translation by eIF2 phosphorylation can also lead to activation of stress-related transcription factors, such as NF-kappaB (nuclear factor kappaB), by lowering the steady-state levels of short-lived regulatory proteins such as IkappaB (inhibitor of NF-kappaB). While many of the genes induced by eIF2 phosphorylation are shared between different environmental stresses, eIF2 kinases function in conjunction with other stress-response pathways, such as those regulated by mitogen-activated protein kinases, to elicit gene expression programmes that are tailored for the specific stress condition. Loss of eIF2 kinase pathways can have important health consequences. Mice devoid of the eIF2 kinase GCN2 [general control non-derepressible-2 or EIF2AK4 (eIF2alpha kinase 4)] show sensitivity to nutritional deficiencies and aberrant eating behaviours, and deletion of PEK [pancreatic eIF2alpha kinase or PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase) or EIF2AK3] leads to neonatal insulin-dependent diabetes, epiphyseal dysplasia and hepatic and renal complications.
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            Initiation of translation in prokaryotes and eukaryotes.

            M. Kozák (1999)
            The mechanisms whereby ribosomes engage a messenger RNA and select the start site for translation differ between prokaryotes and eukaryotes. Initiation sites in polycistronic prokaryotic mRNAs are usually selected via base pairing with ribosomal RNA. That straightforward mechanism is made complicated and interesting by cis- and trans-acting elements employed to regulate translation. Initiation sites in eukaryotic mRNAs are reached via a scanning mechanism which predicts that translation should start at the AUG codon nearest the 5' end of the mRNA. Interest has focused on mechanisms that occasionally allow escape from this first-AUG rule. With natural mRNAs, three escape mechanisms - context-dependent leaky scanning, reinitiation, and possibly direct internal initiation - allow access to AUG codons which, although not first, are still close to the 5' end of the mRNA. This constraint on the initiation step of translation in eukaryotes dictates the location of transcriptional promoters and may have contributed to the evolution of splicing.The binding of Met-tRNA to ribosomes is mediated by a GTP-binding protein in both prokaryotes and eukaryotes, but the more complex structure of the eukaryotic factor (eIF-2) and its association with other proteins underlie some aspects of initiation unique to eukaryotes. Modulation of GTP hydrolysis by eIF-2 is important during the scanning phase of initiation, while modulating the release of GDP from eIF-2 is a key mechanism for regulating translation in eukaryotes. Our understanding of how some other protein factors participate in the initiation phase of translation is in flux. Genetic tests suggest that some proteins conventionally counted as eukaryotic initiation factors may not be required for translation, while other tests have uncovered interesting new candidates. Some popular ideas about the initiation pathway are predicated on static interactions between isolated factors and mRNA. The need for functional testing of these complexes is discussed. Interspersed with these theoretical topics are some practical points concerning the interpretation of cDNA sequences and the use of in vitro translation systems. Some human diseases resulting from defects in the initiation step of translation are also discussed.
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              Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.

              Ebola virus (EBOV) infection causes a severe and fatal hemorrhagic disease that in many ways appears to be similar in humans and nonhuman primates; however, little is known about the development of EBOV hemorrhagic fever. In the present study, 21 cynomolgus monkeys were experimentally infected with EBOV and examined sequentially over a 6-day period to investigate the pathological events of EBOV infection that lead to death. Importantly, dendritic cells in lymphoid tissues were identified as early and sustained targets of EBOV, implicating their important role in the immunosuppression characteristic of EBOV infections. Bystander lymphocyte apoptosis, previously described in end-stage tissues, occurred early in the disease-course in intravascular and extravascular locations. Of note, apoptosis and loss of NK cells was a prominent finding, suggesting the importance of innate immunity in determining the fate of the host. Analysis of peripheral blood mononuclear cell gene expression showed temporal increases in tumor necrosis factor-related apoptosis-inducing ligand and Fas transcripts, revealing a possible mechanism for the observed bystander apoptosis, while up-regulation of NAIP and cIAP2 mRNA suggest that EBOV has evolved additional mechanisms to resist host defenses by inducing protective transcripts in cells that it infects. The sequence of pathogenetic events identified in this study should provide new targets for rational prophylactic and chemotherapeutic interventions.

                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                January 2013
                January 2013
                31 January 2013
                : 9
                : 1
                : e1003147
                [1 ]Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America
                [2 ]Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, United States of America
                [3 ]Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, United States of America
                [4 ]Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [5 ]Biochemistry Graduate Program, Iowa State University, Ames, Iowa, United States of America
                Harvard Medical School, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RSS TH GKA OJ CFB. Performed the experiments: RSS TH AG JMB GKA. Analyzed the data: RSS TH CFB OJ GKA HF. Contributed reagents/materials/analysis tools: CFB HF OJ GKA. Wrote the paper: RSS TH AG GKA HF CFB.


                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                : 25 April 2012
                : 6 December 2012
                Page count
                Pages: 18
                This work is supported by National Institutes of Health grants [AI059536, U54 AI 057158] (Northeast Biodefense Center-Lipkin) to CFB, [5F32AI084453] to RSS, and [AI081914] to GKA. This research was supported in part by the Intramural Research Program of the NIH, NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Gene Expression
                Protein Translation
                Innate Immunity
                Viral Classification
                RNA viruses
                Molecular Cell Biology

                Infectious disease & Microbiology
                Infectious disease & Microbiology


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