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      RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

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          Abstract

          Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell’s RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5′ end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3′ end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction’s fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production.

          DOI: http://dx.doi.org/10.7554/eLife.01892.001

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          More than 40% of people around the globe are at risk of being bitten by mosquitoes infected with the virus that causes Dengue fever. Every year, more than 100 million of these individuals are infected. Many develop severe headaches, pain, and fever, but some develop a life-threatening condition where tiny blood vessels in the body begin to leak. If not treated quickly, this more severe manifestation of the illness can lead to death.

          There are currently no specific therapies or vaccines against Dengue or many other closely related viruses such as West Nile and Japanese Encephalitis. These viruses use instructions encoded in a single strand of RNA to take over an infected cell and to reproduce. The viruses also exploit an enzyme that cells use to destroy RNA to instead produce short stretches of RNA called sfRNAs that, among other things, may help the virus to avoid the immune system of its host. Understanding exactly how Dengue and other viruses thwart this enzyme—which is called Xrn1—may help scientists develop treatments or vaccines for these diseases.

          Chapman et al. have now shown that Dengue virus RNA contains a number of RNA elements that prevent it being completely degraded by the Xrn1 enzyme. In particular, a junction formed by three RNA helixes is critical for stopping the enzyme in its tracks, leaving the disease-associated sfRNA behind. A single mutation in the Dengue RNA disrupts the structure of the three-helix junction and allows the enzyme to completely destroy the RNA. A similar mutation was also made in the West Nile virus RNA and when human cells were infected with the mutated West Nile virus, the short sfRNAs were not produced. Treatments or vaccines targeting this structure may therefore help reduce illness associated with Dengue and related viruses.

          DOI: http://dx.doi.org/10.7554/eLife.01892.002

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          Most cited references31

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          Epidemiology of dengue: past, present and future prospects

          Dengue is currently regarded globally as the most important mosquito-borne viral disease. A history of symptoms compatible with dengue can be traced back to the Chin Dynasty of 265–420 AD. The virus and its vectors have now become widely distributed throughout tropical and subtropical regions of the world, particularly over the last half-century. Significant geographic expansion has been coupled with rapid increases in incident cases, epidemics, and hyperendemicity, leading to the more severe forms of dengue. Transmission of dengue is now present in every World Health Organization (WHO) region of the world and more than 125 countries are known to be dengue endemic. The true impact of dengue globally is difficult to ascertain due to factors such as inadequate disease surveillance, misdiagnosis, and low levels of reporting. Currently available data likely grossly underestimates the social, economic, and disease burden. Estimates of the global incidence of dengue infections per year have ranged between 50 million and 200 million; however, recent estimates using cartographic approaches suggest this number is closer to almost 400 million. The expansion of dengue is expected to increase due to factors such as the modern dynamics of climate change, globalization, travel, trade, socioeconomics, settlement and also viral evolution. No vaccine or specific antiviral therapy currently exists to address the growing threat of dengue. Prompt case detection and appropriate clinical management can reduce the mortality from severe dengue. Effective vector control is the mainstay of dengue prevention and control. Surveillance and improved reporting of dengue cases is also essential to gauge the true global situation as indicated in the objectives of the WHO Global Strategy for Dengue Prevention and Control, 2012–2020. More accurate data will inform the prioritization of research, health policy, and financial resources toward reducing this poorly controlled disease. The objective of this paper is to review historical and current epidemiology of dengue worldwide and, additionally, reflect on some potential reasons for expansion of dengue into the future.
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            Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots.

            A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified.
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              Human impacts have shaped historical and recent evolution in Aedes aegypti, the dengue and yellow fever mosquito.

              Although anthropogenic impacts are often considered harmful to species, human modifications to the landscape can actually create novel niches to which other species can adapt. These "domestication" processes are especially important in the context of arthropod disease vectors, where ecological overlap of vector and human populations may lead to epidemics. Here, we present results of a global genetic study of one such species, the dengue and yellow fever mosquito, Aedes aegypti, whose evolutionary history and current distribution have been profoundly shaped by humans. We used DNA sequences of four nuclear genes and 1504 single nucleotide polymorphism (SNP) markers developed with restriction-site associated DNA (RAD) sequencing to test the hypothesis that Ae. aegypti originated in Africa, where a domestic form arose and spread throughout the tropical and subtropical world with human trade and movement. Results confirmed African ancestry of the species, and supported a single subspeciation event leading to the pantropical domestic form. In addition, genetic data strongly supported the hypothesis that human trade routes first moved domestic Ae. aegypti out of Africa into the New World, followed by a later invasion from the New World into Southeast Asia and the Pacific. These patterns of domestication and invasion are relevant to many species worldwide, as anthropogenic forces increasingly impact evolutionary processes. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                01 April 2014
                2014
                : 3
                : e01892
                Affiliations
                [1 ]Department of Biochemistry and Molecular Genetics, Howard Hughes Medical Institute, University of Colorado Denver School of Medicine , Aurora, United States
                [2 ]Department of Microbiology, Immunology and Pathology, Colorado State University , Fort Collins, United States
                Case Western Reserve University , United States
                Case Western Reserve University , United States
                Author notes
                [* ]For correspondence: jeffrey.kieft@ 123456ucdenver.edu
                Article
                01892
                10.7554/eLife.01892
                3968743
                24692447
                581bac86-4463-40b1-9c89-f02ab5b057b6
                Copyright © 2014, Chapman et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 15 November 2013
                : 25 February 2014
                Funding
                Funded by: Howard Hughes Medical Institute FundRef identification ID: http://dx.doi.org/10.13039/100000011
                Award Recipient :
                Funded by: National Institutes of Health FundRef identification ID: http://dx.doi.org/10.13039/100000002
                Award ID: GM081346, GM097333
                Award Recipient :
                Funded by: National Institutes of Health FundRef identification ID: http://dx.doi.org/10.13039/100000002
                Award ID: U54 AI-065357
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biochemistry
                Microbiology and Infectious Disease
                Custom metadata
                0.7
                The Dengue virus contains discretely folded RNA structures that confer the ability to resist a powerful host cell enzyme and produce a disease-inducing noncoding RNA.

                Life sciences
                xrn1,dengue virus,exonuclease resistance,xrrna,small flavivirus rna,3′utr,viruses
                Life sciences
                xrn1, dengue virus, exonuclease resistance, xrrna, small flavivirus rna, 3′utr, viruses

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