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      N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

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          Summary

          The RNA modification N6-methyladenosine (m 6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m 6A includes methyltransferases and demethylases that add or remove this modification, as well as m 6A-binding YTHDF proteins that promote the translation or degradation of m 6A-modified mRNA. We demonstrate that m 6A modulates infection by hepatitis C virus (HCV). Depletion of m 6A methyltransferases or an m 6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m 6A sites across the HCV genome and determined that inactivating m 6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m 6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m 6A. Altogether, this work identifies m 6A as a conserved regulatory mark across Flaviviridae genomes.

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          Highlights

          • The RNA genomes of HCV, ZIKV, DENV, YFV, and WNV contain m 6A modification

          • The cellular m 6A machinery regulates HCV infectious particle production

          • YTHDF proteins reduce HCV particle production and localize at viral assembly sites

          • m 6A-abrogating mutations in HCV E1 increase infectious particle production

          Abstract

          N6-methyladenosine (m 6A) post-transcriptionally regulates RNA function. Gokhale et al. demonstrate that the RNA genomes of HCV, ZIKV, DENV, YFV, and WNV are modified by m 6A. Depletion of cellular machinery that regulates m 6A or introduction of m 6A-abrogating mutations within HCV RNA increase viral particle production, suggesting that m 6A negatively regulates HCV.

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          Most cited references31

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          The lipid droplet is an important organelle for hepatitis C virus production.

          The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.
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            Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.

            N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).
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              m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency.

              N(6)-methyladenosine (m(6)A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m(6)A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m(6)A, especially gene- and cell-type-specific m(6)A mRNA modifications. We also show that microRNAs (miRNAs) regulate m(6)A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m(6)A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m(6)A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m(6)A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m(6)A formation of mRNAs and provide a foundation for future functional studies of m(6)A modification in cell reprogramming.
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                Author and article information

                Contributors
                Journal
                Cell Host Microbe
                Cell Host Microbe
                Cell Host & Microbe
                Cell Press
                1931-3128
                1934-6069
                09 November 2016
                09 November 2016
                : 20
                : 5
                : 654-665
                Affiliations
                [1 ]Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
                [2 ]Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
                [3 ]Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10021, USA
                [4 ]Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
                [5 ]Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
                [6 ]Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA 30329, USA
                [7 ]Duke Molecular Physiology Institute, Duke University, Durham NC 27701, USA
                [8 ]Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
                [9 ]The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA
                [10 ]The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021, USA
                [11 ]Programme in Emerging Infectious Disease, Duke-NUS Medical School, Singapore 169857, Singapore
                [12 ]Tri-Institutional Program in Computational Biology and Medicine, New York City, NY 10065, USA
                Author notes
                []Corresponding author chm2042@ 123456med.cornell.edu
                [∗∗ ]Corresponding author stacy.horner@ 123456duke.edu
                [13]

                Lead Contact

                [14]

                Twitter: @mason_lab

                [15]

                Twitter: @thehornerlab

                Article
                S1931-3128(16)30393-6
                10.1016/j.chom.2016.09.015
                5123813
                27773535
                b64d4d32-15df-433a-9979-eed15f4006d5
                © 2016 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                Categories
                Article

                Microbiology & Virology
                rna-modifications,m6a,n6-methyladenosine,hcv,flaviviridae,viral particle production,zika,dengue,west nile,yellow fever

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