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      High-Throughput SHAPE Analysis Reveals Structures in HIV-1 Genomic RNA Strongly Conserved across Distinct Biological States

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          Abstract

          Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower ( p-value < 0.0001) SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome.

          Author Summary

          The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis, we invent a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. We find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of the nucleocapsid protein. We directly map RNA–protein interactions inside virions and discover that the nucleocapsid prottein interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. Further application of the high-throughput RNA-structure analysis tools described here will make it possible to address diverse structure–function relationships in intact cellular and viral RNAs.

          Abstract

          Development of novel, quantitative, high-throughput RNA structure analysis tools allows the outline of structure-function relationships for the first 10% of an HIV genome, discovery of structural differences between regulatory and coding regions, and analysis of protein-RNA interactions inside authentic virions.

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          Most cited references54

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          R: A language and environment for statistical computing

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            HIV-1: fifteen proteins and an RNA.

            Human immunodeficiency virus type 1 is a complex retrovirus encoding 15 distinct proteins. Substantial progress has been made toward understanding the function of each protein, and three-dimensional structures of many components, including portions of the RNA genome, have been determined. This review describes the function of each component in the context of the viral life cycle: the Gag and Env structural proteins MA (matrix), CA (capsid), NC (nucleocapsid), p6, SU (surface), and TM (transmembrane); the Pol enzymes PR (protease), RT (reverse transcriptase), and IN (integrase); the gene regulatory proteins Tat and Rev; and the accessory proteins Nef, Vif, Vpr, and Vpu. The review highlights recent biochemical and structural studies that help clarify the mechanisms of viral assembly, infection, and replication.
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              A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry.

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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                pbio
                plbi
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                April 2008
                29 April 2008
                : 6
                : 4
                : e96
                Affiliations
                [1 ] Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [2 ] AIDS Vaccine Program, SAIC-Frederick, National Cancer Institute-Fredrick, Frederick, Maryland, United States of America
                [3 ] Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [4 ] Swiss Institute of Bioinformatics, Lausanne, Switzerland
                [5 ] HIV Drug Resistance Program, National Cancer Institute-Fredrick, Frederick, Maryland, United States of America
                [6 ] Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, United States of America
                [7 ] Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [8 ] Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina, United States of America
                Stanford University, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: weeks@ 123456unc.edu
                Article
                07-PLBI-RA-2987R2 plbi-06-04-22
                10.1371/journal.pbio.0060096
                2689691
                18447581
                50b17baa-7def-467b-b68a-9cb0693ca5e4
                Copyright: © 2008 Wilkinson et al. This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                History
                : 14 September 2007
                : 5 March 2008
                Page count
                Pages: 17
                Categories
                Research Article
                Biochemistry
                Infectious Diseases
                Virology
                Custom metadata
                Wilkinson KA, Gorelick RJ, Vasa SM, Guex N, Rein A, et al. (2008) High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. PLoS Biol 6(4): e96. doi: 10.1371/journal.pbio.0060096

                Life sciences
                Life sciences

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