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1
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Hanson WA, Romero Agosto GA, Rouskin S. Viral RNA Interactome: The Ultimate Researcher's Guide to RNA-Protein Interactions. Viruses 2024; 16:1702. [PMID: 39599817 PMCID: PMC11599142 DOI: 10.3390/v16111702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Revised: 10/18/2024] [Accepted: 10/25/2024] [Indexed: 11/29/2024] Open
Abstract
RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA-protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA-protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.
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Affiliation(s)
| | | | - Silvi Rouskin
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; (W.A.H.); (G.A.R.A.)
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2
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Li Y, Zhang L, Wang L, Li J, Zhao Y, Liu F, Wang Q. Structure and function of type IV IRES in picornaviruses: a systematic review. Front Microbiol 2024; 15:1415698. [PMID: 38855772 PMCID: PMC11157119 DOI: 10.3389/fmicb.2024.1415698] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2024] [Accepted: 05/13/2024] [Indexed: 06/11/2024] Open
Abstract
The Picornaviridae is a family of icosahedral viruses with single-stranded, highly diverse positive-sense RNA genomes. Virions consist of a capsid, without envelope, surrounding a core of RNA genome. A typical genome of picornavirus harbors a well-conserved and highly structured RNA element known as the internal ribosome entry site (IRES), functionally essential for viral replication and protein translation. Based on differences in their structures and mechanisms of action, picornaviral IRESs have been categorized into five types: type I, II, III, IV, and V. Compared with the type IV IRES, the others not only are structurally complicated, but also involve multiple initiation factors for triggering protein translation. The type IV IRES, often referred to as hepatitis C virus (HCV)-like IRES due to its structural resemblance to the HCV IRES, exhibits a simpler and more compact structure than those of the other four. The increasing identification of picornaviruses with the type IV IRES suggests that this IRES type seems to reveal strong retention and adaptation in terms of viral evolution. Here, we systematically reviewed structural features and biological functions of the type IV IRES in picornaviruses. A comprehensive understanding of the roles of type IV IRESs will contribute to elucidating the replication mechanism and pathogenesis of picornaviruses.
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Affiliation(s)
- Yan Li
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China
- Qingdao Center for Animal Disease Control and Prevention, Qingdao, China
| | - Lei Zhang
- Shandong New Hope Liuhe Group Co., Ltd., Qingdao, China
| | - Ling Wang
- University Hospital, Qingdao Agricultural University, Qingdao, China
| | - Jing Li
- Market Supervision Administration of Huangdao District, Qingdao, China
| | - Yanwei Zhao
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China
| | - Fuxiao Liu
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China
| | - Qianqian Wang
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China
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3
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Kallert E, Almena Rodriguez L, Husmann JÅ, Blatt K, Kersten C. Structure-based virtual screening of unbiased and RNA-focused libraries to identify new ligands for the HCV IRES model system. RSC Med Chem 2024; 15:1527-1538. [PMID: 38784459 PMCID: PMC11110755 DOI: 10.1039/d3md00696d] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2023] [Accepted: 03/16/2024] [Indexed: 05/25/2024] Open
Abstract
Targeting RNA including viral RNAs with small molecules is an emerging field. The hepatitis C virus internal ribosome entry site (HCV IRES) is a potential target for translation inhibitor development to raise drug resistance mutation preparedness. Using RNA-focused and unbiased molecule libraries, a structure-based virtual screening (VS) by molecular docking and pharmacophore analysis was performed against the HCV IRES subdomain IIa. VS hits were validated by a microscale thermophoresis (MST) binding assay and a Förster resonance energy transfer (FRET) assay elucidating ligand-induced conformational changes. Ten hit molecules were identified with potencies in the high to medium micromolar range proving the suitability of structure-based virtual screenings against RNA-targets. Hit compounds from a 2-guanidino-quinazoline series, like the strongest binder, compound 8b with an EC50 of 61 μM, show low molecular weight, moderate lipophilicity and reduced basicity compared to previously reported IRES ligands. Therefore, it can be considered as a potential starting point for further optimization by chemical derivatization.
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Affiliation(s)
- Elisabeth Kallert
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Staudingerweg 5 55128 Mainz Germany
| | - Laura Almena Rodriguez
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Staudingerweg 5 55128 Mainz Germany
| | - Jan-Åke Husmann
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Staudingerweg 5 55128 Mainz Germany
| | - Kathrin Blatt
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Staudingerweg 5 55128 Mainz Germany
| | - Christian Kersten
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Staudingerweg 5 55128 Mainz Germany
- Institute for Quantitative and Computational Biosciences, Johannes Gutenberg-University BioZentrum I, Hanns-Dieter-Hüsch-Weg 15 55128 Mainz Germany
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4
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Parker MD, Brunk ES, Getzler AJ, Karbstein K. The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage. PLoS Biol 2024; 22:e3001767. [PMID: 39038273 PMCID: PMC11045238 DOI: 10.1371/journal.pbio.3001767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Accepted: 03/05/2024] [Indexed: 07/24/2024] Open
Abstract
The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.
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Affiliation(s)
- Melissa D. Parker
- The Skaggs Graduate School of Chemical and Biological Sciences, The
Scripps Research Institute, La Jolla, California, United States of
America
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and
Technology, Jupiter, Florida, United States of America
| | - Elise S. Brunk
- The Skaggs Graduate School of Chemical and Biological Sciences, The
Scripps Research Institute, La Jolla, California, United States of
America
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and
Technology, Jupiter, Florida, United States of America
| | - Adam J. Getzler
- The Skaggs Graduate School of Chemical and Biological Sciences, The
Scripps Research Institute, La Jolla, California, United States of
America
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and
Technology, Jupiter, Florida, United States of America
| | - Katrin Karbstein
- The Skaggs Graduate School of Chemical and Biological Sciences, The
Scripps Research Institute, La Jolla, California, United States of
America
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and
Technology, Jupiter, Florida, United States of America
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5
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Janvier A, Hayek H, Alghoul F, Gross L, Allmang C, Martin F, Eriani G. Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 3234:17-29. [PMID: 38507197 DOI: 10.1007/978-3-031-52193-5_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/22/2024]
Abstract
Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.
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Affiliation(s)
- Aurélie Janvier
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Hassan Hayek
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Fatima Alghoul
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Lauriane Gross
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Christine Allmang
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Franck Martin
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Gilbert Eriani
- Architecture et Réactivité de l'ARN, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France.
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6
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Cao Y, Liu H, Lu SS, Jones KA, Govind AP, Jeyifous O, Simmons CQ, Tabatabaei N, Green WN, Holder JL, Tahmasebi S, George AL, Dickinson BC. RNA-based translation activators for targeted gene upregulation. Nat Commun 2023; 14:6827. [PMID: 37884512 PMCID: PMC10603104 DOI: 10.1038/s41467-023-42252-z] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2023] [Accepted: 10/04/2023] [Indexed: 10/28/2023] Open
Abstract
Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.
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Affiliation(s)
- Yang Cao
- Department of Chemistry, The University of Chicago, Chicago, IL, USA
| | - Huachun Liu
- Department of Chemistry, The University of Chicago, Chicago, IL, USA
| | - Shannon S Lu
- Department of Chemistry, The University of Chicago, Chicago, IL, USA
| | - Krysten A Jones
- Department of Chemistry, The University of Chicago, Chicago, IL, USA
| | - Anitha P Govind
- Department of Neurobiology, The University of Chicago, Chicago, IL, USA
| | - Okunola Jeyifous
- Department of Neurobiology, The University of Chicago, Chicago, IL, USA
| | - Christine Q Simmons
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Negar Tabatabaei
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
| | - William N Green
- Department of Neurobiology, The University of Chicago, Chicago, IL, USA
| | - Jimmy L Holder
- Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
- Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA
| | - Soroush Tahmasebi
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
| | - Alfred L George
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Bryan C Dickinson
- Department of Chemistry, The University of Chicago, Chicago, IL, USA.
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7
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Volegova MP, Hermosillo C, Cate JHD. The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs. PLoS One 2023; 18:e0292080. [PMID: 37768948 PMCID: PMC10538695 DOI: 10.1371/journal.pone.0292080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Accepted: 09/12/2023] [Indexed: 09/30/2023] Open
Abstract
Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.
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Affiliation(s)
- Marina P. Volegova
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, United States of America
| | - Cynthia Hermosillo
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, United States of America
| | - Jamie H. D. Cate
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, United States of America
- Department of Chemistry, University of California, Berkeley, CA, United States of America
- Molecular Biosciences and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, United States of America
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8
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Wang Q, Zhao D, Wang L, Sang Y, Meng H, Wang Q, Shan H, Liu F, Geri L. Translation of Senecavirus A polyprotein is initiated from the IRES-proximal initiation codon. Virology 2023; 579:67-74. [PMID: 36608596 DOI: 10.1016/j.virol.2022.12.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Revised: 12/19/2022] [Accepted: 12/19/2022] [Indexed: 12/31/2022]
Abstract
To clarify whether Senecavirus A (SVA) has the potential of alternative translation, an extra G residue was inserted into an SVA cDNA clone, resultantly generating an "AUGAUG" motif. The second AUG is the authentic SVA initiation codon, whereas the first AUG is a putative one. Subsequently, eighteen nucleotides were inserted one by one between AUG and AUG for reconstructing cDNA clones. The test of virus recovery showed that three replication-competent SVAs, whose AUG/AUG-flanked sequences were not multiples of three nucleotides, were successfully rescued from their individual cDNA clones. The wild-type SVA possesses a UUUUU motif within the polyprotein-encoding region. Sanger sequencing showed that these three replication-competent SVAs harbored one or two extra U residues in the UUUUU motif, implying that polyprotein translation was initiated from the putative AUG, and the authentic AUG would be inactivated. This is probably attributed to the lack of ribosome scanning along an SVA genome.
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Affiliation(s)
- Qianqian Wang
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010011, China
| | - Di Zhao
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China
| | - Ling Wang
- University Hospital, Qingdao Agricultural University, Qingdao, 266109, China
| | - Yuxuan Sang
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China
| | - Hailan Meng
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China
| | - Qi Wang
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China
| | - Hu Shan
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China
| | - Fuxiao Liu
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
| | - Letu Geri
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010011, China.
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9
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Awadh AA. The Role of Cytosolic Lipid Droplets in Hepatitis C Virus Replication, Assembly, and Release. BIOMED RESEARCH INTERNATIONAL 2023; 2023:5156601. [PMID: 37090186 PMCID: PMC10121354 DOI: 10.1155/2023/5156601] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/20/2022] [Revised: 03/02/2023] [Accepted: 03/09/2023] [Indexed: 04/25/2023]
Abstract
The hepatitis C virus (HCV) causes chronic hepatitis by establishing a persistent infection. Patients with chronic hepatitis frequently develop hepatic cirrhosis, which can lead to liver cancer-the progressive liver damage results from the host's immune response to the unresolved infection. The HCV replication process, including the entry, replication, assembly, and release stages, while the virus circulates in the bloodstream, it is intricately linked to the host's lipid metabolism, including the dynamic of the cytosolic lipid droplets (cLDs). This review article depicts how this interaction regulates viral cell tropism and aids immune evasion by coining viral particle characteristics. cLDs are intracellular organelles that store most of the cytoplasmic components of neutral lipids and are assumed to play an increasingly important role in the pathophysiology of lipid metabolism and host-virus interactions. cLDs are involved in the replication of several clinically significant viruses, where viruses alter the lipidomic profiles of host cells to improve viral life cycles. cLDs are involved in almost every phase of the HCV life cycle. Indeed, pharmacological modulators of cholesterol synthesis and intracellular trafficking, lipoprotein maturation, and lipid signaling molecules inhibit the assembly of HCV virions. Likewise, small-molecule inhibitors of cLD-regulating proteins inhibit HCV replication. Thus, addressing the molecular architecture of HCV replication will aid in elucidating its pathogenesis and devising preventive interventions that impede persistent infection and prevent disease progression. This is possible via repurposing the available therapeutic agents that alter cLDs metabolism. This review highlights the role of cLD in HCV replication.
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Affiliation(s)
- Abdullah A. Awadh
- Department of Basic Medical Sciences, College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Jeddah 21423, Saudi Arabia
- King Abdullah International Medical Research Center, Jeddah 21423, Saudi Arabia
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10
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Childs-Disney JL, Yang X, Gibaut QMR, Tong Y, Batey RT, Disney MD. Targeting RNA structures with small molecules. Nat Rev Drug Discov 2022; 21:736-762. [PMID: 35941229 PMCID: PMC9360655 DOI: 10.1038/s41573-022-00521-4] [Citation(s) in RCA: 271] [Impact Index Per Article: 90.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/17/2022] [Indexed: 01/07/2023]
Abstract
RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing - by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade.
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Affiliation(s)
| | - Xueyi Yang
- Department of Chemistry, Scripps Research, Jupiter, FL, USA
| | | | - Yuquan Tong
- Department of Chemistry, Scripps Research, Jupiter, FL, USA
| | - Robert T Batey
- Department of Biochemistry, University of Colorado, Boulder, CO, USA.
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11
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Brown ZP, Abaeva IS, De S, Hellen CUT, Pestova TV, Frank J. Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES. EMBO J 2022; 41:e110581. [PMID: 35822879 DOI: 10.15252/embj.2022110581] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Revised: 06/01/2022] [Accepted: 06/14/2022] [Indexed: 02/05/2023] Open
Abstract
Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.
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Affiliation(s)
- Zuben P Brown
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Irina S Abaeva
- Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY, USA
| | - Swastik De
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Christopher U T Hellen
- Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY, USA
| | - Tatyana V Pestova
- Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY, USA
| | - Joachim Frank
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.,Department of Biological Sciences, Columbia University, New York, NY, USA
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12
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Li HC, Yang CH, Lo SY. Cellular factors involved in the hepatitis C virus life cycle. World J Gastroenterol 2021; 27:4555-4581. [PMID: 34366623 PMCID: PMC8326260 DOI: 10.3748/wjg.v27.i28.4555] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/04/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 970, Taiwan
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan
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13
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Hepatitis C Virus Translation Regulation. Int J Mol Sci 2020; 21:ijms21072328. [PMID: 32230899 PMCID: PMC7178104 DOI: 10.3390/ijms21072328] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 03/18/2020] [Accepted: 03/25/2020] [Indexed: 12/12/2022] Open
Abstract
Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.
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14
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Romero-López C, Berzal-Herranz A. The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle. Int J Mol Sci 2020; 21:1479. [PMID: 32098260 PMCID: PMC7073135 DOI: 10.3390/ijms21041479] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 02/05/2023] Open
Abstract
RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.
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Affiliation(s)
- Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
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15
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Di Giorgio A, Duca M. Synthetic small-molecule RNA ligands: future prospects as therapeutic agents. MEDCHEMCOMM 2019; 10:1242-1255. [PMID: 31534649 PMCID: PMC6748380 DOI: 10.1039/c9md00195f] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/31/2019] [Accepted: 04/30/2019] [Indexed: 12/17/2022]
Abstract
RNA is one of the most intriguing and promising biological targets for the discovery of innovative drugs in many pathologies and various biologically relevant RNAs that could serve as drug targets have already been identified. Among the most important ones, one can mention prokaryotic ribosomal RNA which is the target of several marketed antibiotics, viral RNAs or oncogenic microRNAs that are tightly involved in the development and progression of various cancers. Oligonucleotides are efficient and specific RNA targeting agents but suffer from poor pharmacodynamic and pharmacokinetic properties. For this reason, a number of synthetic small-molecule ligands have been identified and studied upon screening of chemical libraries or focused design of RNA binders. In this review, we report the most relevant examples of synthetic compounds bearing sufficient selectivity to envisage clinical studies and future therapeutic applications with a particular attention for the main strategies that can be undertaken toward the improvement of selectivity and biological activity.
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Affiliation(s)
- A Di Giorgio
- Université Côte d'Azur , CNRS , Institute of Chemistry of Nice (ICN) , Nice , France .
| | - M Duca
- Université Côte d'Azur , CNRS , Institute of Chemistry of Nice (ICN) , Nice , France .
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16
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Johnson AG, Petrov AN, Fuchs G, Majzoub K, Grosely R, Choi J, Puglisi JD. Fluorescently-tagged human eIF3 for single-molecule spectroscopy. Nucleic Acids Res 2019; 46:e8. [PMID: 29136179 PMCID: PMC5778468 DOI: 10.1093/nar/gkx1050] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Accepted: 10/24/2017] [Indexed: 01/09/2023] Open
Abstract
Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ∼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
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Affiliation(s)
- Alex G Johnson
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.,Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Alexey N Petrov
- Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
| | - Gabriele Fuchs
- The RNA Institute, Department of Biological Sciences, University of Albany, Albany, NY 12222, USA
| | - Karim Majzoub
- Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA
| | - Rosslyn Grosely
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Junhong Choi
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Joseph D Puglisi
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
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17
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Sam68 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 2 of Viral 5' Untranslated Region. J Virol 2019; 93:JVI.00693-19. [PMID: 31068419 DOI: 10.1128/jvi.00693-19] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Accepted: 04/26/2019] [Indexed: 12/12/2022] Open
Abstract
The Src-associated in mitosis 68-kDa (Sam68) protein is a highly conserved nuclear protein and is involved in a series of cellular processes, including transcription and signal transduction. Sam68 is comprised of 443 amino acids and contains an RGG box domain, a KH domain, and a tyrosine-rich domain. Its role in hepatitis C virus (HCV) replication is unknown. Here, we find that Sam68 promotes HCV replication without affecting viral translation. The RNA immunoprecipitation experiments show that the positive strand of HCV RNA interacts with Sam68. HCV infection triggers the translocation of the Sam68 protein from the nucleus to the cytoplasm, where it interacts with the HCV genome. Further study shows that the region of Sam68 spanning amino acids 1 to 157 is the pivotal domain to interact with the stem-loop 2 of the HCV 5' untranslated region (5' UTR) and is responsible for the enhancement of HCV replication. These data suggested that Sam68 may serve as a proviral factor of HCV to facilitate viral replication through interaction with the viral genome.IMPORTANCE Hepatitis C virus (HCV) is a member of the Flaviviridae family, and its infection causes chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma. No vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. This study discloses a new host factor that binds to the HCV 5' UTR and promotes HCV replication. Sam68 may play an important role in HCV-related diseases, and further investigation is highly encouraged to explore its specific actions in HCV pathogenesis.
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18
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Ullah H, Hou W, Dakshanamurthy S, Tang Q. Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation. Oncotarget 2019; 10:3209-3226. [PMID: 31143369 PMCID: PMC6524932 DOI: 10.18632/oncotarget.26907] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 04/21/2019] [Indexed: 12/11/2022] Open
Abstract
Due to the small number of molecular targets in viruses and the rapid evolution of viral genes, it is very challenging to develop specific antiviral drugs. Viruses require host factors to translate their transcripts, and targeting the host factor(s) offers a unique opportunity to develop broad antiviral drugs. It is well documented that some viruses utilize a host protein, Receptor for Activated C Kinase 1 (RACK1), to translate their mRNAs using a viral mRNA secondary structure known as the Internal Ribosomal Entry Site (IRES). RACK1 is essential for the translation of many viruses including hepatitis C (HCV), polio, Drosophila C (DCV), Dengue, Cricket Paralysis (CrpV), and vaccinia viruses. In addition, HIV-1 and Herpes Simplex virus (HSV-1) are known to use IRES as well. Therefore, host RACK1 protein is an attractive target for developing broad antiviral drugs. Depletion of the host's RACK1 will potentially inhibit virus replication. This background study has led us to the development of novel antiviral therapeutics, such as RACK1 inhibitors. By utilizing the crystal structure of the RACK1A protein from the model plant Arabidopsis and using a structure based drug design method, dozens of small compounds were identified that could potentially bind to the experimentally determined functional site of the RACK1A protein. The SPR assays showed that the small compounds bound strongly to recombinant RACK1A protein. Here we provide evidence that the drugs show high efficacy in inhibition of HSV-1 proliferation in a HEp-2 cell line. The drug showed similar efficacy as the available anti-herpes drug acyclovir and showed supralinear effect when applied in a combinatorial manner. As an increasing number of viruses are reported to use host RACK1 proteins, and more than 100 diverse animals and plant disease-causing viruses are known to use IRES-based translation, these drugs can be established as host-targeted broad antiviral drugs.
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Affiliation(s)
- Hemayet Ullah
- Department of Biology, Howard University, Washington, DC 20059, USA
| | - Wangheng Hou
- Department of Microbiology, Howard University College of Medicine, Washington, DC 20059, USA
| | - Sivanesan Dakshanamurthy
- Department of Oncology, Clinical and Experimental Therapeutics Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Qiyi Tang
- Department of Microbiology, Howard University College of Medicine, Washington, DC 20059, USA
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19
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James CC, Smyth JW. Alternative mechanisms of translation initiation: An emerging dynamic regulator of the proteome in health and disease. Life Sci 2018; 212:138-144. [PMID: 30290184 DOI: 10.1016/j.lfs.2018.09.054] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 09/18/2018] [Accepted: 09/27/2018] [Indexed: 01/06/2023]
Abstract
Eukaryotic mRNAs were historically thought to rely exclusively on recognition and binding of their 5' cap by initiation factors to effect protein translation. While internal ribosome entry sites (IRESs) are well accepted as necessary for the cap-independent translation of many viral genomes, there is now recognition that eukaryotic mRNAs also undergo non-canonical modes of translation initiation. Recently, high-throughput assays have identified thousands of mammalian transcripts with translation initiation occurring at non-canonical start codons, upstream of and within protein coding regions. In addition to IRES-mediated events, regulatory mechanisms of translation initiation have been described involving alternate 5' cap recognition, mRNA sequence elements, and ribosome selection. These mechanisms ensure translation of specific mRNAs under conditions where cap-dependent translation is shut down and contribute to pathological states including cardiac hypertrophy and cancer. Such global and gene-specific dynamic regulation of translation presents us with an increasing number of novel therapeutic targets. While these newly discovered modes of translation initiation have been largely studied in isolation, it is likely that several act on the same mRNA and exquisite coordination is necessary to maintain 'normal' translation. In this short review, we summarize the current state of knowledge of these alternative mechanisms of eukaryotic protein translation, their contribution to normal and pathological cell biology, and the potential of targeting translation initiation therapeutically in human disease.
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Affiliation(s)
- Carissa C James
- Virginia Tech Carilion Research Institute and School of Medicine, Roanoke, VA, USA; Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, USA; Center for Heart and Regenerative Medicine, Virginia Tech Carilion Research Institute, Roanoke, VA, USA
| | - James W Smyth
- Virginia Tech Carilion Research Institute and School of Medicine, Roanoke, VA, USA; Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; Center for Heart and Regenerative Medicine, Virginia Tech Carilion Research Institute, Roanoke, VA, USA.
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20
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Maryam M, Idrees M. Study of promoter hypomethylation profiles ofRASoncogenes in hepatocellular carcinoma derived from hepatitis C virus genotype 3a in Pakistani population. J Med Virol 2018; 90:1516-1523. [DOI: 10.1002/jmv.25221] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 05/03/2018] [Indexed: 02/06/2023]
Affiliation(s)
- Maria Maryam
- National Centre of Excellence in Molecular Biology; University of the Punjab; Lahore Pakistan
| | - Muhammad Idrees
- National Centre of Excellence in Molecular Biology; University of the Punjab; Lahore Pakistan
- Vice Chancellor Office, Hazara University; Mansehra Pakistan
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21
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Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication. J Virol 2018; 92:JVI.01997-17. [PMID: 29343570 DOI: 10.1128/jvi.01997-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Accepted: 01/09/2018] [Indexed: 12/26/2022] Open
Abstract
The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.
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22
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Mailliot J, Martin F. Viral internal ribosomal entry sites: four classes for one goal. WILEY INTERDISCIPLINARY REVIEWS. RNA 2018; 9. [PMID: 29193740 DOI: 10.1002/wrna.1458] [Citation(s) in RCA: 83] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/17/2017] [Revised: 09/19/2017] [Accepted: 10/02/2017] [Indexed: 12/22/2022]
Abstract
To ensure efficient propagation, viruses need to rapidly produce viral proteins after cell entrance. Since viral genomes do not encode any components of the protein biosynthesis machinery, viral proteins must be produced by the host cell. To hi-jack the host cellular translation, viruses use a great variety of distinct strategies. Many single-stranded positive-sensed RNA viruses contain so-called internal ribosome entry sites (IRESs). IRESs are structural RNA motifs that have evolved to specific folds that recruit the host ribosomes on the viral coding sequences in order to synthesize viral proteins. In host canonical translation, recruitment of the translation machinery components is essentially guided by the 5' cap (m7 G) of mRNA. In contrast, IRESs are able to promote efficient ribosome assembly internally and in cap-independent manner. IRESs have been categorized into four classes, based on their length, nucleotide sequence, secondary and tertiary structures, as well as their mode of action. Classes I and II require the assistance of cellular auxiliary factors, the eukaryotic intiation factors (eIF), for efficient ribosome assembly. Class III IRESs require only a subset of eIFs whereas Class IV, which are the more compact, can promote translation without any eIFs. Extensive functional and structural investigations of IRESs over the past decades have allowed a better understanding of their mode of action for viral translation. Because viral translation has a pivotal role in the infectious program, IRESs are therefore attractive targets for therapeutic purposes. WIREs RNA 2018, 9:e1458. doi: 10.1002/wrna.1458 This article is categorized under: Translation > Ribosome Structure/Function Translation > Translation Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
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Affiliation(s)
- Justine Mailliot
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, INSERM U964, Illkirch-Graffenstaden, France
| | - Franck Martin
- Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN" CNRS UPR9002, Université De Strasbourg, Strasbourg, France
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23
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González-Almela E, Williams H, Sanz MA, Carrasco L. The Initiation Factors eIF2, eIF2A, eIF2D, eIF4A, and eIF4G Are Not Involved in Translation Driven by Hepatitis C Virus IRES in Human Cells. Front Microbiol 2018; 9:207. [PMID: 29487587 PMCID: PMC5816946 DOI: 10.3389/fmicb.2018.00207] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2017] [Accepted: 01/30/2018] [Indexed: 12/22/2022] Open
Abstract
Animal viruses have evolved a variety of strategies to ensure the efficient translation of their mRNAs. One such strategy is the use of internal ribosome entry site (IRES) elements, which circumvent the requirement for some eukaryotic initiation factors (eIFs). Much effort has been directed to unravel the precise mechanism of translation initiation by hepatitis C virus (HCV) mRNA. In the present study, we examined the involvement of several eIFs in HCV IRES-driven translation in human cells in a comparative analysis with mRNAs bearing the encephalomyocarditis virus or the Cricket paralysis virus IRES element. Consistent with previous findings, several inhibitors of eIF2 activity, including sodium arsenite, thapsigargin, tunicamycin, and salubrinal, had no inhibitory effect on the translation of an mRNA bearing the HCV IRES, and all induced the phosphorylation of eIF2α. In addition, hippuristanol and pateamine A, two known inhibitors of eIF4A, failed to block HCV IRES-directed translation. To test the release of nuclear proteins to the cytoplasm and to analyze the formation of stress granules, the location of the nuclear protein TIA1 was tested by immunocytochemistry. Both arsenite and pateamine A could efficiently induce the formation of stress granules containing TIA1 and eIF4G, whereas eIF3 and eIF2 failed to localize to these cytoplasmic bodies. The finding of eIF4A and eIF4G in stress granules suggests that they do not participate in mRNA translation. Human HAP1 cells depleted for eIF2A, eIF2D, or both factors, were able to synthesize luciferase from an mRNA bearing the HCV IRES even when eIF2α was phosphorylated. Overall, these results demonstrate that neither eIF2A nor eIF2D does not participate in the translation directed by HCV IRES. We conclude that eIF2, eIF4A, eIF2A, and eIF2D do not participate in the initiation of translation of HCV mRNA.
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Affiliation(s)
- Esther González-Almela
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain
| | - Hugh Williams
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain
| | - Miguel A Sanz
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain
| | - Luis Carrasco
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain
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24
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Willcocks MM, Zaini S, Chamond N, Ulryck N, Allouche D, Rajagopalan N, Davids NA, Fahnøe U, Hadsbjerg J, Rasmussen TB, Roberts LO, Sargueil B, Belsham GJ, Locker N. Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites. Nucleic Acids Res 2018; 45:13016-13028. [PMID: 29069411 PMCID: PMC5727462 DOI: 10.1093/nar/gkx991] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2017] [Accepted: 10/12/2017] [Indexed: 01/23/2023] Open
Abstract
Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles.
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Affiliation(s)
- Margaret M Willcocks
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Salmah Zaini
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Nathalie Chamond
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Nathalie Ulryck
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Delphine Allouche
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Noemie Rajagopalan
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Nana A Davids
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Ulrik Fahnøe
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Johanne Hadsbjerg
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Thomas Bruun Rasmussen
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Lisa O Roberts
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK.,School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Bruno Sargueil
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Graham J Belsham
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Nicolas Locker
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
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25
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Johnson AG, Grosely R, Petrov AN, Puglisi JD. Dynamics of IRES-mediated translation. Philos Trans R Soc Lond B Biol Sci 2017; 372:rstb.2016.0177. [PMID: 28138065 DOI: 10.1098/rstb.2016.0177] [Citation(s) in RCA: 66] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/16/2016] [Indexed: 12/19/2022] Open
Abstract
Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning. Owing to their simplicity and ribosomal affinity, the CrPV and HCV IRES have been important models for structural and functional studies of the eukaryotic ribosome during initiation, serving as excellent targets for recent technological breakthroughs in cryogenic electron microscopy (cryo-EM) and single-molecule analysis. High-resolution structural models of ribosome : IRES complexes, coupled with dynamics studies, have clarified decades of biochemical research and provided an outline of the conformational and compositional trajectory of the ribosome during initiation. Here we review recent progress in the study of HCV- and CrPV-type IRESs, highlighting important structural and dynamics insights and the synergy between cryo-EM and single-molecule studies.This article is part of the themed issue 'Perspectives on the ribosome'.
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Affiliation(s)
- Alex G Johnson
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.,Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Rosslyn Grosely
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Alexey N Petrov
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
| | - Joseph D Puglisi
- Department of Structural Biology, Stanford University, Stanford, CA 94305, USA
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Jaafar ZA, Oguro A, Nakamura Y, Kieft JS. Translation initiation by the hepatitis C virus IRES requires eIF1A and ribosomal complex remodeling. eLife 2016; 5. [PMID: 28009256 PMCID: PMC5238962 DOI: 10.7554/elife.21198] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2016] [Accepted: 12/22/2016] [Indexed: 12/16/2022] Open
Abstract
Internal ribosome entry sites (IRESs) are important RNA-based translation initiation signals, critical for infection by many pathogenic viruses. The hepatitis C virus (HCV) IRES is the prototype for the type 3 IRESs and is also invaluable for exploring principles of eukaryotic translation initiation, in general. Current mechanistic models for the type 3 IRESs are useful but they also present paradoxes, including how they can function both with and without eukaryotic initiation factor (eIF) 2. We discovered that eIF1A is necessary for efficient activity where it stabilizes tRNA binding and inspects the codon-anticodon interaction, especially important in the IRES' eIF2-independent mode. These data support a model in which the IRES binds preassembled translation preinitiation complexes and remodels them to generate eukaryotic initiation complexes with bacterial-like features. This model explains previous data, reconciles eIF2-dependent and -independent pathways, and illustrates how RNA structure-based control can respond to changing cellular conditions.
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Affiliation(s)
- Zane A Jaafar
- Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, United States
| | - Akihiro Oguro
- Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | | | - Jeffrey S Kieft
- Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, United States.,RNA BioScience Initiative, University of Colorado Denver School of Medicine, Aurora, United States
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Charrette BP, Boerneke MA, Hermann T. Ligand Optimization by Improving Shape Complementarity at a Hepatitis C Virus RNA Target. ACS Chem Biol 2016; 11:3263-3267. [PMID: 27775338 DOI: 10.1021/acschembio.6b00687] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Crystal structure analysis revealed key interactions of a 2-amino-benzimidazole viral translation inhibitor that captures an elongated conformation of an RNA switch target in the internal ribosome entry site (IRES) of hepatitis C virus (HCV). Here, we have designed and synthesized quinazoline derivatives with improved shape complementarity at the ligand binding site of the viral RNA target. A spiro-cyclopropyl modification aimed at filling a pocket in the back of the RNA binding site led to a 5-fold increase of ligand affinity while a slightly more voluminous dimethyl substitution at the same position did not improve binding. We demonstrate that precise shape complementarity based solely on hydrophobic interactions contributes significantly to ligand binding even at a hydrophilic RNA target site such as the HCV IRES conformational switch.
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Affiliation(s)
- Brian P. Charrette
- Department
of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093, United States
| | - Mark A. Boerneke
- Department
of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093, United States
| | - Thomas Hermann
- Department
of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093, United States
- Center
for Drug Discovery Innovation, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093, United States
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28
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Abstract
Ligand-responsive RNA mechanical switches represent a new class of simple switching modules that adopt well-defined ligand-free and bound conformational states, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), these RNA switch motifs were found in the genome of diverse other viruses. Although large variations are seen in sequence and local secondary structure of the switches, their function in viral translation initiation that requires selective ligand recognition is conserved. We recently determined the crystal structure of an RNA switch from Seneca Valley virus (SVV) which is able to functionally replace the switch of HCV. The switches from both viruses recognize identical cognate ligands despite their sequence dissimilarity. Here, we describe the discovery of 7 new switches in addition to the previously established 5 examples. We highlight structural and functional features unique to this class of ligand-responsive RNA mechanical switches and discuss implications for therapeutic development and the construction of RNA nanostructures.
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Key Words
- AEV, avian encephalomyelitis virus
- BDV, border disease virus
- BVDV, bovine viral diarrhea virus
- CSFV, classical swine fever virus
- DHV, Duck hepatitis virus
- DPV, duck picornavirus
- GBV, GB virus
- GPV, giraffe pestivirus
- HCV, hepatitis C virus
- IRES
- IRES, internal ribosome entry site
- IVT, in vitro translation
- NPHV, non-primate hepacivirus
- RNA switch
- SPV, simian picornavirus
- SVV, Seneca Valley virus
- conformational switch
- hepatitis C virus
- riboswitch
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Affiliation(s)
- Mark A Boerneke
- a Department of Chemistry and Biochemistry ; University of California, San Diego ; La Jolla , CA USA
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Shier MK, El-Wetidy MS, Ali HH, Al-Qattan MM. Hepatitis c virus genotype 4 replication in the hepatocellular carcinoma cell line HepG2/C3A. Saudi J Gastroenterol 2016; 22:240-8. [PMID: 27184644 PMCID: PMC4898095 DOI: 10.4103/1319-3767.182461] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND/AIMS The lack of a reliable cell culture system allowing persistent in vitro hepatitis C virus (HCV) propagation is still restraining the search for novel antiviral strategies. HepG2 cells transfection with HCV allows for viral replication. However, the replication is weak presumably because of HepG2 lack of miRNA-122, which is essential for viral replication. Other agents such as polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) have been shown to increase the efficiency of infection with other viruses. This study included comparison of HCV genotype 4 5'UTR and core RNA levels and HCV core protein expression at different time intervals in the absence or presence of PEG and/or DMSO postinfection. MATERIALS AND METHODS We used serum with native HCV particles in infecting HepG2 cells in vitro. HCV replication was assessed by reverse transcriptase polymerase chain reaction for detection of HCV RNA and immunofluorescence and flow cytometry for detection of HCV core protein. RESULTS HCV 5'UTR and core RNA expression was evident at different time intervals after viral infection, especially after cells were treated with PEG. HCV core protein was also evident at different time intervals using both immunofluorescence and flow cytometry. PEG, not DMSO, has increased the HCV core protein expression in the treated cells, similar to its effect on viral RNA expression. CONCLUSIONS These expression profiles suggest that the current model of cultured HepG2 cells allows the study of HCV genotype 4 replication and different stages of the viral life cycle.
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Affiliation(s)
- Medhat K. Shier
- College of Medicine Research Center, King Saud University, Riyadh, Saudi Arabia,Department of Medical Microbiology and Immunology, College of Medicine, Menofia University, Egypt,Address for correspondence: Dr. Medhat K. Shier, College of Medicine Research Center, King Saud University, PO Box 2925 (74), Riyadh - 11461, Saudi Arabia. E-mail:
| | | | - Hebatallah H. Ali
- College of Medicine Research Center, King Saud University, Riyadh, Saudi Arabia
| | - Mohammad M. Al-Qattan
- College of Medicine Research Center, King Saud University, Riyadh, Saudi Arabia,Department of Surgery, College of Medicine, King Saud University, Riyadh, Saudi Arabia
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30
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Sabila M, Kundu N, Smalls D, Ullah H. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast. FRONTIERS IN PLANT SCIENCE 2016; 7:176. [PMID: 26941753 PMCID: PMC4764707 DOI: 10.3389/fpls.2016.00176] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2015] [Accepted: 02/02/2016] [Indexed: 05/21/2023]
Abstract
Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways.
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HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5' Untranslated Region. J Virol 2015; 90:2332-44. [PMID: 26656705 DOI: 10.1128/jvi.02795-15] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2015] [Accepted: 12/04/2015] [Indexed: 12/12/2022] Open
Abstract
UNLABELLED High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5' untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome. IMPORTANCE Hepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5' untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.
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32
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Angulo J, Ulryck N, Deforges J, Chamond N, Lopez-Lastra M, Masquida B, Sargueil B. LOOP IIId of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex. Nucleic Acids Res 2015; 44:1309-25. [PMID: 26626152 PMCID: PMC4756818 DOI: 10.1093/nar/gkv1325] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Accepted: 11/11/2015] [Indexed: 12/14/2022] Open
Abstract
As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a ‘kissing complex’ between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation.
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Affiliation(s)
- Jenniffer Angulo
- CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
| | - Nathalie Ulryck
- CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France
| | - Jules Deforges
- CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France
| | - Nathalie Chamond
- CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France
| | - Marcelo Lopez-Lastra
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
| | - Benoît Masquida
- UMR 7156 Génétique Moléculaire Génomique Microbiologie, CNRS - Université de Strasbourg, Strasbourg, France
| | - Bruno Sargueil
- CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France
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Yamamoto H, Collier M, Loerke J, Ismer J, Schmidt A, Hilal T, Sprink T, Yamamoto K, Mielke T, Bürger J, Shaikh TR, Dabrowski M, Hildebrand PW, Scheerer P, Spahn CMT. Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA. EMBO J 2015; 34:3042-58. [PMID: 26604301 DOI: 10.15252/embj.201592469] [Citation(s) in RCA: 65] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Accepted: 10/29/2015] [Indexed: 12/12/2022] Open
Abstract
Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation.
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Affiliation(s)
- Hiroshi Yamamoto
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Marianne Collier
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Justus Loerke
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Jochen Ismer
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Andrea Schmidt
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Tarek Hilal
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Thiemo Sprink
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Kaori Yamamoto
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Thorsten Mielke
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany
| | - Jörg Bürger
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany
| | - Tanvir R Shaikh
- Structural Biology Programme, CEITEC, Masaryk University, Brno, Czech Republic
| | - Marylena Dabrowski
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Peter W Hildebrand
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Patrick Scheerer
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
| | - Christian M T Spahn
- Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin, Berlin, Germany
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Mauro VP, Matsuda D. Translation regulation by ribosomes: Increased complexity and expanded scope. RNA Biol 2015; 13:748-55. [PMID: 26513496 DOI: 10.1080/15476286.2015.1107701] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The primary function of ribosomes is to decode mRNAs into polypeptide chains; however, this description is overly simplistic. Accumulating evidence shows that ribosomes themselves can affect the relative efficiency with which various mRNAs are translated and indicates that these effects can be modulated by ribosome heterogeneity. The notion that ribosomes have regulatory capabilities was elaborated more than a decade ago in the ribosome filter hypothesis. Various lines of evidence support this idea and have shown that the translation of some mRNAs is affected by discrete binding interactions with rRNA or ribosomal proteins. Recent work from our laboratory has demonstrated that base-pairing of the Hepatitis C Virus (HCV) internal ribosome entry site (IRES) to 18S rRNA is required for IRES function, but only in the context of more complex ribosomal interactions. The HCV IRES provides an example of the ribosome filter that involves multiple binding interactions between mRNAs and ribosomal subunits.
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Affiliation(s)
- Vincent P Mauro
- a Promosome, LLC , San Diego , CA , USA.,b The Scripps Research Institute , La Jolla , CA , USA
| | - Daiki Matsuda
- b The Scripps Research Institute , La Jolla , CA , USA
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35
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Swiatkowska A, Zydowicz P, Gorska A, Suchacka J, Dutkiewicz M, Ciesiołka J. The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach. PLoS One 2015; 10:e0141676. [PMID: 26513723 PMCID: PMC4626026 DOI: 10.1371/journal.pone.0141676] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2015] [Accepted: 10/12/2015] [Indexed: 02/05/2023] Open
Abstract
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5'-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2'-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers' binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.
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Affiliation(s)
- Agata Swiatkowska
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Paulina Zydowicz
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Agnieszka Gorska
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Julia Suchacka
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Mariola Dutkiewicz
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Jerzy Ciesiołka
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
- * E-mail:
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Abstract
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis and infects approximately three to four million people per year, about 170 million infected people in total, making it one of the major global health problems. In a minority of cases HCV is cleared spontaneously, but in most of the infected individuals infection progresses to a chronic state associated with high risk to develop liver cirrhosis, hepatocellular cancer, or liver failure. The treatment of HCV infection has evolved over the years. Interferon (IFN)-α in combination with ribavirin has been used for decades as standard therapy. More recently, a new standard-of-care treatment has been approved based on a triple combination with either HCV protease inhibitor telaprevir or boceprevir. In addition, various options for all-oral, IFN-free regimens are currently being evaluated. Despite substantial improvement of sustained virological response rates, some intrinsic limitations of these new direct-acting antivirals, including serious side effects, the risk of resistance development and high cost, urge the development of alternative or additional therapeutic strategies. Gene therapy represents a feasible alternative treatment. Small RNA technology, including RNA interference (RNAi) techniques and antisense approaches, is one of the potentially promising ways to investigate viral and host cell factors that are involved in HCV infection and replication. With this, newly developed gene therapy regimens will be provided to treat HCV. In this chapter, a comprehensive overview guides you through the current developments and applications of RNAi and microRNA-based gene therapy strategies in HCV treatment.
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Abstract
Pestiviruses are among the economically most important pathogens of livestock. The biology of these viruses is characterized by unique and interesting features that are both crucial for their success as pathogens and challenging from a scientific point of view. Elucidation of these features at the molecular level has made striking progress during recent years. The analyses revealed that major aspects of pestivirus biology show significant similarity to the biology of human hepatitis C virus (HCV). The detailed molecular analyses conducted for pestiviruses and HCV supported and complemented each other during the last three decades resulting in elucidation of the functions of viral proteins and RNA elements in replication and virus-host interaction. For pestiviruses, the analyses also helped to shed light on the molecular basis of persistent infection, a special strategy these viruses have evolved to be maintained within their host population. The results of these investigations are summarized in this chapter.
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Affiliation(s)
- Norbert Tautz
- Institute for Virology and Cell Biology, University of Lübeck, Lübeck, Germany
| | - Birke Andrea Tews
- Institut für Immunologie, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany
| | - Gregor Meyers
- Institut für Immunologie, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
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38
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Nawtaisong P, Fraser ME, Carter JR, Fraser MJ. Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells. Virology 2015; 481:223-34. [PMID: 25840398 DOI: 10.1016/j.virol.2015.02.023] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2014] [Revised: 11/25/2014] [Accepted: 02/09/2015] [Indexed: 01/17/2023]
Abstract
The HCV-IRES sequence is vital for both protein translation and genome replication and serves as a potential target for anti-HCV therapy. We constructed a series of anti-HCV group I introns (αHCV-GrpIs) to attack conserved target sites within the HCV IRES. These αHCV-GrpIs were designed to mediate a trans-splicing reaction that replaces the viral RNA genome downstream of the 5' splice site with a 3' exon that encodes an apoptosis-inducing gene. Pro-active forms of the apoptosis inducing genes BID, Caspase 3, Caspase 8, or tBax were modified by incorporation of the HCV NS5A/5B cleavage sequence in place of their respective endogenous cleavage sites to ensure that only HCV infected cells would undergo apoptosis following splicing and expression. Huh7.5 cells transfected with each intron were challenged at MOI 0.1 with HCV-Jc1FLAG2 which expresses a Gaussia Luciferase (GLuc) marker. Virus-containing supernatants were then assayed for GLuc expression as a measure of viral replication inhibition. Cellular extracts were analyzed for the presence of correct splice products by RT-PCR and DNA sequencing. We also measured levels of Caspase 3 activity as a means of quantifying apoptotic cell death. Each of these αHCV-GrpI introns was able to correctly splice their 3' apoptotic exons onto the virus RNA genome at the targeted Uracil, and resulted in greater than 80% suppression of the GLuc marker. A more pronounced suppression effect was observed with TCID₅₀ virus titrations, which demonstrated that these αHCV-GrpIs were able to suppress viral replication by more than 2 logs, or greater than 99%. Robust activation of the apoptotic factor within the challenged cells was evidenced by a significant increase of Caspase 3 activity upon viral infection compared to non-challenged cells. This novel genetic intervention tool may prove beneficial in certain HCV subjects.
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Affiliation(s)
- Pruksa Nawtaisong
- Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States
| | - Mark E Fraser
- Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States
| | - James R Carter
- Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States
| | - Malcolm J Fraser
- Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, United States.
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Lloyd RE. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses. Virology 2015; 479-480:457-74. [PMID: 25818028 PMCID: PMC4426963 DOI: 10.1016/j.virol.2015.03.001] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2014] [Revised: 01/12/2015] [Accepted: 03/03/2015] [Indexed: 01/18/2023]
Abstract
Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups.
Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.
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Affiliation(s)
- Richard E Lloyd
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, United States.
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Boerneke MA, Dibrov SM, Gu J, Wyles DL, Hermann T. Functional conservation despite structural divergence in ligand-responsive RNA switches. Proc Natl Acad Sci U S A 2014; 111:15952-7. [PMID: 25349403 PMCID: PMC4234586 DOI: 10.1073/pnas.1414678111] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.
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Affiliation(s)
| | | | - Jing Gu
- Department of Chemistry and Biochemistry
| | - David L Wyles
- Division of Infectious Diseases, Department of Medicine, and
| | - Thomas Hermann
- Department of Chemistry and Biochemistry, Center for Drug Discovery Innovation, University of California, San Diego, La Jolla, CA 92093
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Khawaja A, Vopalensky V, Pospisek M. Understanding the potential of hepatitis C virus internal ribosome entry site domains to modulate translation initiation via their structure and function. WILEY INTERDISCIPLINARY REVIEWS-RNA 2014; 6:211-24. [PMID: 25352252 PMCID: PMC4361049 DOI: 10.1002/wrna.1268] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/14/2014] [Revised: 08/31/2014] [Accepted: 09/02/2014] [Indexed: 12/16/2022]
Abstract
Translation initiation in the hepatitis C virus (HCV) occurs through a cap-independent mechanism that involves an internal ribosome entry site (IRES) capable of interacting with and utilizing the eukaryotic translational machinery. In this review, we focus on the structural configuration of the different HCV IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. In some cases, multiple mutations, even those scattered across different domains, led to restoration of the translational activity of the HCV IRES, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long-range inter- and/or intra-domain functional interactions. The precise functioning of the HCV IRES requires the specific interaction of its domains with ribosomal subunits and a subset of eukaryotic translation initiation factors (eIFs). The structural conformation, sequence preservation and variability, and translational machinery association with the HCV IRES regions are also thoroughly discussed, along with other factors that can affect and influence the formation of translation initiation complexes. WIREs RNA 2015, 6:211–224. doi: 10.1002/wrna.1268
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Affiliation(s)
- Anas Khawaja
- Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic
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Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo. Proc Natl Acad Sci U S A 2014; 111:15385-9. [PMID: 25313046 DOI: 10.1073/pnas.1413472111] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example--translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine-Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.
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Romero-López C, Berzal-Herranz A. Structure-function relationship in viral RNA genomes: The case of hepatitis C virus. World J Med Genet 2014; 4:6-18. [DOI: 10.5496/wjmg.v4.i2.6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/23/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus (HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5’-untranslatable regions (5’UTRs) and 3’UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5’ terminus of the viral genome and regulated by distal functional RNA domains placed at the 3’ end. Subsequent RNA replication strongly depends on the 3’UTR folding and is also influenced by the 5’ end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNA-RNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.
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Chan SW. Establishment of chronic hepatitis C virus infection: Translational evasion of oxidative defence. World J Gastroenterol 2014; 20:2785-2800. [PMID: 24659872 PMCID: PMC3961964 DOI: 10.3748/wjg.v20.i11.2785] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Revised: 12/03/2013] [Accepted: 01/15/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) causes a clinically important disease affecting 3% of the world population. HCV is a single-stranded, positive-sense RNA virus belonging to the genus Hepacivirus within the Flaviviridae family. The virus establishes a chronic infection in the face of an active host oxidative defence, thus adaptation to oxidative stress is key to virus survival. Being a small RNA virus with a limited genomic capacity, we speculate that HCV deploys a different strategy to evade host oxidative defence. Instead of counteracting oxidative stress, it utilizes oxidative stress to facilitate its own survival. Translation is the first step in the replication of a plus strand RNA virus so it would make sense if the virus can exploit the host oxidative defence in facilitating this very first step. This is particularly true when HCV utilizes an internal ribosome entry site element in translation, which is distinctive from that of cap-dependent translation of the vast majority of cellular genes, thus allowing selective translation of genes under conditions when global protein synthesis is compromised. Indeed, we were the first to show that HCV translation was stimulated by an important pro-oxidant-hydrogen peroxide in hepatocytes, suggesting that HCV is able to adapt to and utilize the host anti-viral response to facilitate its own translation thus allowing the virus to thrive under oxidative stress condition to establish chronicity. Understanding how HCV translation is regulated under oxidative stress condition will advance our knowledge on how HCV establishes chronicity. As chronicity is the initiator step in disease progression this will eventually lead to a better understanding of pathogenicity, which is particularly relevant to the development of anti-virals and improved treatments of HCV patients using anti-oxidants.
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Dibrov SM, Parsons J, Carnevali M, Zhou S, Rynearson KD, Ding K, Garcia Sega E, Brunn ND, Boerneke MA, Castaldi MP, Hermann T. Hepatitis C virus translation inhibitors targeting the internal ribosomal entry site. J Med Chem 2013; 57:1694-707. [PMID: 24138284 DOI: 10.1021/jm401312n] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.
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Affiliation(s)
- Sergey M Dibrov
- Department of Chemistry and Biochemistry, University of California, San Diego , 9500 Gilman Drive, La Jolla, California 92093, United States
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Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 2013; 503:539-43. [PMID: 24185006 DOI: 10.1038/nature12658] [Citation(s) in RCA: 149] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2013] [Accepted: 09/13/2013] [Indexed: 12/12/2022]
Abstract
Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.
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Hepatitis C virus NS3 inhibitors: current and future perspectives. BIOMED RESEARCH INTERNATIONAL 2013; 2013:467869. [PMID: 24282816 PMCID: PMC3825274 DOI: 10.1155/2013/467869] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/29/2013] [Accepted: 09/08/2013] [Indexed: 12/24/2022]
Abstract
Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.
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Imran M, Manzoor S, Ashraf J, Khalid M, Tariq M, Khaliq HM, Azam S. Role of viral and host factors in interferon based therapy of hepatitis C virus infection. Virol J 2013; 10:299. [PMID: 24079723 PMCID: PMC3849893 DOI: 10.1186/1743-422x-10-299] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2013] [Accepted: 09/24/2013] [Indexed: 02/07/2023] Open
Abstract
The current standard of care (SOC) for hepatitis C virus (HCV) infection is the combination of pegylated interferon (PEG-IFN), Ribavirin and protease inhibitor for HCV genotype 1. Nevertheless, this treatment is successful only in 70-80% of the patients. In addition, the treatment is not economical and is of immense physical burden for the subject. It has been established now, that virus-host interactions play a significant role in determining treatment outcomes. Therefore identifying biological markers that may predict the treatment response and hence treatment outcome would be useful. Both IFN and Ribavirin mainly act by modulating the immune system of the patient. Therefore, the treatment response is influenced by genetic variations of the human as well as the HCV genome. The goal of this review article is to summarize the impact of recent scientific advances in this area regarding the understanding of human and HCV genetic variations and their effect on treatment outcomes. Google scholar and PubMed have been used for literature research. Among the host factors, the most prominent associations are polymorphisms within the region of the interleukin 28B (IL28B) gene, but variations in other cytokine genes have also been linked with the treatment outcome. Among the viral factors, HCV genotypes are noteworthy. Moreover, for sustained virological responses (SVR), variations in core, p7, non-structural 2 (NS2), NS3 and NS5A genes are also important. However, all considered single nucleotide polymorphisms (SNPs) of IL28B and viral genotypes are the most important predictors for interferon based therapy of HCV infection.
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Affiliation(s)
- Muhammad Imran
- Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology (NUST), 44000 Islamabad, Pakistan.
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Malygin AA, Kossinova OA, Shatsky IN, Karpova GG. HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation. Nucleic Acids Res 2013; 41:8706-14. [PMID: 23873958 PMCID: PMC3794592 DOI: 10.1093/nar/gkt632] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.
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Affiliation(s)
- Alexey A Malygin
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
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Bai Y, Zhou K, Doudna JA. Hepatitis C virus 3'UTR regulates viral translation through direct interactions with the host translation machinery. Nucleic Acids Res 2013; 41:7861-74. [PMID: 23783572 PMCID: PMC3763534 DOI: 10.1093/nar/gkt543] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.
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Affiliation(s)
- Yun Bai
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of California, Berkeley, CA 94720, USA and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
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