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Kibe A, Buck S, Gribling-Burrer AS, Gilmer O, Bohn P, Koch T, Mireisz CNM, Schlosser A, Erhard F, Smyth RP, Caliskan N. The translational landscape of HIV-1 infected cells reveals key gene regulatory principles. Nat Struct Mol Biol 2025; 32:841-852. [PMID: 39815046 PMCID: PMC12086091 DOI: 10.1038/s41594-024-01468-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Accepted: 12/03/2024] [Indexed: 01/18/2025]
Abstract
Human immunodeficiency virus-1 (HIV-1) uses a number of strategies to modulate viral and host gene expression during its life cycle. To characterize the transcriptional and translational landscape of HIV-1 infected cells, we used a combination of ribosome profiling, disome sequencing and RNA sequencing. We show that HIV-1 messenger RNAs are efficiently translated at all stages of infection, despite evidence for a substantial decrease in the translational efficiency of host genes that are implicated in host cell translation. Our data identify upstream open reading frames in the HIV-1 5'-untranslated region as well as internal open reading frames in the Vif and Pol coding domains. We also observed ribosomal collisions in Gag-Pol upstream of the ribosome frameshift site that we attributed to an RNA structural fold using RNA structural probing and functional analysis. Antisense oligonucleotides designed to alter the base of this structure decreased frameshift efficiency. Overall, our data highlight the complexity of HIV-1 gene regulation and provide a key resource for decoding of host-pathogen interactions upon HIV-1 infection. Furthermore, we provide evidence for a RNA structural fold including the frameshift site that could serve as a target for antiviral therapy.
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Affiliation(s)
- Anuja Kibe
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
| | - Stefan Buck
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
- Faculty of Informatics and Data Science, University of Regensburg, Regensburg, Germany
| | - Anne-Sophie Gribling-Burrer
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
- Institute of Molecular and Cellular Biology (CNRS), UPR 9002, University of Strasbourg, Strasbourg, France
| | - Orian Gilmer
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
| | - Patrick Bohn
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
| | - Tatyana Koch
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
| | - Chiara Noemi-Marie Mireisz
- Institute of Molecular and Cellular Biology (CNRS), UPR 9002, University of Strasbourg, Strasbourg, France
| | - Andreas Schlosser
- Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany
| | - Florian Erhard
- Faculty of Informatics and Data Science, University of Regensburg, Regensburg, Germany
- Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany
| | - Redmond P Smyth
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany
- Institute of Molecular and Cellular Biology (CNRS), UPR 9002, University of Strasbourg, Strasbourg, France
| | - Neva Caliskan
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HIRI-HZI), Würzburg, Germany.
- Faculty of Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany.
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Labaronne E, Décimo D, Bertrand L, Guiguettaz L, Sohier TJM, Cluet D, Vivet-Boudou V, Chaves Valadão AL, Dahoui C, François P, Hatin I, Lambotte O, Samri A, Autran B, Etienne L, Goujon C, Paillart JC, Namy O, Ramirez BC, Ohlmann T, Moris A, Ricci EP. Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression. Nat Commun 2025; 16:1706. [PMID: 39966383 PMCID: PMC11836288 DOI: 10.1038/s41467-025-56772-3] [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: 08/11/2023] [Accepted: 01/29/2025] [Indexed: 02/20/2025] Open
Abstract
Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus that relies on alternative splicing, translational, and post-translational mechanisms to produce over 15 functional proteins from its single ~10 kb transcriptional unit. Using ribosome profiling, nascent protein labeling, RNA sequencing, and whole-proteomics of infected CD4 + T lymphocytes, we characterized the transcriptional, translational, and post-translational landscape during infection. While viral infection exerts a significant impact on host transcript abundance, global translation rates are only modestly affected. Proteomics data reveal extensive transcriptional and post-translational regulation, with many genes showing opposing trends between transcript/ribosome profiling and protein abundance. These findings highlight a complex regulatory network orchestrating gene expression at multiple levels. Viral ribosome profiling further uncovered extensive non-AUG translation of small peptides from upstream open reading frames (uORFs) within the 5' long terminal repeat, which elicit specific T cell responses in people living with HIV. Conservation of uORF translation among retroviruses, along with TAR sequences, shapes DDX3 dependency for efficient translation of the main viral open reading frames.
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Affiliation(s)
- Emmanuel Labaronne
- Laboratoire de Biologie et Modelisation de la Cellule, Ecole Normale Superieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Universite Claude Bernard Lyon 1, 46 allee d'Italie F-69364, Lyon, France
- ADLIN Science, Evry-Courcouronnes, France
| | - Didier Décimo
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
| | - Lisa Bertrand
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Laura Guiguettaz
- Laboratoire de Biologie et Modelisation de la Cellule, Ecole Normale Superieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Universite Claude Bernard Lyon 1, 46 allee d'Italie F-69364, Lyon, France
| | - Thibault J M Sohier
- Laboratoire de Biologie et Modelisation de la Cellule, Ecole Normale Superieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Universite Claude Bernard Lyon 1, 46 allee d'Italie F-69364, Lyon, France
| | - David Cluet
- Laboratoire de Biologie et Modelisation de la Cellule, Ecole Normale Superieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Universite Claude Bernard Lyon 1, 46 allee d'Italie F-69364, Lyon, France
| | - Valérie Vivet-Boudou
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | | | - Clara Dahoui
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
| | - Pauline François
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Isabelle Hatin
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Olivier Lambotte
- Université Paris Saclay, Inserm, CEA, AP-HP, UMR1184 IDMIT, Department of Internal Medicine & Clinical Immunology, Bicêtre Hospital, Le Kremlin Bicêtre, Paris, France
- CEA, DSV/iMETI, Division of Immuno-Virology, IDMIT, Fontenay-aux-Roses, France
| | - Assia Samri
- Sorbonne Université, Inserm U1135, CNRS ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France
| | - Brigitte Autran
- Sorbonne Université, Inserm U1135, CNRS ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France
| | - Lucie Etienne
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
| | | | - Jean-Christophe Paillart
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Olivier Namy
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Bertha Cecilia Ramirez
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
- Mucosal entry, persistance and neuro-immune control of HIV and other viruses. Institut Cochin, INSERM U1016, CNRS UMR8104, Université de Paris Cité, Paris, France
| | - Théophile Ohlmann
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France
| | - Arnaud Moris
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Emiliano P Ricci
- Laboratoire de Biologie et Modelisation de la Cellule, Ecole Normale Superieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Universite Claude Bernard Lyon 1, 46 allee d'Italie F-69364, Lyon, France.
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Woolley CR, Chariker JH, Rouchka EC, Ford EE, Hudson E, Rasche KM, Whitley CS, Vanwinkle Z, Casella CR, Smith ML, Mitchell TC. Full-length mRNA sequencing resolves novel variation in 5' UTR length for genes expressed during human CD4 T-cell activation. Immunogenetics 2025; 77:14. [PMID: 39904916 PMCID: PMC11794378 DOI: 10.1007/s00251-025-01371-1] [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: 11/14/2024] [Accepted: 01/23/2025] [Indexed: 02/06/2025]
Abstract
Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5' untranslated region (UTR). Differential 5' UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5' UTR lengthened by over 100 bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.
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Affiliation(s)
- Cassandra R Woolley
- Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA
| | - Julia H Chariker
- Department of Neuroscience Training, University of Louisville School of Medicine, KY, Louisville, USA
| | - Eric C Rouchka
- Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA
- KY INBRE Bioinformatics Core, University of Louisville School of Medicine, Louisville, KY, USA
| | - Easton E Ford
- Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA
| | - Elizabeth Hudson
- Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA
| | - Kamille M Rasche
- Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA
| | - Caleb S Whitley
- Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA
| | - Zachary Vanwinkle
- Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA
| | - Carolyn R Casella
- Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA
| | - Melissa L Smith
- Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA
| | - Thomas C Mitchell
- Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA.
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Buckingham AB, Ho S, Knops-Mckim F, Ingemarsdotter CK, Lever AM. Optimization of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102341. [PMID: 39434850 PMCID: PMC11491724 DOI: 10.1016/j.omtn.2024.102341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/26/2024] [Accepted: 09/12/2024] [Indexed: 10/23/2024]
Abstract
Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.
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Affiliation(s)
- Amanda B. Buckingham
- University of Cambridge, Department of Medicine, Level 5 Addenbrooke’s Hospital, Hills Rd, Cambridge CB2 0QQ, UK
| | - Sophia Ho
- University of Cambridge, Department of Medicine, Level 5 Addenbrooke’s Hospital, Hills Rd, Cambridge CB2 0QQ, UK
| | | | - Carin K. Ingemarsdotter
- University of Cambridge, Department of Medicine, Level 5 Addenbrooke’s Hospital, Hills Rd, Cambridge CB2 0QQ, UK
| | - Andrew M.L. Lever
- University of Cambridge, Department of Medicine, Level 5 Addenbrooke’s Hospital, Hills Rd, Cambridge CB2 0QQ, UK
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5
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Li W, Huang Y, Yuan H, Han J, Li Z, Tong A, Li Y, Li H, Liu Y, Jia L, Wang X, Li J, Zhang B, Li L. Characterizing transcripts of HIV-1 different substrains using direct RNA sequencing. Heliyon 2024; 10:e39474. [PMID: 39512311 PMCID: PMC11541491 DOI: 10.1016/j.heliyon.2024.e39474] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2024] [Revised: 10/07/2024] [Accepted: 10/15/2024] [Indexed: 11/15/2024] Open
Abstract
Post-transcriptional processing and modification of viral RNA, including alternative splicing, polyadenylation, and methylation, play crucial roles in regulating viral gene expression, enhancing genomic stability, and increasing replication efficiency. These processes have significant implications for viral biology and antiviral therapies. In this study, using Oxford Nanopore Technology (ONT) direct RNA sequencing (DRS), we provided a comprehensive analysis of the transcriptome and epitranscriptome features of the HIV-1 B (NL4-3) subtype strain and, for the first time, characterized these features in the CRF01_AE (GX2005002) subtype strain. We identified 11 novel splicing sites among the 61 RNA isoforms in NL4-3 and defined the splicing sites for GX2005002 based on its 63 RNA isoforms. Furthermore, we identified 74 and 79 chemically modified sites in the transcripts of NL4-3 and GX2005002, respectively. Although differences in poly(A) tail length were observed between the two HIV-1 strains, no specific correlation was detected between poly(A) tail length and the number of modification sites. Additionally, three distinct N6-methyladenosine (m6A) modification sites were identified in both NL4-3 and GX2005002 transcripts. This study provides a detailed analysis of post-transcriptional processing modifications in HIV-1 and suggests promising avenues for future research that could potentially be applied as new therapeutic targets in HIV treatment.
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Affiliation(s)
- Weizhen Li
- School of Public Health and Health Management, Gannan Medical University, Ganzhou, Jiangxi, 341000, China
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Yong Huang
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Haowen Yuan
- Department of Microbiological Laboratory Technology, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, 250012, China
| | - Jingwan Han
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Zhengyang Li
- School of Public Health and Health Management, Gannan Medical University, Ganzhou, Jiangxi, 341000, China
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Aiping Tong
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Yating Li
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Hanping Li
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Yongjian Liu
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Lei Jia
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Xiaolin Wang
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Jingyun Li
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Bohan Zhang
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
| | - Lin Li
- School of Public Health and Health Management, Gannan Medical University, Ganzhou, Jiangxi, 341000, China
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
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6
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Roesmann F, Müller L, Klaassen K, Heß S, Widera M. Interferon-Regulated Expression of Cellular Splicing Factors Modulates Multiple Levels of HIV-1 Gene Expression and Replication. Viruses 2024; 16:938. [PMID: 38932230 PMCID: PMC11209495 DOI: 10.3390/v16060938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 05/31/2024] [Accepted: 06/03/2024] [Indexed: 06/28/2024] Open
Abstract
Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication.
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Affiliation(s)
- Fabian Roesmann
- Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
| | - Lisa Müller
- Institute of Virology, Medical Faculty, University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
| | - Katleen Klaassen
- Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
| | - Stefanie Heß
- Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
| | - Marek Widera
- Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
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7
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Zhong X, Zhou Z, Yang G. The Functions of N-methyladenosine (m6A) Modification on HIV-1 mRNA. Cell Biochem Biophys 2024; 82:561-574. [PMID: 38753251 DOI: 10.1007/s12013-024-01280-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/17/2024] [Indexed: 08/25/2024]
Abstract
In recent years, there has been a growing interest in the study of RNA modifications, with some researchers focusing specifically on the connection between these modifications and viruses, as well as the impact they have on viral mRNA and its functionality. The most common type of RNA chemical modification is m6A, which involves the addition of a methyl group covalently to the N6 position of adenosine. It is a widely observed and evolutionarily conserved RNA modification. The regulation of m6A modification primarily involves methyltransferases (writers) and demethylases (erasers) and is mediated by m6A-binding proteins (readers). In HIV-1, m6A sites are predominantly located in the 5' untranslated region (5'UTR) and 3' untranslated region (3'UTR). Additionally, m6A modifications are also present in the RRE RNA of HIV-1. This review provides a detailed account of the effects of these m6A modifications on HIV-1 functionality.
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Affiliation(s)
- XinYu Zhong
- College of Pharmacy, Zhejiang University of Technology, Hangzhou, 310013, China
- Department of Clinical Medicine, School of Medicine, Hangzhou City University, Hangzhou, 310013, China
| | - ZhuJiao Zhou
- College of Pharmacy, Zhejiang University of Technology, Hangzhou, 310013, China
- Department of Clinical Medicine, School of Medicine, Hangzhou City University, Hangzhou, 310013, China
| | - Geng Yang
- Department of Clinical Medicine, School of Medicine, Hangzhou City University, Hangzhou, 310013, China.
- Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Hangzhou City University, Hangzhou, 310013, China.
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8
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Gribling-Burrer AS, Bohn P, Smyth RP. Isoform-specific RNA structure determination using Nano-DMS-MaP. Nat Protoc 2024; 19:1835-1865. [PMID: 38347203 DOI: 10.1038/s41596-024-00959-3] [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: 08/01/2023] [Accepted: 12/12/2023] [Indexed: 06/12/2024]
Abstract
RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.
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Affiliation(s)
- Anne-Sophie Gribling-Burrer
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
| | - Patrick Bohn
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
| | - Redmond P Smyth
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
- Faculty of Medicine, University of Würzburg, Würzburg, Germany.
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9
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Wattanasombat S, Tongjai S. Easing genomic surveillance: A comprehensive performance evaluation of long-read assemblers across multi-strain mixture data of HIV-1 and Other pathogenic viruses for constructing a user-friendly bioinformatic pipeline. F1000Res 2024; 13:556. [PMID: 38984017 PMCID: PMC11231628 DOI: 10.12688/f1000research.149577.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 05/14/2024] [Indexed: 07/11/2024] Open
Abstract
Background Determining the appropriate computational requirements and software performance is essential for efficient genomic surveillance. The lack of standardized benchmarking complicates software selection, especially with limited resources. Methods We developed a containerized benchmarking pipeline to evaluate seven long-read assemblers-Canu, GoldRush, MetaFlye, Strainline, HaploDMF, iGDA, and RVHaplo-for viral haplotype reconstruction, using both simulated and experimental Oxford Nanopore sequencing data of HIV-1 and other viruses. Benchmarking was conducted on three computational systems to assess each assembler's performance, utilizing QUAST and BLASTN for quality assessment. Results Our findings show that assembler choice significantly impacts assembly time, with CPU and memory usage having minimal effect. Assembler selection also influences the size of the contigs, with a minimum read length of 2,000 nucleotides required for quality assembly. A 4,000-nucleotide read length improves quality further. Canu was efficient among de novo assemblers but not suitable for multi-strain mixtures, while GoldRush produced only consensus assemblies. Strainline and MetaFlye were suitable for metagenomic sequencing data, with Strainline requiring high memory and MetaFlye operable on low-specification machines. Among reference-based assemblers, iGDA had high error rates, RVHaplo showed the best runtime and accuracy but became ineffective with similar sequences, and HaploDMF, utilizing machine learning, had fewer errors with a slightly longer runtime. Conclusions The HIV-64148 pipeline, containerized using Docker, facilitates easy deployment and offers flexibility to select from a range of assemblers to match computational systems or study requirements. This tool aids in genome assembly and provides valuable information on HIV-1 sequences, enhancing viral evolution monitoring and understanding.
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Affiliation(s)
- Sara Wattanasombat
- Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand
| | - Siripong Tongjai
- Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand
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10
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Baek A, Lee GE, Golconda S, Rayhan A, Manganaris AA, Chen S, Tirumuru N, Yu H, Kim S, Kimmel C, Zablocki O, Sullivan MB, Addepalli B, Wu L, Kim S. Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m 6As. Nat Microbiol 2024; 9:1340-1355. [PMID: 38605174 PMCID: PMC11087264 DOI: 10.1038/s41564-024-01638-5] [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: 03/10/2023] [Accepted: 02/15/2024] [Indexed: 04/13/2024]
Abstract
Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.
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Affiliation(s)
- Alice Baek
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA
| | - Ga-Eun Lee
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA
- Translational Data Analytics Institute, Ohio State University, Columbus, OH, USA
| | - Sarah Golconda
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA
| | - Asif Rayhan
- Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA
| | - Anastasios A Manganaris
- Translational Data Analytics Institute, Ohio State University, Columbus, OH, USA
- Department of Computer Science and Engineering, Ohio State University, Columbus, OH, USA
| | - Shuliang Chen
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
| | - Nagaraja Tirumuru
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
| | - Hannah Yu
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA
| | - Shihyoung Kim
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA
| | - Christopher Kimmel
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA
- Translational Data Analytics Institute, Ohio State University, Columbus, OH, USA
| | - Olivier Zablocki
- Center of Microbiome Science, Ohio State University, Columbus, OH, USA
- Department of Microbiology, Ohio State University, Columbus, OH, USA
| | - Matthew B Sullivan
- Center of Microbiome Science, Ohio State University, Columbus, OH, USA
- Department of Microbiology, Ohio State University, Columbus, OH, USA
- Department of Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, OH, USA
| | - Balasubrahmanyam Addepalli
- Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA
| | - Li Wu
- Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Sanggu Kim
- Center for Retrovirus Research, Ohio State University, Columbus, OH, USA.
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA.
- Infectious Diseases Institute, Ohio State University, Columbus, OH, USA.
- Translational Data Analytics Institute, Ohio State University, Columbus, OH, USA.
- Center for RNA Biology, Ohio State University, Columbus, OH, USA.
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11
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Vanegas-Torres CA, Schindler M. HIV-1 Vpr Functions in Primary CD4 + T Cells. Viruses 2024; 16:420. [PMID: 38543785 PMCID: PMC10975730 DOI: 10.3390/v16030420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 03/05/2024] [Accepted: 03/05/2024] [Indexed: 05/23/2024] Open
Abstract
HIV-1 encodes four accesory proteins in addition to its structural and regulatory genes. Uniquely amongst them, Vpr is abundantly present within virions, meaning it is poised to exert various biological effects on the host cell upon delivery. In this way, Vpr contributes towards the establishment of a successful infection, as evidenced by the extent to which HIV-1 depends on this factor to achieve full pathogenicity in vivo. Although HIV infects various cell types in the host organism, CD4+ T cells are preferentially targeted since they are highly permissive towards productive infection, concomitantly bringing about the hallmark immune dysfunction that accompanies HIV-1 spread. The last several decades have seen unprecedented progress in unraveling the activities Vpr possesses in the host cell at the molecular scale, increasingly underscoring the importance of this viral component. Nevertheless, it remains controversial whether some of these advances bear in vivo relevance, since commonly employed cellular models significantly differ from primary T lymphocytes. One prominent example is the "established" ability of Vpr to induce G2 cell cycle arrest, with enigmatic physiological relevance in infected primary T lymphocytes. The objective of this review is to present these discoveries in their biological context to illustrate the mechanisms whereby Vpr supports HIV-1 infection in CD4+ T cells, whilst identifying findings that require validation in physiologically relevant models.
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Affiliation(s)
| | - Michael Schindler
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tuebingen, 72076 Tuebingen, Germany;
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12
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Yandrapally S, Sarkar S, Banerjee S. HIV-1 Tat commandeers nuclear export of Rev-viral RNA complex by controlling hnRNPA2-mediated splicing. J Virol 2023; 97:e0104423. [PMID: 37905837 PMCID: PMC10688328 DOI: 10.1128/jvi.01044-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Accepted: 09/26/2023] [Indexed: 11/02/2023] Open
Abstract
IMPORTANCE HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.
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Affiliation(s)
- Sriram Yandrapally
- Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
| | - Satarupa Sarkar
- Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
| | - Sharmistha Banerjee
- Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
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13
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Yu R, Abdullah SMU, Sun Y. HMMPolish: a coding region polishing tool for TGS-sequenced RNA viruses. Brief Bioinform 2023; 24:bbad264. [PMID: 37478372 PMCID: PMC10516367 DOI: 10.1093/bib/bbad264] [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: 03/25/2023] [Revised: 06/05/2023] [Accepted: 06/29/2023] [Indexed: 07/23/2023] Open
Abstract
Access to accurate viral genomes is important to downstream data analysis. Third-generation sequencing (TGS) has recently become a popular platform for virus sequencing because of its long read length. However, its per-base error rate, which is higher than next-generation sequencing, can lead to genomes with errors. Polishing tools are thus needed to correct errors either before or after sequence assembly. Despite promising results of available polishing tools, there is still room to improve the error correction performance to perform more accurate genome assembly. The errors, particularly those in coding regions, can hamper analysis such as linage identification and variant monitoring. In this work, we developed a novel pipeline, HMMPolish, for correcting (polishing) errors in protein-coding regions of known RNA viruses. This tool can be applied to either raw TGS reads or the assembled sequences of the target virus. By utilizing profile Hidden Markov Models of protein families/domains in known viruses, HMMPolish can correct errors that are ignored by available polishers. We extensively validated HMMPolish on 34 datasets that covered four clinically important viruses, including HIV-1, influenza-A, norovirus, and severe acute respiratory syndrome coronavirus 2. These datasets contain reads with different properties, such as sequencing depth and platforms (PacBio or Nanopore). The benchmark results against popular/representative polishers show that HMMPolish competes favorably on error correction in coding regions of known RNA viruses.
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Affiliation(s)
- Runzhou Yu
- Electrical Engineering, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
| | | | - Yanni Sun
- Electrical Engineering, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
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14
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Bohn P, Gribling-Burrer AS, Ambi UB, Smyth RP. Nano-DMS-MaP allows isoform-specific RNA structure determination. Nat Methods 2023; 20:849-859. [PMID: 37106231 PMCID: PMC10250195 DOI: 10.1038/s41592-023-01862-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Accepted: 03/21/2023] [Indexed: 04/29/2023]
Abstract
Genome-wide measurements of RNA structure can be obtained using reagents that react with unpaired bases, leading to adducts that can be identified by mutational profiling on next-generation sequencing machines. One drawback of these experiments is that short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, information is acquired as a population average in regions that are shared between transcripts, thus blurring the underlying structural landscape. Here, we present nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP)-a method that exploits long-read sequencing to provide isoform-resolved structural information of highly similar RNA molecules. We demonstrate the value of Nano-DMS-MaP by resolving the complex structural landscape of human immunodeficiency virus-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures at the packaging site within the common 5' untranslated region, likely explaining why spliced viral RNAs are excluded from viral particles. Thus, Nano-DMS-MaP is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short-read ensemble analyses.
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Affiliation(s)
- Patrick Bohn
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany
| | - Anne-Sophie Gribling-Burrer
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany
| | - Uddhav B Ambi
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany
| | - Redmond P Smyth
- Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
- Julius-Maximilians-Universität Würzburg, Faculty of Medicine, Würzburg, Germany.
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15
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Gallardo CM, Nguyen AVT, Routh AL, Torbett BE. Selective ablation of 3' RNA ends and processive RTs facilitate direct cDNA sequencing of full-length host cell and viral transcripts. Nucleic Acids Res 2022; 50:e98. [PMID: 35736235 PMCID: PMC9508845 DOI: 10.1093/nar/gkac516] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 04/25/2022] [Accepted: 06/01/2022] [Indexed: 11/13/2022] Open
Abstract
Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3' RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.
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Affiliation(s)
- Christian M Gallardo
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.,Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Anh-Viet T Nguyen
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Andrew L Routh
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.,Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Bruce E Torbett
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.,Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.,Institute for Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA 98109, USA.,Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98101, USA.,Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, USA
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16
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Chazot-Franguiadakis L, Eid J, Socol M, Molcrette B, Guégan P, Mougel M, Salvetti A, Montel F. Optical Quantification by Nanopores of Viruses, Extracellular Vesicles, and Nanoparticles. NANO LETTERS 2022; 22:3651-3658. [PMID: 35475610 DOI: 10.1021/acs.nanolett.2c00253] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 105 particles/mL for an acquisition time of 60 s with the ability to increase the acquisition time to achieve a lower limit.
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Affiliation(s)
| | - Joelle Eid
- Institut de Recherche en Infectiologie de Montpellier, UMR CNRS 9004, Université de Montpellier, Montpellier 34965, France
| | - Marius Socol
- Institut de Recherche en Infectiologie de Montpellier, UMR CNRS 9004, Université de Montpellier, Montpellier 34965, France
| | - Bastien Molcrette
- Laboratoire de Physique, UMR CNRS 5672, ENS de Lyon, Université de Lyon, Lyon 69007, France
| | - Philippe Guégan
- Institut Parisien de Chimie Moléculaire, UMR CNRS 8232, Sorbonne Université, Paris 75252, France
| | - Marylène Mougel
- Institut de Recherche en Infectiologie de Montpellier, UMR CNRS 9004, Université de Montpellier, Montpellier 34965, France
| | - Anna Salvetti
- Centre International de Recherche en Infectiologie, UMR CNRS 5308, Université de Lyon, INSERM, Lyon 69007, France
| | - Fabien Montel
- Laboratoire de Physique, UMR CNRS 5672, ENS de Lyon, Université de Lyon, Lyon 69007, France
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17
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van Heuvel Y, Schatz S, Rosengarten JF, Stitz J. Infectious RNA: Human Immunodeficiency Virus (HIV) Biology, Therapeutic Intervention, and the Quest for a Vaccine. Toxins (Basel) 2022; 14:toxins14020138. [PMID: 35202165 PMCID: PMC8876946 DOI: 10.3390/toxins14020138] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 02/02/2022] [Accepted: 02/09/2022] [Indexed: 11/16/2022] Open
Abstract
Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus (HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells. To do so, the genomic RNA undergoes complex alternative splicing to facilitate the synthesis of the structural, accessory, and regulatory proteins. However, HIV strongly relies on the host cell machinery recruiting cellular factors to complete its replication cycle. Antiretroviral therapy (ART) targets different steps in the cycle, preventing disease progression to the acquired immunodeficiency syndrome (AIDS). The comprehension of the host immune system interaction with the virus has fostered the development of a variety of vaccine platforms. Despite encouraging provisional results in vaccine trials, no effective vaccine has been developed, yet. However, novel promising vaccine platforms are currently under investigation.
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Affiliation(s)
- Yasemin van Heuvel
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln—University of Applied Sciences, Chempark Leverkusen, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany; (Y.v.H.); (S.S.); (J.F.R.)
- Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 3-9, 30167 Hannover, Germany
| | - Stefanie Schatz
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln—University of Applied Sciences, Chempark Leverkusen, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany; (Y.v.H.); (S.S.); (J.F.R.)
- Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 3-9, 30167 Hannover, Germany
| | - Jamila Franca Rosengarten
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln—University of Applied Sciences, Chempark Leverkusen, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany; (Y.v.H.); (S.S.); (J.F.R.)
- Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 3-9, 30167 Hannover, Germany
| | - Jörn Stitz
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln—University of Applied Sciences, Chempark Leverkusen, Kaiser-Wilhelm-Allee, 51368 Leverkusen, Germany; (Y.v.H.); (S.S.); (J.F.R.)
- Correspondence:
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18
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N'Da Konan S, Ségéral E, Bejjani F, Bendoumou M, Ait Said M, Gallois-Montbrun S, Emiliani S. YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity. Retrovirology 2022; 19:4. [PMID: 35101069 PMCID: PMC8805373 DOI: 10.1186/s12977-022-00589-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Accepted: 01/11/2022] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m6A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m6A writers and readers on the biogenesis of HIV-1 RNAs. RESULTS We showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic writer down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear writer YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity. CONCLUSIONS Our findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m6A nuclear reader YTHDC1 is required for efficient production of infectious viral particles.
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Affiliation(s)
- Sarah N'Da Konan
- Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France
| | - Emmanuel Ségéral
- Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France
| | - Fabienne Bejjani
- Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France
| | - Maryam Bendoumou
- Service of Molecular Virology, Department of Molecular Biology, Université Libre de Bruxelles, 6041, Gosselies, Belgium
| | - Mélissa Ait Said
- Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France
| | | | - Stéphane Emiliani
- Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France.
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19
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Affiliation(s)
- Alejandro R Gener
- Integrative Molecular and Biomedical Sciences Program, Graduate School of Biomedical Sciences, Baylor College of Medicine
- Margaret M. and Albert B. Alkek Department of Medicine, Nephrology, Baylor College of Medicine
- Department of Genetics, MD Anderson Cancer Center, Houston, Texas
- School of Medicine, Universidad Central del Caribe, Bayamón, Puerto Rico, USA
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20
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Müller L, Moskorz W, Brillen AL, Hillebrand F, Ostermann PN, Kiel N, Walotka L, Ptok J, Timm J, Lübke N, Schaal H. Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b. Int J Mol Sci 2021; 23:ijms23010156. [PMID: 35008581 PMCID: PMC8745674 DOI: 10.3390/ijms23010156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Revised: 12/18/2021] [Accepted: 12/22/2021] [Indexed: 12/02/2022] Open
Abstract
The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.
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21
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de Oliveira Andrade F, Cucco MS, Borba MMN, Neto RC, Gois LL, de Almeida Rego FF, Santos LA, Barreto FK. An overview of sequencing technology platforms applied to HTLV-1 studies: a systematic review. Arch Virol 2021; 166:3037-3048. [PMID: 34415436 PMCID: PMC8377154 DOI: 10.1007/s00705-021-05204-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2021] [Accepted: 06/20/2021] [Indexed: 11/28/2022]
Abstract
Human T-lymphotropic virus type 1 (HTLV-1) was the first human retrovirus described. The viral factors involved in the different clinical manifestations of infected individuals are still unknown, and in this sense, sequencing technologies can support viral genome studies, contributing to a better understanding of infection outcome. Currently, several sequencing technologies are available with different approaches. To understand the methodological advances in the HTLV-1 field, it is necessary to organize a synthesis by a rigorous review. This systematic literature review describes different technologies used to generate HTLV-1 sequences. The review follows the PRISMA guidelines, and the search for articles was performed in PubMed, Lilacs, Embase, and SciELO databases. From the 574 articles found in search, 62 were selected. The articles showed that, even with the emergence of new sequencing technologies, the traditional Sanger method continues to be the most commonly used methodology for generating HTLV-1 genome sequences. There are many questions that remain unanswered in the field of HTLV-1 research, and this reflects on the small number of studies using next-generation sequencing technologies, which could help address these gaps. The data compiled and analyzed here can help research on HTLV-1, assisting in the choice of sequencing technologies.
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Affiliation(s)
- Felipe de Oliveira Andrade
- Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Campus Anísio Teixeira, Rua Hormindo Barros, 58. Bairro Candeias, Vitória da Conquista, 45.029-094, Brazil
| | - Marina Silveira Cucco
- Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil
- Faculdade de Medicina da Bahia, Programa de Pós-graduação em Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil
| | | | | | - Luana Leandro Gois
- Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil
- Escola Bahiana de Medicina e Saúde Pública, Salvador, Brazil
- Universidade Católica do Salvador, Salvador, Brazil
| | | | - Luciane Amorim Santos
- Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil
- Faculdade de Medicina da Bahia, Programa de Pós-graduação em Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil
- Escola Bahiana de Medicina e Saúde Pública, Salvador, Brazil
- Universidade Católica do Salvador, Salvador, Brazil
| | - Fernanda Khouri Barreto
- Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Campus Anísio Teixeira, Rua Hormindo Barros, 58. Bairro Candeias, Vitória da Conquista, 45.029-094, Brazil.
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22
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Zhang Y, Ma L. Application of high-throughput sequencing technology in HIV drug resistance detection. BIOSAFETY AND HEALTH 2021. [DOI: 10.1016/j.bsheal.2021.06.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
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23
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Abstract
After human immunodeficiency virus type 1 (HIV-1) was identified in the early 1980s, intensive work began to understand the molecular basis of HIV-1 gene expression. Subgenomic HIV-1 RNA regions, spread throughout the viral genome, were described to have a negative impact on the nuclear export of some viral transcripts. Those studies revealed an intrinsic RNA code as a new form of nuclear export regulation. Since such regulatory regions were later also identified in other viruses, as well as in cellular genes, it can be assumed that, during evolution, viruses took advantage of them to achieve more sophisticated replication mechanisms. Here, we review HIV-1 cis-acting repressive sequences that have been identified, and we discuss their possible underlying mechanisms and importance. Additionally, we show how current bioinformatic tools might allow more predictive approaches to identify and investigate them.
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Grewe B, Vogt C, Horstkötter T, Tippler B, Xiao H, Müller B, Überla K, Wagner R, Asbach B, Bohne J. The HIV 5' Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity. Viruses 2021; 13:v13060997. [PMID: 34071819 PMCID: PMC8227319 DOI: 10.3390/v13060997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 05/17/2021] [Accepted: 05/22/2021] [Indexed: 11/16/2022] Open
Abstract
Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.
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Affiliation(s)
- Bastian Grewe
- Department of Molecular and Medical Virology, Ruhr-University, 44801 Bochum, Germany; (B.G.); (B.T.); (H.X.); (B.M.); (K.Ü.)
| | - Carolin Vogt
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany; (C.V.); (T.H.)
| | - Theresa Horstkötter
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany; (C.V.); (T.H.)
| | - Bettina Tippler
- Department of Molecular and Medical Virology, Ruhr-University, 44801 Bochum, Germany; (B.G.); (B.T.); (H.X.); (B.M.); (K.Ü.)
- Department of Biochemistry, Ruhr-University, 44780 Bochum, Germany
| | - Han Xiao
- Department of Molecular and Medical Virology, Ruhr-University, 44801 Bochum, Germany; (B.G.); (B.T.); (H.X.); (B.M.); (K.Ü.)
- Institute of Clinical and Molecular Virology, University Clinics Erlangen, 91054 Erlangen, Germany
| | - Bianca Müller
- Department of Molecular and Medical Virology, Ruhr-University, 44801 Bochum, Germany; (B.G.); (B.T.); (H.X.); (B.M.); (K.Ü.)
| | - Klaus Überla
- Department of Molecular and Medical Virology, Ruhr-University, 44801 Bochum, Germany; (B.G.); (B.T.); (H.X.); (B.M.); (K.Ü.)
- Institute of Clinical and Molecular Virology, University Clinics Erlangen, 91054 Erlangen, Germany
| | - Ralf Wagner
- Institute of Medical Microbiology and Hygiene, University Regensburg, 93053 Regensburg, Germany; (R.W.); (B.A.)
- Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, 93053 Regensburg, Germany
| | - Benedikt Asbach
- Institute of Medical Microbiology and Hygiene, University Regensburg, 93053 Regensburg, Germany; (R.W.); (B.A.)
| | - Jens Bohne
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany; (C.V.); (T.H.)
- Correspondence: ; Tel.: +49-511-532-4308
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25
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Emery A, Swanstrom R. HIV-1: To Splice or Not to Splice, That Is the Question. Viruses 2021; 13:181. [PMID: 33530363 PMCID: PMC7912102 DOI: 10.3390/v13020181] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 01/21/2021] [Accepted: 01/22/2021] [Indexed: 02/05/2023] Open
Abstract
The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.
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MESH Headings
- Alternative Splicing
- Exons
- Gene Expression Regulation, Viral
- HIV-1/genetics
- Nucleic Acid Conformation
- RNA Splicing
- RNA, Messenger/chemistry
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Viral/chemistry
- RNA, Viral/genetics
- RNA, Viral/metabolism
- Regulatory Sequences, Nucleic Acid
- Virus Latency/genetics
- rev Gene Products, Human Immunodeficiency Virus/genetics
- rev Gene Products, Human Immunodeficiency Virus/metabolism
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Affiliation(s)
- Ann Emery
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA;
| | - Ronald Swanstrom
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA;
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
- Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27599, USA
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