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Bhattarai K, Holcik M. Diverse roles of heterogeneous nuclear ribonucleoproteins in viral life cycle. FRONTIERS IN VIROLOGY 2022. [DOI: 10.3389/fviro.2022.1044652] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Understanding the host-virus interactions helps to decipher the viral replication strategies and pathogenesis. Viruses have limited genetic content and rely significantly on their host cell to establish a successful infection. Viruses depend on the host for a broad spectrum of cellular RNA-binding proteins (RBPs) throughout their life cycle. One of the major RBP families is the heterogeneous nuclear ribonucleoproteins (hnRNPs) family. hnRNPs are typically localized in the nucleus, where they are forming complexes with pre-mRNAs and contribute to many aspects of nucleic acid metabolism. hnRNPs contain RNA binding motifs and frequently function as RNA chaperones involved in pre-mRNA processing, RNA splicing, and export. Many hnRNPs shuttle between the nucleus and the cytoplasm and influence cytoplasmic processes such as mRNA stability, localization, and translation. The interactions between the hnRNPs and viral components are well-known. They are critical for processing viral nucleic acids and proteins and, therefore, impact the success of the viral infection. This review discusses the molecular mechanisms by which hnRNPs interact with and regulate each stage of the viral life cycle, such as replication, splicing, translation, and assembly of virus progeny. In addition, we expand on the role of hnRNPs in the antiviral response and as potential targets for antiviral drug research and development.
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Angulo J, Cáceres CJ, Contreras N, Fernández-García L, Chamond N, Ameur M, Sargueil B, López-Lastra M. Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site. Viruses 2022; 15:8. [PMID: 36680049 PMCID: PMC9864772 DOI: 10.3390/v15010008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 12/03/2022] [Accepted: 12/16/2022] [Indexed: 12/24/2022] Open
Abstract
Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.
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Affiliation(s)
- Jenniffer Angulo
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Facultad de Odontología, Universidad Finis Terrae, Santiago 7501015, Chile
| | - C. Joaquín Cáceres
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA
| | - Nataly Contreras
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Santiago 7500975, Chile
| | - Leandro Fernández-García
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
| | - Nathalie Chamond
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Melissa Ameur
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Bruno Sargueil
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Marcelo López-Lastra
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
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Wang J, Sun D, Wang M, Cheng A, Zhu Y, Mao S, Ou X, Zhao X, Huang J, Gao Q, Zhang S, Yang Q, Wu Y, Zhu D, Jia R, Chen S, Liu M. Multiple functions of heterogeneous nuclear ribonucleoproteins in the positive single-stranded RNA virus life cycle. Front Immunol 2022; 13:989298. [PMID: 36119073 PMCID: PMC9478383 DOI: 10.3389/fimmu.2022.989298] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2022] [Accepted: 08/12/2022] [Indexed: 11/13/2022] Open
Abstract
The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.
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Affiliation(s)
- Jingming Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Di Sun
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- *Correspondence: Anchun Cheng,
| | - Yukun Zhu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Sai Mao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Xuming Ou
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Xinxin Zhao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Juan Huang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Qun Gao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Shaqiu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Qiao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Ying Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Dekang Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Renyong Jia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Mafeng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China
- Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
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Zhang A, Sun Y, Jing H, Liu J, Duan E, Ke W, Tao R, Li Y, Wang J, Cao S, Zhao P, Wang H, Zhang Y. Interaction of HnRNP F with the guanine-rich segments in viral antigenomic RNA enhances porcine reproductive and respiratory syndrome virus-2 replication. Virol J 2022; 19:82. [PMID: 35570267 PMCID: PMC9107676 DOI: 10.1186/s12985-022-01811-4] [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: 01/09/2022] [Accepted: 05/05/2022] [Indexed: 11/12/2022] Open
Abstract
Background Heterogeneous nuclear ribonucleoprotein (HnRNP) F is a member of HnRNP family proteins that participate in splicing of cellular newly synthesized mRNAs by specifically recognizing tandem guanine-tracts (G-tracts) RNA sequences. Whether HnRNP F could recognize viral-derived tandem G-tracts and affect virus replication remain poorly defined. Methods The effect of HnRNP F on porcine reproductive and respiratory syndrome virus (PRRSV) propagation was evaluated by real-time PCR, western blotting, and plaque-forming unit assay. The association between HnRNP F and PRRSV guanine-rich segments (GRS) were analyzed by RNA pulldown and RNA immunoprecipitation. The expression pattern of HnRNP F was investigated by western blotting and nuclear and cytoplasmic fractionation. Results Knockdown of endogenous HnRNP F effectively blocks the synthesis of viral RNA and nucleocapsid (N) protein. Conversely, overexpression of porcine HnRNP F has the opposite effect. Moreover, RNA pulldown and RNA immunoprecipitation assays reveal that the qRMM1 and qRRM2 domains of HnRNP F recognize the GRS in PRRSV antigenomic RNA. Finally, HnRNP F is redistributed into the cytoplasm and forms a complex with guanine-quadruplex (G4) helicase DHX36 during PRRSV infection. Conclusions These findings elucidate the potential functions of HnRNP F in regulating the proliferation of PRRSV and contribute to a better molecular understanding of host-PRRSV interactions.
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Vassilaki N, Frakolaki E, Kalliampakou KI, Sakellariou P, Kotta-Loizou I, Bartenschlager R, Mavromara P. A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF. Int J Mol Sci 2020; 21:ijms21186974. [PMID: 32972019 PMCID: PMC7554737 DOI: 10.3390/ijms21186974] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 09/04/2020] [Accepted: 09/18/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.
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Affiliation(s)
- Niki Vassilaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
- Correspondence: (N.V.); (P.M.)
| | - Efseveia Frakolaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Katerina I. Kalliampakou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Panagiotis Sakellariou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Ioly Kotta-Loizou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany;
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Penelope Mavromara
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
- Laboratory of Biochemistry and Molecular Virology, Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Thrace, Greece
- Correspondence: (N.V.); (P.M.)
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Wan Q, Song D, Li H, He ML. Stress proteins: the biological functions in virus infection, present and challenges for target-based antiviral drug development. Signal Transduct Target Ther 2020; 5:125. [PMID: 32661235 PMCID: PMC7356129 DOI: 10.1038/s41392-020-00233-4] [Citation(s) in RCA: 95] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 05/26/2020] [Accepted: 06/13/2020] [Indexed: 02/06/2023] Open
Abstract
Stress proteins (SPs) including heat-shock proteins (HSPs), RNA chaperones, and ER associated stress proteins are molecular chaperones essential for cellular homeostasis. The major functions of HSPs include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. Regarded as a double-edged sword, HSPs also cooperate with numerous viruses and cancer cells to promote their survival. RNA chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnRNPs), which are essential factors for manipulating both the functions and metabolisms of pre-mRNAs/hnRNAs transcribed by RNA polymerase II. hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. Dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., Parkinson’s diseases, Alzheimer disease), stroke and infectious diseases. In this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. As SPs also attract a great interest as potential antiviral targets (e.g., COVID-19), we also discuss the present progress and challenges in this area of HSP-based drug development, as well as with compounds already under clinical evaluation.
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Affiliation(s)
- Qianya Wan
- Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong, China
| | - Dan Song
- Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong, China
| | - Huangcan Li
- Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong, China
| | - Ming-Liang He
- Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong, China. .,CityU Shenzhen Research Institute, Shenzhen, China.
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Barrera A, Olguín V, Vera-Otarola J, López-Lastra M. Cap-independent translation initiation of the unspliced RNA of retroviruses. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1863:194583. [PMID: 32450258 DOI: 10.1016/j.bbagrm.2020.194583] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Revised: 05/12/2020] [Accepted: 05/18/2020] [Indexed: 12/12/2022]
Abstract
Retroviruses are a unique family of RNA viruses that utilize a virally encoded reverse transcriptase (RT) to replicate their genomic RNA (gRNA) through a proviral DNA intermediate. The provirus is permanently integrated into the host cell chromosome and is expressed by the host cell transcription, RNA processing, and translation machinery. Retroviral messenger RNAs (mRNAs) entirely resemble a cellular mRNA as they have a 5'cap structure, 5'untranslated region (UTR), an open reading frame (ORF), 3'UTR, and a 3'poly(A) tail. The primary transcription product interacts with the cellular RNA processing machinery and is spliced, exported to the cytoplasm, and translated. However, a proportion of the pre-mRNA subverts typical RNA processing giving rise to the full-length RNA. In the cytoplasm, the full-length retroviral RNA fulfills a dual role acting as mRNA and as the gRNA. Simple retroviruses generate two pools of full-length RNA, one for each purpose. However, complex retroviruses have a single pool of full-length RNA, which is destined for translation or encapsidation. As for eukaryotic mRNAs, translational control of retroviral protein synthesis is mostly exerted at the step of initiation. Interestingly, some retroviral mRNAs, both simple and complex, use a dual mechanism to initiate protein synthesis, a cap-dependent initiation mechanism, or via internal initiation using an internal ribosome entry site (IRES). In this review, we describe and discuss data regarding the molecular mechanism driving the canonical cap-dependent and IRES-mediated translation initiation for retroviral mRNA, focusing the discussion mainly on the most studied retroviral mRNA, the HIV-1 mRNA.
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Affiliation(s)
- Aldo Barrera
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
| | - Valeria Olguín
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
| | - Jorge Vera-Otarola
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
| | - Marcelo López-Lastra
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile.
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Gu J, Chen Z, Chen X, Wang Z. Heterogeneous nuclear ribonucleoprotein (hnRNPL) in cancer. Clin Chim Acta 2020; 507:286-294. [PMID: 32376323 DOI: 10.1016/j.cca.2020.04.040] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2019] [Revised: 04/30/2020] [Accepted: 04/30/2020] [Indexed: 12/15/2022]
Abstract
Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a type of RNA binding protein that is mainly located in the nucleus. hnRNPL protein, encoded by the gene located at 19q13.2, is an important member of the hnRNP family. In recent years, studies have shown that hnRNPL is highly expressed in a variety of tumors and plays a vital role in tumor progression. hnRNPL promotes various biological processes of tumor cells, including proliferation, migration and invasion. In this review, we discuss the clinical significance of hnRNPL by reviewing the mechanism of hnRNPL in the tumorigenesis of various cancers.
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Affiliation(s)
- Jingyao Gu
- Cancer Medical Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Zhenyao Chen
- Cancer Medical Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Xin Chen
- Cancer Medical Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Zhaoxia Wang
- Cancer Medical Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, PR China.
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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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Xu L, Shen J, Jia J, Jia R. Inclusion of hnRNP L Alternative Exon 7 Is Associated with Good Prognosis and Inhibited by Oncogene SRSF3 in Head and Neck Squamous Cell Carcinoma. BIOMED RESEARCH INTERNATIONAL 2019; 2019:9612425. [PMID: 31828152 PMCID: PMC6885243 DOI: 10.1155/2019/9612425] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/31/2019] [Revised: 08/22/2019] [Accepted: 10/11/2019] [Indexed: 12/31/2022]
Abstract
BACKGROUND AND OBJECTIVES Alternative splicing is increasingly associated with cancers. HnRNP L is a splicing factor that promotes carcinogenesis in head and neck squamous cell carcinoma (HNSCC) and other cancers. Alternative exon 7 of hnRNP L contains an in-frame stop codon. Exon 7-included transcripts can be degraded via nonsense-mediated decay or encode a truncated hnRNP L protein. Exon 7-excluded transcripts can encode full-length functional hnRNP L protein. HnRNP L has an autoregulation mechanism by promoting the inclusion of its own exon 7. This study aimed to understand the relationship between the alternative splicing of exon 7 and HNSCC. Oncogenic splicing factor SRSF3 has an alternative exon 4 and similar autoregulation mechanism. HnRNP L promotes SRSF3 exon 4 inclusion and then inhibits SRSF3 autoregulation. MATERIALS AND METHODS The relationship between alternative splicing of hnRNP L exon 7 and clinical characteristics of HNSCC in a TCGA dataset was analyzed and confirmed by RT-PCR in a cohort of 61 oral squamous cell carcinoma (OSCC) patients. The regulators of exon 7 splicing were screened in 29 splicing factors and confirmed by overexpression or silencing assay in HEK 293, CAL 27, and SCC-9 cell lines. RESULTS The inclusion of hnRNP L exon 7 was significantly negatively associated with the progression and prognosis of HNSCC, which was confirmed in the cohort of 61 OSCC patients. SRSF3 inhibited exon 7 inclusion and hnRNP L autoregulation and then promoted the expression of full-length functional hnRNP L protein. SRSF3 exon 4 inclusion was correlated with hnRNP L exon 7 inclusion in both HNSCC and breast cancer. HNSCC patients with both low hnRNP L exon 7 and SRSF3 exon 4 inclusion show poor overall survival. CONCLUSIONS Inclusion of hnRNP L alternative exon 7 is associated with good prognosis and inhibited by oncogene SRSF3 in HNSCC.
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Affiliation(s)
- Lingfeng Xu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Jiaoxiang Shen
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
- Department of Orthodontics, Xiamen Stomatology Hospital, Hospital and School of Stomatology, Xiamen Medical University, Xiamen, China
| | - Jun Jia
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China
| | - Rong Jia
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
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11
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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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12
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Sharma G, Raheja H, Das S. Hepatitis C virus: Enslavement of host factors. IUBMB Life 2018; 70:41-49. [PMID: 29281185 DOI: 10.1002/iub.1702] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Revised: 11/20/2017] [Accepted: 11/28/2017] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus (HCV) has infected over 170 million people world-wide. This infection causes severe liver damage that can progress to hepatocellular carcinoma leading to death of the infected patients. Development of a cell culture model system for the study of HCV infection in the recent past has helped the researchers world-wide to understand the biology of this virus. Studies over the past decade have revealed the tricks played by the virus to sustain itself, for as long as 40 years, in the host setup without being eliminated by the immune system. Today we understand that the host organelles and different cellular proteins are affected during HCV infection. This cytoplasmic virus has all the cellular organelles at its disposal to successfully replicate, from ribosomes and intracellular membranous structures to the nucleus. It modulates these organelles at both the structural and the functional levels. The vast knowledge about the viral genome and viral proteins has also helped in the development of drugs against the virus. Despite the achieved success rate to cure the infected patients, we struggle to eliminate the cases of recurrence and the non-responders. Such cases might emerge owing to the property of the viral genome to accumulate mutations during its succeeding replication cycles which favours its survival. The current situation calls an urgent need for alternate therapeutic strategies to counter this major problem of human health. © 2017 IUBMB Life, 70(1):41-49, 2018.
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Affiliation(s)
- Geetika Sharma
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Harsha Raheja
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, Karnataka, India
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13
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Jost I, Shalamova LA, Gerresheim GK, Niepmann M, Bindereif A, Rossbach O. Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges. RNA Biol 2018; 15:1032-1039. [PMID: 29486652 PMCID: PMC6161685 DOI: 10.1080/15476286.2018.1435248] [Citation(s) in RCA: 88] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Circular RNAs (circRNAs) were recently described as a novel class of cellular RNAs. Two circRNAs were reported to function as molecular sponges, sequestering specific microRNAs, thereby de-repressing target mRNAs. Due to their elevated stability in comparison to linear RNA, circRNAs may be an interesting tool in molecular medicine and biology. In this study, we provide a proof-of-principle that circRNAs can be engineered as microRNA sponges. As a model system, we used the Hepatitis C Virus (HCV), which requires cellular microRNA-122 for its life cycle. We produced artificial circRNA sponges in vitro that efficiently sequester microRNA-122, thereby inhibiting viral protein production in an HCV cell culture system. These circRNAs are more stable than their linear counterparts, and localize both to the cytoplasm and to the nucleus, opening up a wide range of potential applications.
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Affiliation(s)
- Isabelle Jost
- a Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, Heinrich-Buff-Ring 17 , Giessen , Germany
| | - Lyudmila A Shalamova
- a Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, Heinrich-Buff-Ring 17 , Giessen , Germany.,b Institute of Biochemistry, Faculty of Medicine, University of Giessen , Friedrichstrasse 24, Giessen , Germany
| | - Gesche K Gerresheim
- a Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, Heinrich-Buff-Ring 17 , Giessen , Germany.,b Institute of Biochemistry, Faculty of Medicine, University of Giessen , Friedrichstrasse 24, Giessen , Germany
| | - Michael Niepmann
- b Institute of Biochemistry, Faculty of Medicine, University of Giessen , Friedrichstrasse 24, Giessen , Germany
| | - Albrecht Bindereif
- a Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, Heinrich-Buff-Ring 17 , Giessen , Germany
| | - Oliver Rossbach
- a Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, Heinrich-Buff-Ring 17 , Giessen , Germany
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14
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Lv D, Wu H, Xing R, Shu F, Lei B, Lei C, Zhou X, Wan B, Yang Y, Zhong L, Mao X, Zou Y. HnRNP-L mediates bladder cancer progression by inhibiting apoptotic signaling and enhancing MAPK signaling pathways. Oncotarget 2017; 8:13586-13599. [PMID: 28088793 PMCID: PMC5355122 DOI: 10.18632/oncotarget.14600] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Accepted: 01/03/2017] [Indexed: 12/28/2022] Open
Abstract
Heterogeneous nuclear ribonucleoprotein L (hnRNP-L) is a promoter of various kinds of cancers, but its actions in bladder cancer (BC) are unclear. In this study, we investigated the function and the underlying mechanism of hnRNP-L in bladder carcinogenesis. Our results demonstrated that enhanced hnRNP-L expression in BC tissues was associated with poor overall survival of BC patients. Depletion of hnRNP-L significantly suppressed cell proliferation in vitro and inhibited xenograft tumor growth in vivo. Furthermore, downregulation of hnRNP-L resulted in G1-phase cell cycle arrest and enhanced apoptosis accompanied by inhibition of EMT and cell migration. All these cellular changes were reversed by ectopic expression of hnRNP-L. Deletion of hnRNP-L resulted in decreased expression of Bcl-2, enhanced expression of caspases-3, -6 and -9 and inhibition of the MAPK signaling pathway. These findings demonstrate that hnRNP-L contributes to poor prognosis and tumor progression of BC by inhibiting the intrinsic apoptotic signaling and enhancing MAPK signaling pathways.
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Affiliation(s)
- Daojun Lv
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Huayan Wu
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Rongwei Xing
- Department of Urology, Affiliated Weihai Second Municipal Hospital of Qingdao University, Weihai 264200, P. R. China
| | - Fangpeng Shu
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Bin Lei
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Chengyong Lei
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Xumin Zhou
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Bo Wan
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Yu Yang
- Department of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, Guangdong Province, 518036, China
| | - Liren Zhong
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
| | - Xiangming Mao
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China.,Department of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, Guangdong Province, 518036, China
| | - Yaguang Zou
- Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China
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15
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Seo JY, Kim DY, Kim SH, Kim HJ, Ryu HG, Lee J, Lee KH, Kim KT. Heterogeneous nuclear ribonucleoprotein (hnRNP) L promotes DNA damage-induced cell apoptosis by enhancing the translation of p53. Oncotarget 2017; 8:51108-51122. [PMID: 28881634 PMCID: PMC5584235 DOI: 10.18632/oncotarget.17003] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 02/06/2017] [Indexed: 12/22/2022] Open
Abstract
The tumor suppressor p53 is an essential gene in the induction of cell cycle arrest, DNA repair, and apoptosis. p53 protein is induced under cellular stress, blocking cell cycle progression and inducing DNA repair. Under DNA damage conditions, it has been reported that post-transcriptional regulation of p53 mRNA contributes to the increase in p53 protein level. Here we demonstrate that heterogeneous nuclear ribonucleoprotein (hnRNP) L enhances p53 mRNA translation. We found that hnRNP L is increased and binds to the 5'UTR of p53 mRNA in response to DNA damage. Increased hnRNP L caused enhancement of p53 mRNA translation. Conversely, p53 protein levels were decreased following hnRNP L knock-down, rendering them resistant to apoptosis and arrest in the G2/M phase after DNA damage. Thus, our findings suggest that hnRNP L functions as a positive regulator of p53 translation and promotes cell cycle arrest and apoptosis.
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Affiliation(s)
- Ji-Young Seo
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
| | - Do-Yeon Kim
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea.,Department of Pharmacology, School of Dentistry, Kyungpook National University (KNU), Daegu, Gyeongbuk, Republic of Korea
| | - Seong-Hoon Kim
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
| | - Hyo-Jin Kim
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
| | - Hye Guk Ryu
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
| | - Juhyun Lee
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
| | - Kyung-Ha Lee
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea.,Division of Bio-technology and Convergence, Daegu Haany University (DHU), Gyeongsan-si, Gyeongbuk, Republic of Korea
| | - Kyong-Tai Kim
- Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea.,Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Republic of Korea
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16
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Regulation Mechanisms of Viral IRES-Driven Translation. Trends Microbiol 2017; 25:546-561. [PMID: 28242053 DOI: 10.1016/j.tim.2017.01.010] [Citation(s) in RCA: 117] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Revised: 01/10/2017] [Accepted: 01/30/2017] [Indexed: 02/06/2023]
Abstract
Internal ribosome entry sites (IRESs) can be found in the mRNA of many viruses as well as in cellular genes involved in the stress response, cell cycle, and apoptosis. IRES-mediated translation can occur when dominant cap-dependent translation is inhibited, and viruses can take advantage of this to subvert host translation machinery. In this review, we focus on the four major types of IRES identified in RNA viruses, and outline their distinct structural properties and requirements of translational factors. We further discuss auxiliary host factors known as IRES trans-acting factors (ITAFs), which are involved in the modulation of optimal IRES activity. Currently known strategies employed by viruses to harness ITAFs and regulate IRES activity are also highlighted.
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17
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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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18
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Loh TJ, Cho S, Moon H, Jang HN, Williams DR, Jung DW, Kim IC, Ghigna C, Biamonti G, Zheng X, Shen H. hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2015; 1849:743-50. [DOI: 10.1016/j.bbagrm.2015.01.004] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/29/2014] [Revised: 01/16/2015] [Accepted: 01/19/2015] [Indexed: 12/27/2022]
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19
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Brunetti JE, Scolaro LA, Castilla V. The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a host factor required for dengue virus and Junín virus multiplication. Virus Res 2015; 203:84-91. [DOI: 10.1016/j.virusres.2015.04.001] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Revised: 03/27/2015] [Accepted: 04/01/2015] [Indexed: 02/05/2023]
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20
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Lenarcic EM, Ziehr BJ, Moorman NJ. An unbiased proteomics approach to identify human cytomegalovirus RNA-associated proteins. Virology 2015; 481:13-23. [PMID: 25765003 DOI: 10.1016/j.virol.2015.02.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2014] [Revised: 11/18/2014] [Accepted: 02/06/2015] [Indexed: 12/13/2022]
Abstract
Post-transcriptional events regulate herpesvirus gene expression, yet few herpesvirus RNA-binding proteins have been identified. We used an unbiased approach coupling oligo(dT) affinity capture with proteomics to identify viral RNA-associated proteins during infection. Using this approach, we identified and confirmed changes in the abundance or activity of two host RNA-associated proteins, DHX9 and DDX3, in cells infected with human cytomegalovirus (HCMV). We also identified and confirmed previously unreported activities for the HCMV US22 and pp71 proteins as RNA-associated viral proteins and confirmed that a known viral RNA-binding protein, pTRS1, associates with RNA in infected cells. Further, we found that HCMV pp71 co-sedimented with polysomes, associated with host and viral RNAs, and stimulated the overall rate of protein synthesis. These results demonstrate that oligo(dT) affinity capture coupled with proteomics provides a rapid and straightforward means to identify RNA-associated viral proteins during infection that may participate in the post-transcriptional control of gene expression.
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Affiliation(s)
- Erik M Lenarcic
- Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States
| | - Benjamin J Ziehr
- Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States
| | - Nathaniel J Moorman
- Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States.
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21
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Sagan SM, Chahal J, Sarnow P. cis-Acting RNA elements in the hepatitis C virus RNA genome. Virus Res 2015; 206:90-8. [PMID: 25576644 DOI: 10.1016/j.virusres.2014.12.029] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2014] [Revised: 12/12/2014] [Accepted: 12/24/2014] [Indexed: 12/22/2022]
Abstract
Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with an estimated 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. As a positive-sense RNA virus, the HCV genome itself must serve as a template for translation, replication and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate structural changes to expose different regions of the genome to viral and cellular proteins to carry out the HCV life cycle. The ∼ 9600 nucleotide viral genome contains a single long open reading frame flanked by 5' and 3' non-coding regions that contain cis-acting RNA elements important for viral translation, replication and stability. Additional cis-acting RNA elements have also been identified in the coding sequences as well as in the 3' end of the negative-strand replicative intermediate. Herein, we provide an overview of the importance of these cis-acting RNA elements in the HCV life cycle.
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Affiliation(s)
- Selena M Sagan
- Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada
| | - Jasmin Chahal
- Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada
| | - Peter Sarnow
- Department of Microbiology and Immunology, Stanford University, Stanford, CA, United States.
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22
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hnRNP L and NF90 interact with hepatitis C virus 5'-terminal untranslated RNA and promote efficient replication. J Virol 2014; 88:7199-209. [PMID: 24719423 DOI: 10.1128/jvi.00225-14] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
UNLABELLED The 5'-terminal sequence of the hepatitis C virus (HCV) positive-strand RNA genome is essential for viral replication. Critical host factors, including a miR-122/Ago2 complex and poly(rC)-binding protein 2 (PCBP2), associate with this RNA segment. We used a biotinylated RNA pulldown approach to isolate host factors binding to the HCV 5' terminal 47 nucleotides and, in addition to Ago2 and PCBP2, identified several novel proteins, including IGF2BP1, hnRNP L, DHX9, ADAR1, and NF90 (ILF3). PCBP2, IGF2BP1, and hnRNP L bound single-stranded RNA, while DHX9, ADAR1, and NF90 bound a cognate double-stranded RNA bait. PCBP2, IGF2BP1, and hnRNP L binding were blocked by preannealing the single-stranded RNA bait with miR-122, indicating that they bind the RNA in competition with miR-122. However, IGF2BP1 binding was also inhibited by high concentrations of heparin, suggesting that it bound the bait nonspecifically. Among these proteins, small interfering RNA-mediated depletion of hnRNP L and NF90 significantly impaired viral replication and reduced infectious virus yields without substantially affecting HCV internal ribosome entry site-mediated translation. hnRNP L and NF90 were found to associate with HCV RNA in infected cells and to coimmunoprecipitate with NS5A in an RNA-dependent manner. Both also associate with detergent-resistant membranes where viral replication complexes reside. We conclude that hnRNP and NF90 are important host factors for HCV replication, at least in cultured cells, and may be present in the replication complex. IMPORTANCE Although HCV replication has been intensively studied in many laboratories, many aspects of the viral life cycle remain obscure. Here, we use a novel RNA pulldown strategy coupled with mass spectrometry to identify host cell proteins that interact functionally with regulatory RNA elements located at the extreme 5' end of the positive-strand RNA genome. We identify two, primarily nuclear RNA-binding proteins, hnRNP L and NF90, with previously unrecognized proviral roles in HCV replication. The data presented add to current understanding of the replication cycle of this pathogenic human virus.
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23
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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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Rossbach O, Hung LH, Khrameeva E, Schreiner S, König J, Curk T, Zupan B, Ule J, Gelfand MS, Bindereif A. Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L. RNA Biol 2014; 11:146-55. [PMID: 24526010 DOI: 10.4161/rna.27991] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a multifunctional RNA-binding protein that is involved in many different processes, such as regulation of transcription, translation, and RNA stability. We have previously characterized hnRNP L as a global regulator of alternative splicing, binding to CA-repeat, and CA-rich RNA elements. Interestingly, hnRNP L can both activate and repress splicing of alternative exons, but the precise mechanism of hnRNP L-mediated splicing regulation remained unclear. To analyze activities of hnRNP L on a genome-wide level, we performed individual-nucleotide resolution crosslinking-immunoprecipitation in combination with deep-sequencing (iCLIP-Seq). Sequence analysis of the iCLIP crosslink sites showed significant enrichment of C/A motifs, which perfectly agrees with the in vitro binding consensus obtained earlier by a SELEX approach, indicating that in vivo hnRNP L binding targets are mainly determined by the RNA-binding activity of the protein. Genome-wide mapping of hnRNP L binding revealed that the protein preferably binds to introns and 3' UTR. Additionally, position-dependent splicing regulation by hnRNP L was demonstrated: The protein represses splicing when bound to intronic regions upstream of alternative exons, and in contrast, activates splicing when bound to the downstream intron. These findings shed light on the longstanding question of differential hnRNP L-mediated splicing regulation. Finally, regarding 3' UTR binding, hnRNP L binding preferentially overlaps with predicted microRNA target sites, indicating global competition between hnRNP L and microRNA binding. Translational regulation by hnRNP L was validated for a subset of predicted target 3'UTRs.
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Affiliation(s)
- Oliver Rossbach
- Institute of Biochemistry; University of Giessen; Giessen, Germany
| | - Lee-Hsueh Hung
- Institute of Biochemistry; University of Giessen; Giessen, Germany
| | - Ekaterina Khrameeva
- Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia
| | - Silke Schreiner
- Institute of Biochemistry; University of Giessen; Giessen, Germany
| | - Julian König
- Institute of Molecular Biology (IMB); Mainz, Germany; Institute of Neurology; University College London; London, United Kingdom
| | - Tomaž Curk
- Faculty of Computer and Information Science; University of Ljubljana; Ljubljana, Slovenia
| | - Blaž Zupan
- Faculty of Computer and Information Science; University of Ljubljana; Ljubljana, Slovenia
| | - Jernej Ule
- Institute of Neurology; University College London; London, United Kingdom
| | - Mikhail S Gelfand
- Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia
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Abstract
ABSTRACT: The Caliciviridae includes small positive-sense, ssRNA viruses, which infect both animals and humans and cause a wide range of diseases. Human caliciviruses are considered the leading cause of outbreaks and sporadic cases of viral gastroenteritis worldwide. Caliciviruses are nonenveloped with a positive-sense, ssRNA genome. As with other positive-sense, ssRNA viruses, they require interactions between viral components and host-cellular factors at different steps along the viral life cycle. Although knowledge about the role of host-cell proteins in the Caliciviridae life cycle remains modest, evidence on this topic is rapidly emerging. This article compiles and discusses the information regarding the involvement of host-cellular factors in the various stages of the calicivirus replication process, emphasizing factors that might be involved in viral translation and/or RNA replication.
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Affiliation(s)
- Ana Lorena Gutiérrez-Escolano
- *Department of Infectomics & Molecular Pathogenesis, Center for Research & Advanced Studies (CINVESTAV), Mexico City, Mexico
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Real CI, Megger DA, Sitek B, Jahn-Hofmann K, Ickenstein LM, John MJ, Walker A, Timm J, Kuhlmann K, Eisenacher M, Meyer HE, Gerken G, Broering R, Schlaak JF. Identification of proteins that mediate the pro-viral functions of the interferon stimulated gene 15 in hepatitis C virus replication. Antiviral Res 2013; 100:654-61. [DOI: 10.1016/j.antiviral.2013.10.009] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
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Yau WY, Shih HC, Tsai MH, Sheu JC, Chen CH, Chow LP. Autoantibody recognition of an N-terminal epitope of hnRNP L marks the risk for developing HBV-related hepatocellular carcinoma. J Proteomics 2013; 94:346-58. [PMID: 24125732 DOI: 10.1016/j.jprot.2013.10.003] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2013] [Revised: 09/06/2013] [Accepted: 10/04/2013] [Indexed: 02/07/2023]
Abstract
UNLABELLED Hepatocellular carcinoma (HCC) is associated with a poor prognosis and remains one of the leading causes of cancer death worldwide. Tumor-associated antigens (TAAs) and autoantibodies have been reported as potential markers in different cancers. Here, we employed an immunoproteomic approach to identify TAAs in the sera of patients with hepatitis B virus-related HCC (HBV-HCC). Immunoreactive spots were excised from 2-DE and analyzed by nano-LC-MS/MS. This analysis identified 16 HCC-associated antigens, including hnRNP L. The antigenicity of hnRNP L was further validated by immunoblotting using recombinant proteins. Autoantibodies against hnRNP L were found in 60% patients with HBV-HCC. Using sera from hnRNP L-positive patients, we found that most of these antibodies recognized glycine-rich region in the N-terminus of hnRNP L. In addition, high titers of autoantibodies against hnRNP L were found in HBV-HCC patients' sera and were associated with increased tumor size and reduced survival rate. hnRNP L protein was also found highly expressed in HCC tissue. Knockdown of hnRNP L significantly suppressed cell growth, migration, and invasion in vitro. Our results indicate that an N-terminal epitope of hnRNP L is a potential biomarker for the diagnosis of HBV-HCC and show that hnRNP L contributes to HCC progression. BIOLOGICAL SIGNIFICANCE In this paper, we employed an immunoproteomic approach to identify TAAs in the sera of patients with hepatitis B virus-related HCC (HBV-HCC). We identified hnRNP L as a tumor-associated antigen in HBV-relative HCC patients. Glycine-rich region located at the N-terminus of hnRNP L constitutes the major epitope. We also demonstrated that hnRNP L is involved in cell proliferation and metastasis.
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Affiliation(s)
- Wen-Yea Yau
- Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
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28
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Wang C, Zhang L, Shen X. Development of a nucleic acid lateral flow strip for detection of hepatitis C virus (HCV) core antigen. NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 2013; 32:59-68. [PMID: 23448141 DOI: 10.1080/15257770.2013.763976] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10(4) copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.
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Affiliation(s)
- Chunfeng Wang
- Digestive System Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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29
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Zhang W, Zeng F, Liu Y, Zhao Y, Lv H, Niu L, Teng M, Li X. Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation. J Biol Chem 2013; 288:22636-49. [PMID: 23782695 DOI: 10.1074/jbc.m113.463901] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.
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Affiliation(s)
- Wenjuan Zhang
- Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China
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Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification. J Virol 2013; 87:8697-712. [PMID: 23740976 DOI: 10.1128/jvi.00950-13] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.
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31
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32
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Reinboth J, Ascierto ML, Chen NG, Zhang Q, Yu YA, Aguilar RJ, Carretero R, Worschech A, Zhao Y, Wang E, Marincola FM, Szalay AA. Correlates between host and viral transcriptional program associated with different oncolytic vaccinia virus isolates. Hum Gene Ther Methods 2012; 23:285-96. [PMID: 23131031 DOI: 10.1089/hgtb.2012.057] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Vaccinia virus (VACV) has emerged as an attractive tool in oncolytic virotherapy. VACV replication efficiency plays a crucial role in the therapeutic outcome. However, little is known about the influence of host factors on viral replication efficiency and permissiveness of a host cell line to infection and oncolysis. In this study, replication of the attenuated VACV GLV-1h68 strain and three wild-type VACV isolates was determined in two autologous human melanoma cell lines (888-MEL and 1936-MEL). Host gene expression and viral gene expression in infected cells were evaluated via respective expression array platforms. Microarray analyses followed by sequential statistical approaches characterized human genes that change specifically due to virus infection. Viral gene transcription correlated with viral replication in a time-dependent manner. A set of human genes revealed strong correlations with the respective viral gene expression. Finally we identified a set of human genes with possible predictive value for viral replication in an independent dataset. The results demonstrate a probable correlation between viral replication, early gene expression, and the respective host response, and thus a possible involvement of human host factors in viral early replication. The characterization of human target genes that influence viral replication could help answer the question of host cell permissiveness to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
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Affiliation(s)
- Jennifer Reinboth
- Genelux Corporation, San Diego Science Center, San Diego, CA 92109, USA
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33
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Reinboth J, Ascierto ML, Chen NG, Zhang Q, Yu YA, Aguilar RJ, Carretero R, Worschech A, Zhao Y, Wang E, Marincola FM, Szalay AA. Correlates between host and viral transcriptional program associated with different oncolytic vaccinia virus isolates. Hum Gene Ther Methods 2012. [DOI: 10.1089/hum.2012.057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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34
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Chatel-Chaix L, Germain MA, Götte M, Lamarre D. Direct-acting and host-targeting HCV inhibitors: current and future directions. Curr Opin Virol 2012; 2:588-98. [PMID: 22959589 DOI: 10.1016/j.coviro.2012.08.002] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2012] [Accepted: 08/07/2012] [Indexed: 02/07/2023]
Abstract
The inclusion of NS3 protease inhibitors to the interferon-containing standard of care improved sustained viral response rates in hepatitis C virus (HCV) infected patients. However, there is still an unmet medical need as this drug regimen is poorly tolerated and lacks efficacy, especially in difficult-to-treat patients. Intense drug discovery and development efforts have focused on direct-acting antivirals (DAA) that target NS3 protease, NS5B polymerase and the NS5A protein. DAA combinations are currently assessed in clinical trials. Alternative antivirals have emerged that target host machineries co-opted by HCV. Finally, continuous and better understanding of HCV biology allows speculating on the value of novel classes of DAA required in future personalized all-oral interferon-free combination therapy and for supporting global disease eradication.
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Affiliation(s)
- Laurent Chatel-Chaix
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Montréal, Québec H3T 1J4, Canada
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35
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Castilla V, Scolaro LA. Involvement of heterogeneous nuclear ribonucleoproteins in viral multiplication. Future Virol 2012. [DOI: 10.2217/fvl.12.48] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The study of virus–host interactions is a major goal in molecular virology and provides new effective targets for antiviral therapies. Heterogeneous nuclear ribonucleoproteins (hnRNPs) constitute a group of cellular RNA-binding proteins localized predominantly within the nucleus, which participate in gene transcription and subsequent RNA post-transcriptional modifications. The interaction between hnRNPs and viral components was extensively demonstrated, as well as the ability of virus infections to alter the intracellular localization or the level of expression of different hnRNPs. The involvement of these proteins in the replication of numerous viruses including members from the Retroviridae, Flaviviridae, Coronaviridae, Arenaviridae, Rhabdoviridae, Papillomaviridae, Orthomyxoviridae, Picornaviridae, Togaviridae and Herpesviridae families, has been reported. In order to gain an increased understanding of the interactions between virus and cell that result in the productive infection of the latter, in this review we discuss the main findings about the role of hnRNPs in different steps of viral replication, such as RNA synthesis, translation, RNA processing and egress of newly assembled progeny virus.
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Affiliation(s)
- Viviana Castilla
- Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Luis A Scolaro
- Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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36
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Liu G, Razanau A, Hai Y, Yu J, Sohail M, Lobo VG, Chu J, Kung SKP, Xie J. A conserved serine of heterogeneous nuclear ribonucleoprotein L (hnRNP L) mediates depolarization-regulated alternative splicing of potassium channels. J Biol Chem 2012; 287:22709-16. [PMID: 22570490 DOI: 10.1074/jbc.m112.357343] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.
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Affiliation(s)
- Guodong Liu
- Department of Physiology, Faculty of Medicine, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB R3E 0J9, Canada
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Abstract
Hepatitis C viral protein translation occurs in a cap-independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5'-untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap-dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis-acting viral factors, as well as trans-acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.
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Affiliation(s)
- Brett Hoffman
- Vaccine and Infectious Disease Organization/International Vaccine Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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Yao L, Dong H, Zhu H, Nelson D, Liu C, Lambiase L, Li X. Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line. J Viral Hepat 2011; 18:e523-9. [PMID: 21914072 PMCID: PMC3736357 DOI: 10.1111/j.1365-2893.2011.01452.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)-mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES-mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES-mediated translation.
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Affiliation(s)
- L. Yao
- Division of Gastroenterology, Department of Medicine, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL
| | - H. Dong
- Department of Pathology, College of Medicine, University of Florida, Gainesville, FL, USA
| | - H. Zhu
- Department of Molecular Medicine, College of Biology Hunan University, Changsha, Hunan Province, China
| | - D. Nelson
- Department of Medicine, College of Medicine, University of Florida, Gainesville, FL
| | - C. Liu
- Department of Pathology, College of Medicine, University of Florida, Gainesville, FL, USA
| | - L. Lambiase
- Division of Gastroenterology, Department of Medicine, University of Tennessee College of Medicine, Chattanooga, TN, USA
| | - X. Li
- Division of Gastroenterology, Department of Medicine, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL
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Casaca A, Fardilha M, da Cruz e Silva E, Cunha C. The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen. Virol J 2011; 8:358. [PMID: 21774814 PMCID: PMC3160407 DOI: 10.1186/1743-422x-8-358] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Accepted: 07/20/2011] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. METHODS A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. RESULTS Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. CONCLUSIONS S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.
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Affiliation(s)
- Ana Casaca
- Unidade de Microbiologia Médica, Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal
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Malys N, McCarthy JEG. Translation initiation: variations in the mechanism can be anticipated. Cell Mol Life Sci 2011; 68:991-1003. [PMID: 21076851 PMCID: PMC11115079 DOI: 10.1007/s00018-010-0588-z] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2010] [Revised: 10/25/2010] [Accepted: 10/28/2010] [Indexed: 01/05/2023]
Abstract
Translation initiation is a critical step in protein synthesis. Previously, two major mechanisms of initiation were considered as essential: prokaryotic, based on SD interaction; and eukaryotic, requiring cap structure and ribosomal scanning. Although discovered decades ago, cap-independent translation has recently been acknowledged as a widely spread mechanism in viruses, which may take place in some cellular mRNA translations. Moreover, it has become evident that translation can be initiated on the leaderless mRNA in all three domains of life. New findings demonstrate that other distinguishable types of initiation exist, including SD-independent in Bacteria and Archaea, and various modifications of 5' end-dependent and internal initiation mechanisms in Eukarya. Since translation initiation has developed through the loss, acquisition, and modification of functional elements, all of which have been elevated by competition with viral translation in a large number of organisms of different complexity, more variation in initiation mechanisms can be anticipated.
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Affiliation(s)
- Naglis Malys
- Manchester Centre for Integrative Systems Biology, Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, The University of Manchester, UK.
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41
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Jeong S, Han SR, Lee YJ, Kim JH, Lee SW. Identification of RNA aptamer specific to mutant KRAS protein. Oligonucleotides 2010; 20:155-61. [PMID: 20565241 DOI: 10.1089/oli.2010.0231] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Mutations in the KRAS gene are required for early occurrence and maintenance of tumorigenesis and are the most frequently found in many types of human malignant diseases. Therefore, approaches targeting RAS function have been proposed for cancer therapy. However, no selective and specific inhibitors of KRAS have yet been developed as anticancer agents. In this study, by employing counter-systematic evolution of ligands by exponential enrichment technique, we identified and characterized an RNA aptamer that specifically bound to mutant KRAS protein with a point mutation in codon 12 of the KRAS gene. Real-time polymerase chain reaction analysis, surface plasmon resonance measurements, and competitive precipitation experiments showed that the selected aptamer contained activities of specific and high-affinity binding to the mutant KRAS (K(D) approximately 4.04 nM) but much less binding to the wild type (K(D) approximately 227 nM). This RNA aptamer could be useful as a ligand for specific therapeutics and diagnostics against mutant KRAS-mediated cancers.
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Affiliation(s)
- Sujin Jeong
- Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin, Republic of Korea
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Abstract
The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.
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Yoon S, Lee G, Han D, Song JY, Kang KS, Lee YS. Neutralization of infectivity of porcine circovirus type 2 (PCV2) by capsid-binding 2'F-RNA aptamers. Antiviral Res 2010; 88:19-24. [PMID: 20615431 DOI: 10.1016/j.antiviral.2010.06.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2010] [Revised: 06/17/2010] [Accepted: 06/28/2010] [Indexed: 11/25/2022]
Abstract
Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated diseases (PCVD), which is responsible for economic losses in the swine industry. The capsid protein of PCV2 has important role for virus neutralization that blocks viral infection. To develop the therapeutic agents, two 2'F-RNA aptamers that bound to the PCV2 capsid protein with nanomole affinity were isolated from a 2'F-RNA library by the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The binding affinity of aptamers was analyzed by Electrophoretic Mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis. The RNA aptamers have been shown to exhibit high affinity and specificity to PCV2 capsid protein and to neutralize PCV2 infectivity in PK-15 cells in dose dependent manner. Neutralizing aptamers such as this could be promising candidates in developing efficacious anti-PCV2 drugs as well as therapeutic delivery reagent.
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Affiliation(s)
- Sorah Yoon
- Virology Division, National Veterinary Research and Quarantine Service, 480, Anyang 6-dong, Manan-gu, Anyang, 430-757, Republic of Korea
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Insights into the biology of IRES elements through riboproteomic approaches. J Biomed Biotechnol 2010; 2010:458927. [PMID: 20150968 PMCID: PMC2817807 DOI: 10.1155/2010/458927] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2009] [Accepted: 12/03/2009] [Indexed: 12/11/2022] Open
Abstract
Translation initiation is a highly regulated process that exerts a strong influence on the posttranscriptional control of gene expression. Two alternative mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism operating in most mRNAs, and the internal ribosome entry site (IRES)-dependent mechanism, first discovered in picornaviruses. IRES elements are highly structured RNA sequences that, in most instances, require specific proteins for recruitment of the translation machinery. Some of these proteins are eukaryotic initiation factors. In addition, RNA-binding proteins (RBPs) play a key role in internal initiation control. RBPs are pivotal regulators of gene expression in response to numerous stresses, including virus infection. This review discusses recent advances on riboproteomic approaches to identify IRES transacting factors (ITAFs) and the relationship between RNA-protein interaction and IRES activity, highlighting the most relevant features on picornavirus and hepatitis C virus IRESs.
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Jeong S, Han SR, Lee YJ, Lee SW. Selection of RNA aptamers specific to active prostate-specific antigen. Biotechnol Lett 2009; 32:379-85. [PMID: 19943183 DOI: 10.1007/s10529-009-0168-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2009] [Revised: 10/30/2009] [Accepted: 10/30/2009] [Indexed: 11/28/2022]
Abstract
A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.
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Affiliation(s)
- Sujin Jeong
- Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, 126, Jukjeon-dong, Suji-gu, Yongin, 448-701, Korea
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Lee JH, Kim SH, Pascua PNQ, Song MS, Baek YH, Jin X, Choi JK, Kim CJ, Kim H, Choi YK. Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression. Virology 2009; 397:89-99. [PMID: 19926108 DOI: 10.1016/j.virol.2009.10.041] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2009] [Revised: 09/18/2009] [Accepted: 10/27/2009] [Indexed: 01/01/2023]
Abstract
To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.
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Affiliation(s)
- Jun Han Lee
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea
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Takeshita M, Ishida YI, Akamatsu E, Ohmori Y, Sudoh M, Uto H, Tsubouchi H, Kataoka H. Proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis C virus RNA. J Biol Chem 2009; 284:21165-76. [PMID: 19531480 DOI: 10.1074/jbc.m109.004945] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.
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Affiliation(s)
- Masahiko Takeshita
- Research Division, Minami Nippon Dairy Co-op Co., Ltd., Miyazaki 885-0073, Japan
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Niepmann M. Internal translation initiation of picornaviruses and hepatitis C virus. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2009; 1789:529-41. [PMID: 19439208 DOI: 10.1016/j.bbagrm.2009.05.002] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2009] [Revised: 04/29/2009] [Accepted: 05/02/2009] [Indexed: 01/02/2023]
Abstract
Picornaviruses and other positive-strand RNA viruses like hepatitis C virus (HCV) enter the cell with a single RNA genome that directly serves as the template for translation. Accordingly, the viral RNA genome needs to recruit the cellular translation machinery for viral protein synthesis. By the use of internal ribosome entry site (IRES) elements in their genomic RNAs, these viruses bypass translation competition with the bulk of capped cellular mRNAs and, moreover, establish the option to largely shut-down cellular protein synthesis. In this review, I discuss the structure and function of viral IRES elements, focusing on the recruitment of the cellular translation machinery by the IRES and on factors that may contribute to viral tissue tropism on the level of translation.
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Affiliation(s)
- Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392 Giessen, Germany.
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