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Nunes S, Bastos R, Marinho AI, Vieira R, Benício I, de Noronha MA, Lírio S, Brodskyn C, Tavares NM. Recent advances in the development and clinical application of miRNAs in infectious diseases. Noncoding RNA Res 2025; 10:41-54. [PMID: 39296638 PMCID: PMC11406675 DOI: 10.1016/j.ncrna.2024.09.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 08/06/2024] [Accepted: 09/01/2024] [Indexed: 09/21/2024] Open
Abstract
In the search for new biomarkers and therapeutic targets for infectious diseases, several molecules have been investigated. Small RNAs, known as microRNAs (miRs), are important regulators of gene expression, and have emerged as promising candidates for these purposes. MiRs are a class of small, endogenous non-coding RNAs that play critical roles in several human diseases, including host-pathogen interaction mechanisms. Recently, miRs signatures have been reported in different infectious diseases, opening new perspectives for molecular diagnosis and therapy. MiR profiles can discriminate between healthy individuals and patients, as well as distinguish different disease stages. Furthermore, the possibility of assessing miRs in biological fluids, such as serum and whole blood, renders these molecules feasible for the development of new non-invasive diagnostic and prognostic tools. In this manuscript, we will comprehensively describe miRs as biomarkers and therapeutic targets in infectious diseases and explore how they can contribute to the advance of existing and new tools. Additionally, we will discuss different miR analysis platforms to understand the obstacles and advances of this molecular approach and propose their potential clinical applications and contributions to public health.
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Affiliation(s)
- Sara Nunes
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
| | - Rana Bastos
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Federal University of Bahia (UFBA), Salvador, Brazil
| | - Ananda Isis Marinho
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Federal University of Bahia (UFBA), Salvador, Brazil
| | - Raissa Vieira
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Federal University of Bahia (UFBA), Salvador, Brazil
| | - Ingra Benício
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
| | | | - Sofia Lírio
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Bahiana School of Medicine and Public Health, Salvador, Brazil
| | - Cláudia Brodskyn
- Federal University of Bahia (UFBA), Salvador, Brazil
- Laboratory of Parasite-Host Interaction and Epidemiology (LaIPHE), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Instituto Nacional de Ciência e Tecnologia (INCT) Iii - Instituto de Investigação Em Imunologia, São Paulo, Brazil
| | - Natalia Machado Tavares
- Laboratory of Medicine and Precision Public Health (MeSP), Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
- Federal University of Bahia (UFBA), Salvador, Brazil
- Instituto Nacional de Ciência e Tecnologia (INCT) Iii - Instituto de Investigação Em Imunologia, São Paulo, Brazil
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Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity. Int J Mol Sci 2022; 23:ijms232315059. [PMID: 36499386 PMCID: PMC9740606 DOI: 10.3390/ijms232315059] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2022] [Revised: 11/22/2022] [Accepted: 11/29/2022] [Indexed: 12/04/2022] Open
Abstract
The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with Renilla and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the Renilla gene contained sequences targeted by microRNAs (miRNAs) in the 3'UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter Renilla luciferase synthesis. The mRNA-protein complexes formed by transcripts regulated by different miRNAs differed from each other and were different in different cell types, as shown by sucrose gradient centrifugation. Unexpectedly, the presence of miRNA targets on Renilla transcripts also affected the expression of the co-transfected but non-targeted firefly luciferase gene in both cell types. Renilla and firefly transcripts were found in the same sucrose gradient fractions and specific anti-miRNA oligoribonucleotides, which influenced the expression of the Renilla gene, and also influenced that of firefly gene. These results suggest that, in addition to targeted transcripts, miRNAs may also modulate the expression of non-targeted transcripts, and using the latter to normalize the results may cause bias. We discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.
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USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation. J Virol 2019; 93:JVI.01708-18. [PMID: 30626683 DOI: 10.1128/jvi.01708-18] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2018] [Accepted: 12/23/2018] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.
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Mutational Analysis of the Bovine Hepacivirus Internal Ribosome Entry Site. J Virol 2018; 92:JVI.01974-17. [PMID: 29769341 DOI: 10.1128/jvi.01974-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 05/11/2018] [Indexed: 12/19/2022] Open
Abstract
In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.
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Chan YL, Liao CL, Lin YL. Human Kinase/Phosphatase-Wide RNAi Screening Identified Checkpoint Kinase 2 as a Cellular Factor Facilitating Japanese Encephalitis Virus Infection. Front Cell Infect Microbiol 2018; 8:142. [PMID: 29868498 PMCID: PMC5966567 DOI: 10.3389/fcimb.2018.00142] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Accepted: 04/20/2018] [Indexed: 11/15/2022] Open
Abstract
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans with high mortality. Not much is known about the interactions between viral and cellular factors that regulate JEV infection. By using a kinase/phosphatase-wide RNAi screening approach, we identified a cell cycle-regulating molecule, checkpoint kinase 2 (CHK2), that plays a role in regulating JEV replication. JEV infection induced G1 arrest and activated CHK2. Inactivation of CHK2 and its upstream ataxia-telangiectasia mutated kinase in JEV-infected cells by using inhibitors reduced virus replication. Likewise, JEV replication was significantly decreased by knockdown of CHK2 expression with shRNA-producing lentiviral transduction. We identified CHK2 as a cellular factor participating in JEV replication, for a new strategy in addressing JEV infection.
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Affiliation(s)
- Yi-Lin Chan
- Department of Life Science, Chinese Culture University, Taipei, Taiwan.,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Ching-Len Liao
- Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan.,National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan
| | - Yi-Ling Lin
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan.,Genomics Research Center, Academia Sinica, Taipei, Taiwan
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6
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Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol 2018; 9:395. [PMID: 29593672 PMCID: PMC5857606 DOI: 10.3389/fmicb.2018.00395] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Accepted: 02/21/2018] [Indexed: 12/15/2022] Open
Abstract
Hepatitis C virus (HCV) preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES) in the 5' untranslated region (5' UTR), while also downstream elements like the cis-replication element (CRE) in the coding region and the 3' UTR are involved in translation regulation. The cis-elements controlling replication of the viral RNA genome are located mainly in the 5'- and 3'-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA-RNA interactions (LRIs) are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis-elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122) binds to two target sites at the 5' end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3' UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis-elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis-element in question acts on HCV replication when physically present in the plus strand genome or in the minus strand antigenome. Therefore, it may be required to use reduced systems that selectively focus on the analysis of HCV minus strand initiation and/or plus strand initiation.
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Affiliation(s)
- Michael Niepmann
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Lyudmila A Shalamova
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany.,Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Gesche K Gerresheim
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany.,Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Oliver Rossbach
- Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
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Mengardi C, Limousin T, Ricci EP, Soto-Rifo R, Decimo D, Ohlmann T. microRNAs stimulate translation initiation mediated by HCV-like IRESes. Nucleic Acids Res 2017; 45:4810-4824. [PMID: 28077561 PMCID: PMC5416841 DOI: 10.1093/nar/gkw1345] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 12/22/2016] [Indexed: 01/04/2023] Open
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3΄ untranslated region (UTR) of targeted mRNAs. miRNA-induced inhibition of translation occurs during the initiation step, most probably at the level of ribosome scanning. In this process, the RNA-induced silencing complex interacts both with PABP and the 43S pre-initiation complex to disrupt scanning of the 40S ribosome. However, in some specific cases, miRNAs can stimulate translation. Although the mechanism of miRNA-mediated upregulation is unknown, it appears that the poly(A) tail and the lack of availability of the TNRC6 proteins are amongst major determinants. The genomic RNA of the Hepatitis C Virus is uncapped, non-polyadenylated and harbors a peculiar internal ribosome entry site (IRES) that binds the ribosome directly to the AUG codon. Thus, we have exploited the unique properties of the HCV IRES and other related IRESes (HCV-like) to study how translation initiation can be modulated by miRNAs on these elements. Here, we report that miRNA binding to the 3΄ UTR can stimulate translation of a reporter gene given that its expression is driven by an HCV-like IRES and that it lacks a poly(A) tail at its 3΄ extremity.
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Affiliation(s)
- Chloé Mengardi
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
| | - Taran Limousin
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
| | - Emiliano P Ricci
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
| | - Ricardo Soto-Rifo
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
| | - Didier Decimo
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
| | - Théophile Ohlmann
- CIRI, International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France.,INSERM, U1111, Lyon, France.,Ecole Normale Supérieure de Lyon, Lyon, France.,Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.,CNRS, UMR5308, Lyon, France
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Nieder-Röhrmann A, Dünnes N, Gerresheim GK, Shalamova LA, Herchenröther A, Niepmann M. Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the 5' untranslated region of hepatitis C virus RNA. J Gen Virol 2017; 98:212-224. [PMID: 28008821 DOI: 10.1099/jgv.0.000697] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The liver-specific microRNA-122 (miR-122) binds to two conserved binding sites in the 5' UTR of hepatitis C virus (HCV) RNA. This binding was reported to enhance HCV RNA replication, translation and stability. We have analysed binding of miR-122/Argonaute 2 (Ago2) complexes to these sites using anti-Ago2 co-immunoprecipitation of radioactively labelled HCV RNAs along with ectopic miR-122 in HeLa cells. Our results show that the miR-122 target sites can be addressed separately. When both target sites were addressed simultaneously, we observed a synergistic binding of both miR/Ago2 complexes. Consistently, simultaneous binding of both miR-122/Ago2 complexes results in cooperative translation stimulation. In the binding assays as well as in the translation assays, binding site 1 has a stronger effect than binding site 2. We also analysed the overall RNA stability as well as the 5' end integrity of these HCV RNAs in the presence of miR-122. Surprisingly, using short HCV reporter RNAs, we did not find effects of miR-122 binding on overall RNA stability or 5' end integrity over up to 36 h. In contrast, using full-length HCV genomes that are incapable of replication, we found a positive influence of miR-122 on RNA stability, indicating that features of the full-length HCV genome that do not reside in the 5' and 3' UTRs may render HCV RNA genome stability miR-122 dependent.
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Affiliation(s)
- Anika Nieder-Röhrmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
| | - Nadia Dünnes
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
| | - Gesche K Gerresheim
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
| | - Lyudmila A Shalamova
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
| | - Andreas Herchenröther
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
| | - Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany
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9
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Gerresheim GK, Dünnes N, Nieder-Röhrmann A, Shalamova LA, Fricke M, Hofacker I, Höner Zu Siederdissen C, Marz M, Niepmann M. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure. Cell Mol Life Sci 2017; 74:747-760. [PMID: 27677491 PMCID: PMC11107659 DOI: 10.1007/s00018-016-2377-9] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Revised: 08/29/2016] [Accepted: 09/21/2016] [Indexed: 02/08/2023]
Abstract
We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.
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Affiliation(s)
- Gesche K Gerresheim
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Nadia Dünnes
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Anika Nieder-Röhrmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Lyudmila A Shalamova
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Markus Fricke
- Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany
| | - Ivo Hofacker
- Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria
| | - Christian Höner Zu Siederdissen
- Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria
- Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, Universität Leipzig, 04107, Leipzig, Germany
| | - Manja Marz
- Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany
- FLI Leibniz Institute for Age Research, 07743, Jena, Germany
| | - Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
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Piedade D, Azevedo-Pereira JM. MicroRNAs, HIV and HCV: a complex relation towards pathology. Rev Med Virol 2016; 26:197-215. [PMID: 27059433 DOI: 10.1002/rmv.1881] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Revised: 03/11/2016] [Accepted: 03/15/2016] [Indexed: 12/13/2022]
Abstract
MicroRNAs are small non-coding RNAs that modulate protein production by post-transcriptional gene regulation. They impose gene expression control by interfering with mRNA translation and stability in cell cytoplasm through a mechanism involving specific binding to mRNA based on base pair complementarity. Because of their intracellular replication cycle it is no surprise that viruses evolved in a way that allows them to use microRNAs to infect, replicate and persist in host cells. Several ways of interference between virus and host-cell microRNA machinery have been described. Most of the time, viruses drastically alter host-cell microRNA expression or synthesize their own microRNA to facilitate infection and pathogenesis. HIV and HCV are two prominent examples of this complex interplay revealing how fine-tuning of microRNA expression is crucial for controlling key host pathways that allow viral infection and replication, immune escape and persistence. In this review we delve into the mechanisms underlying cellular and viral-encoded microRNA functions in the context of HIV and HCV infections. We focus on which microRNAs are differently expressed and deregulated upon viral infection and how these alterations dictate the fate of virus and cell. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Diogo Piedade
- Host-Pathogen Interaction Unit, iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa, Portugal
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11
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Murine norovirus replication induces G0/G1 cell cycle arrest in asynchronously growing cells. J Virol 2015; 89:6057-66. [PMID: 25810556 DOI: 10.1128/jvi.03673-14] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Accepted: 03/20/2015] [Indexed: 01/08/2023] Open
Abstract
UNLABELLED Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae. IMPORTANCE Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication.
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Abstract
Hepatitis C virus (HCV) is a global health burden with an estimated 170-200 million peoples chronically infected worldwide. HCV infection remains as an independent risk factor for chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, and a major reason for liver transplantation. Discovery of direct acting antiviral (DAA) drugs have shown promising results with more than 90% success rate in clearing the HCV RNA in patients, although long-term consequences remain to be evaluated. microRNAs (miRNAs) are important players in establishment of HCV infection and target crucial host cellular factors needed for productive HCV replication and augmented cell growth. Altered expression of miRNAs is involved in the pathogenesis associated with HCV infection by controlling signaling pathways such as immune response, proliferation and apoptosis. miRNA is emerging as a means of communication between various cell types inside the liver. There is likely possibility of developing circulating miRNAs as biomarkers of disease progression and can also serve as diagnostic tool with potential of early therapeutic intervention in HCV associated end stage liver disease. This review focuses on recent studies highlighting the contribution of miRNAs in HCV life cycle and their coordinated regulation in HCV mediated liver disease progression.
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Affiliation(s)
| | - Robert Steele
- Departments of Pathology, Saint Louis University, St. Louis, Missouri, USA
| | - Ranjit Ray
- Departments of Internal Medicine, Saint Louis University, St. Louis, Missouri, USA
| | - Ratna B Ray
- Departments of Pathology, Saint Louis University, St. Louis, Missouri, USA
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Zhou Y, Sun L, Wang X, Zhou L, Li J, Liu M, Wang F, Peng J, Gui X, Zhao H, Reichenbach N, Zhou D, Ho WZ. Heroin use promotes HCV infection and dysregulates HCV-related circulating microRNAs. J Neuroimmune Pharmacol 2015; 10:102-10. [PMID: 25572448 DOI: 10.1007/s11481-014-9577-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2014] [Accepted: 12/23/2014] [Indexed: 01/22/2023]
Abstract
Hepatitis C virus (HCV) infection is common among injection drug users (IDUs). There is accumulating evidence that circulating microRNAs (miRNAs) are associated with HCV infection and disease progression. The present study was undertaken to determine the in vivo impact of heroin use on HCV infection and HCV-related circulating miRNA expression. Using the blood specimens from four groups of the study subjects (HCV-infected individuals, heroin users with/without HCV infection, and healthy volunteers), we found that HCV-infected heroin users had significantly higher viral load than HCV-infected non-heroin users (p = 0.0004). Measurement of HCV-related circulating miRNAs in plasma showed that miRs-122, 141, 29a, 29b, and 29c were significantly increased in the heroin users with HCV infection, whereas miR-351, an HCV inhibitory miRNA, was significantly decreased in heroin users as compared to control subjects. Further investigation identified a negative correlation between the plasma levels of miR-29 family members and severity of HCV infection based on aspartate aminotransferase to platelet ratio index (APRI). In addition, heroin use and/or HCV infection also dysregulated a panel of plasma miRNAs. Taken together, these data for the first time revealed in vivo evidence that heroin use and/or HCV infection alter circulating miRNAs, which provides a novel mechanism for the impaired innate anti-HCV immunity among IDUs.
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Affiliation(s)
- Yu Zhou
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3500 N. Broad St., Philadelphia, PA, 19140, USA
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14
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Ohlmann T, Mengardi C, López-Lastra M. Translation initiation of the HIV-1 mRNA. ACTA ACUST UNITED AC 2014; 2:e960242. [PMID: 26779410 DOI: 10.4161/2169074x.2014.960242] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2014] [Revised: 05/23/2014] [Accepted: 06/17/2014] [Indexed: 12/17/2022]
Abstract
Translation initiation of the full-length mRNA of the human immunodeficiency virus can occur via several different mechanisms to maintain production of viral structural proteins throughout the replication cycle. HIV-1 viral protein synthesis can occur by the use of both a cap-dependant and IRES-driven mechanism depending on the physiological conditions of the cell and the status of the ongoing infection. For both of these mechanisms there is a need for several viral and cellular co-factors for optimal translation of the viral mRNA. In this review we will describe the mechanism used by the full-length mRNA to initiate translation highlighting the role of co-factors within this process. A particular emphasis will be given to the role of the DDX3 RNA helicase in HIV-1 mRNA translation initiation.
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Affiliation(s)
- Théophile Ohlmann
- CIRI; International Center for Infectiology Research; Université de Lyon; Lyon, France; Inserm; Lyon, France; Ecole Normale Supérieure de Lyon; Lyon, France; Université Lyon 1; Center International de Recherche en Infectiologie; Lyon, France; CNRS; Lyon, France
| | - Chloé Mengardi
- CIRI; International Center for Infectiology Research; Université de Lyon; Lyon, France; Inserm; Lyon, France; Ecole Normale Supérieure de Lyon; Lyon, France; Université Lyon 1; Center International de Recherche en Infectiologie; Lyon, France; CNRS; Lyon, France
| | - Marcelo López-Lastra
- Laboratorio de Virología Molecular; Instituto Milenio de Inmunología e Inmunoterapia; Centro de Investigaciones Médicas; Escuela de Medicina; Pontificia Universidad Católica de Chile ; Santiago, Chile
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15
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Gupta P, Cairns MJ, Saksena NK. Regulation of gene expression by microRNA in HCV infection and HCV-mediated hepatocellular carcinoma. Virol J 2014; 11:64. [PMID: 24690114 PMCID: PMC3977900 DOI: 10.1186/1743-422x-11-64] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Accepted: 03/27/2014] [Indexed: 02/06/2023] Open
Abstract
MicroRNA (miRNA) exert a profound effect on Hepatitis C virus (HCV) replication and on the manifestation of HCV-associated hepatocellular carcinoma (HCC). miR-122 in particular, is highly enriched in liver and has been shown to interact with HCV, suggesting this virus has evolved to subvert and manipulate the host gene silencing machinery in order to support its life cycle. It is therefore likely that miR-122 and other miRNAs play an important role in the pathophysiology of HCV infection. The changes in post-transcriptional gene regulation by the miRNAs may play a key role in the manifestation of chronic liver disease and hepatocellular carcinoma. Understanding of HCV-host miRNA interactions will ultimately lead to the design of therapeutic modalities against HCV infection and HCV-mediated HCC and may also provide important biomarkers that direct treatment options. Here, we review the current knowledge on the role of miRNA and gene expression on HCV infection and hepatocellular carcinoma, in addition to the possible role of miRNA as future therapeutic targets.
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Affiliation(s)
| | | | - Nitin K Saksena
- Centre for Virus Research, Westmead Millennium Institute, Darcy Road, Sydney, Westmead NSW 2145, Australia.
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16
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Hepatitis C virus (HCV) interaction with astrocytes: nonproductive infection and induction of IL-18. J Neurovirol 2014; 20:278-93. [PMID: 24671718 DOI: 10.1007/s13365-014-0245-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2014] [Revised: 02/10/2014] [Accepted: 02/20/2014] [Indexed: 12/15/2022]
Abstract
Hepatitis C virus (HCV) infection causes the central nervous system (CNS) abnormalities in more than 50 % of chronically infected subjects. However, the underlying mechanisms are largely unknown. In this study, we characterized the HCV interactions with astrocytes, one of the putative HCV target cells in the brain. We demonstrated that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the cell-free form or through cell-cell contact. We then determined the potential restriction steps of HCV infection and replication in these cells. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated RNA translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, we found that HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. Taken together, these results showed that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction.
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Abstract
To replicate their genomes in cells and generate new progeny, viruses typically require factors provided by the cells that they have infected. Subversion of the cellular machinery that controls replication of the infected host cell is a common activity of many viruses. Viruses employ different strategies to deregulate cell cycle checkpoint controls and modulate cell proliferation pathways. A number of DNA and RNA viruses encode proteins that target critical cell cycle regulators to achieve cellular conditions that are beneficial for viral replication. Many DNA viruses induce quiescent cells to enter the cell cycle; this is thought to increase pools of deoxynucleotides and thus, facilitate viral replication. In contrast, some viruses can arrest cells in a particular phase of the cell cycle that is favorable for replication of the specific virus. Cell cycle arrest may inhibit early cell death of infected cells, allow the cells to evade immune defenses, or help promote virus assembly. Although beneficial for the viral life cycle, virus-mediated alterations in normal cell cycle control mechanisms could have detrimental effects on cellular physiology and may ultimately contribute to pathologies associated with the viral infection, including cell transformation and cancer progression and maintenance. In this chapter, we summarize various strategies employed by DNA and RNA viruses to modulate the replication cycle of the virus-infected cell. When known, we describe how these virus-associated effects influence replication of the virus and contribute to diseases associated with infection by that specific virus.
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Affiliation(s)
- Eishi Noguchi
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania USA
| | - Mariana C. Gadaleta
- Dept of Biochemistry & Molecular Biology, Drexel University College of Medicine, Philadelphia, USA
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18
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Conrad KD, Niepmann M. The role of microRNAs in hepatitis C virus RNA replication. Arch Virol 2013; 159:849-62. [PMID: 24158346 DOI: 10.1007/s00705-013-1883-4] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Accepted: 09/28/2013] [Indexed: 12/16/2022]
Abstract
Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.
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Affiliation(s)
- K Dominik Conrad
- Institute of Biochemistry, School of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
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19
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Shrivastava S, Mukherjee A, Ray RB. Hepatitis C virus infection, microRNA and liver disease progression. World J Hepatol 2013; 5:479-486. [PMID: 24073299 PMCID: PMC3782685 DOI: 10.4254/wjh.v5.i9.479] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 07/30/2013] [Accepted: 08/16/2013] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a global health problem with an estimated 170-200 million peoples (approximately 3% of world population) are chronically infected worldwide and new infections are predicted to be on rise in coming years. HCV infection remains categorized as a major risk factor for chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. There has been considerable improvement in our understanding of virus life cycle since, the discovery of HCV two-decades ago. MicroRNAs (miRNAs) are important players in establishment of HCV infection and their propagation in infected hepatocytes. They target crucial host cellular factors needed for productive HCV replication and augmented cell growth. Very first anti-miRNA oligonucleotides, miravirsen has been tested in clinical trial and shown promising results as therapeutic agent in treatment against chronic HCV infection. Deregulated expression of miRNAs has been linked to the pathogenesis associated with HCV infection by controlling signaling pathways such as, proliferation, apoptosis and migration. Circulating miRNAs emerging as growing field in identification of biomarkers in disease progression and their potential as a means of communication between cells inside the liver is an exciting area of research in future. This review focuses on recent studies enforcing the contribution of miRNAs in HCV life cycle and coordinated regulation in HCV mediated liver disease progression.
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Conteduca V, Sansonno D, Russi S, Pavone F, Dammacco F. Therapy of chronic hepatitis C virus infection in the era of direct-acting and host-targeting antiviral agents. J Infect 2013; 68:1-20. [PMID: 24012819 DOI: 10.1016/j.jinf.2013.08.019] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2013] [Revised: 08/07/2013] [Accepted: 08/22/2013] [Indexed: 02/06/2023]
Abstract
OBJECTIVES Chronic hepatitis C virus (HCV) infection represents a leading worldwide medical and social problem. The expanding knowledge of HCV lifecycle has led to the development of novel antiviral agents that: a) specifically target a viral function (direct-acting antivirals), or b) specifically inhibit viral replication. The present review describes the novel anti-HCV drugs that have been better studied at the time of this writing and the current two types of treatment, namely interferon-based and interferon-free regimens. In addition, predictive factors, virological responses, side-effects, and resistance mechanisms of the novel agents are summarized. CONCLUSIONS The introduction of novel antiviral agents is remarkably changing the therapeutic combinations aimed at improving virological responses both for easy-to-cure and difficult-to-treat patients. Since additional, effective drugs are under advanced development, it seems reasonable to expect that further therapeutic and prognostic improvements will be achieved in the near future.
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Affiliation(s)
- Vincenza Conteduca
- Section of Internal Medicine and Clinical Oncology, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
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21
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Hu MC, Di Sole F, Zhang J, McLeroy P, Moe OW. Chronic regulation of the renal Na(+)/H(+) exchanger NHE3 by dopamine: translational and posttranslational mechanisms. Am J Physiol Renal Physiol 2013; 304:F1169-80. [PMID: 23427139 DOI: 10.1152/ajprenal.00630.2012] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The intrarenal autocrine/paracrine dopamine (DA) system contributes to natriuresis in response to both acute and chronic Na(+) loads. While the acute DA effect is well described, how DA induces natriuresis chronically is not known. We used an animal and a cell culture model to study the chronic effect of DA on a principal renal Na(+) transporter, Na(+)/H(+) exchanger-3 (NHE3). Intraperitoneal injection of Gludopa in rats for 2 days elevated DA excretion and decreased total renal cortical and apical brush-border NHE3 antigen. Chronic treatment of an opossum renal proximal cell line with DA decreased NHE3 activity, cell surface and total cellular NHE3 antigen, but not NHE3 transcript. The decrease in NHE3 antigen was dose and time dependent with maximal inhibition at 16-24 h and half maximal effect at 3 × 10(-7) M. This is in contradistinction to the acute effect of DA on NHE3 (half maximal at 2 × 10(-6) M), which was not associated with changes in total cellular NHE3 protein. The DA-induced decrease in total NHE3 protein was associated with decrease in NHE3 translation and mediated by cis-sequences in the NHE3 5'-untranslated region. DA also decreased cell surface and total cellular NHE3 protein half-life. The DA-induced decrease in total cellular NHE3 was partially blocked by proteasome inhibition but not by lysosome inhibition, and DA increased ubiquitylation of total and surface NHE3. In summary, chronic DA inhibits NHE3 with mechanisms distinct from its acute action and involves decreased NHE3 translation and increased NHE3 degradation, which are novel mechanisms for NHE3 regulation.
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Affiliation(s)
- Ming Chang Hu
- Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8885, USA
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22
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Conrad KD, Giering F, Erfurth C, Neumann A, Fehr C, Meister G, Niepmann M. MicroRNA-122 dependent binding of Ago2 protein to hepatitis C virus RNA is associated with enhanced RNA stability and translation stimulation. PLoS One 2013; 8:e56272. [PMID: 23405269 PMCID: PMC3566042 DOI: 10.1371/journal.pone.0056272] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2012] [Accepted: 01/08/2013] [Indexed: 01/16/2023] Open
Abstract
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.
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Affiliation(s)
- K. Dominik Conrad
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Florian Giering
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Corinna Erfurth
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Angelina Neumann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Carmen Fehr
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Gunter Meister
- Institute of Biochemistry, Faculty of Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany
| | - Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
- * E-mail:
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24
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MicroRNA-mediated mRNA translation activation in quiescent cells and oocytes involves recruitment of a nuclear microRNP. Sci Rep 2012; 2:842. [PMID: 23150790 PMCID: PMC3496365 DOI: 10.1038/srep00842] [Citation(s) in RCA: 117] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Accepted: 09/25/2012] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs can promote translation of specific mRNAs in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes. We report that microRNA-mediated upregulation of target mRNAs in oocytes is dependent on nuclear entry of the microRNA; cytoplasmically-injected microRNA repress target mRNAs. Components of the activation microRNP, AGO, FXR1 (FXR1-iso-a) and miR16 are present in the nucleus and cytoplasm. Importantly, microRNA target mRNAs for upregulation, Myt1, TNFα and a reporter bearing the TNFα AU-rich, microRNA target sequence, are associated with AGO in immature oocyte nuclei and AGO2 in G0 human nuclei, respectively. mRNAs that are repressed or lack target sites are not associated with nuclear AGO. Crosslinking-coupled immunopurification revealed greater association of AGO2 with FXR1 in the nucleus compared to cytoplasm. Consistently, overexpression of FXR1-iso-a rescues activation of cytoplasmically-injected RNAs and in low density, proliferating cells. These data indicate the importance of a compartmentalized AGO2-FXR1-iso-a complex for selective recruitment for microRNA-mediated upregulation.
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25
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Abstract
MicroRNAs (miRNAs) can exert a profound effect on Hepatitis C virus (HCV) replication. The interaction of HCV with the highly liver-enriched miRNA, miR-122 represents one such unique example of viruses having evolved mechanism(s) to usurp the host miRNA machinery to support viral life cycle. Furthermore, HCV infection can also trigger changes in the cellular miRNA profile, which may ultimately contribute to the outcome of viral infection. Accumulating knowledge on HCV-host miRNA interactions has ultimately influenced the design of therapeutic interventions against chronic HCV infection. The importance of microRNA modulation in Human Immunodeficiency Virus (HIV-1) replication has been reported, albeit only in the context of HIV-1 mono-infection. The development of HCV infection is dramatically influenced during co-infection with HIV-1. Here, we review the current knowledge on miRNAs in HCV mono-infection. In addition, we discuss the potential role of some miRNAs, identified from the analyses of public data, in HCV/HIV-1 co-infection.
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Vasudevan S. Functional validation of microRNA-target RNA interactions. Methods 2012; 58:126-34. [PMID: 22910526 DOI: 10.1016/j.ymeth.2012.08.002] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2012] [Revised: 08/05/2012] [Accepted: 08/06/2012] [Indexed: 12/15/2022] Open
Abstract
MicroRNAs are small, non-coding RNA regulators of gene expression with important outcomes in cell state, proliferation, metabolism, immunity and development; their deregulation leads to significant clinical consequences. MicroRNAs and their associated target RNAs can be identified by genetic, bioinformatic and biochemical methods. MicroRNAs can recognize target mRNAs via direct base-pairing and recruit effector complexes to modulate their gene expression in a sequence-specific manner. MicroRNA interactions with target RNAs produce their roles in gene expression. The following are some of the validation methods employed to confirm functionally relevant microRNA interactions with their target mRNAs. Each method involves interference with the microRNA or the target mRNA to disable their interaction, which should lead to loss of microRNA-mediated gene expression if the interaction is functionally consequential. Subsequent alleviation of the interference and restoration of productive base-pairing interactions between the microRNA and target should rescue microRNA-mediated gene expression and confirm the functional requirement for direct microRNA-target mRNA interaction. Characterization of functional microRNA interactions with their target mRNAs will provide significant insights into their gene expression regulatory mechanism and lead to the development of potential therapeutic approaches to manipulate these interactions and their consequent gene expression outcomes.
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Affiliation(s)
- S Vasudevan
- Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, United States.
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27
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Goergen D, Niepmann M. Stimulation of Hepatitis C Virus RNA translation by microRNA-122 occurs under different conditions in vivo and in vitro. Virus Res 2012; 167:343-52. [PMID: 22677772 DOI: 10.1016/j.virusres.2012.05.022] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2012] [Revised: 05/18/2012] [Accepted: 05/21/2012] [Indexed: 02/07/2023]
Abstract
Translation of the Hepatitis C Virus (HCV) positive strand RNA genome is directed by an internal ribosome entry site (IRES) in the viral RNA's 5'-untranslated region (5'-UTR). HCV propagates preferentially in the liver, and HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) acting on two target sites in the 5'-UTR. This stimulation is effective in living cells containing miR-122 and also in the rabbit reticulocyte lysate in vitro-translation system after addition of miR-122. Another RNA sequence located in the Core protein coding sequence can base-pair in a long-range RNA-RNA interaction to the HCV 5'-UTR, overlapping with the miR-122 target sites and the short spacer between them, and thereby inhibits HCV translation. Here we show genetic evidence that in reticulocyte lysate single-stranded miR-122 interferes with this inhibitory long-range RNA-RNA interaction and thereby contributes to enhanced HCV translation, involving not only the 5'-seed sequence of miR-122 but also sequences at its 3'-end. Also RNA oligonucleotides shorter than a typical microRNA stimulate HCV translation, confirming that in the reticulocyte lysate the stimulation of HCV translation functions by displacement of the inhibitory long-range interaction by miR-122. In contrast, in transfected HuH-7 hepatoma cells and in HeLa cells this interference of miR-122 with the inhibitory long-range RNA-RNA interaction plays not a major role, but only duplex miR-122 RNAs of the correct length stimulate HCV translation. These results suggest that: (1) the processing of the microRNA precursors and (2) the events occurring at the HCV RNA differ between cells and reticulocyte lysate.
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Affiliation(s)
- Dagmar Goergen
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392 Giessen, Germany
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