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1
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Szymanik KH, Rex EA, Pothireddy VR, Gammon DB, Hancks DC, Sullivan CS. Viral piracy of host RNA phosphatase DUSP11 by avipoxviruses. PLoS Pathog 2025; 21:e1013101. [PMID: 40258008 DOI: 10.1371/journal.ppat.1013101] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Accepted: 04/02/2025] [Indexed: 04/23/2025] Open
Abstract
Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5' end (5'ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5'pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5'ppp generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how experimentally altered DUSP11 activity can impact immune activation, prompting further exploration into natural contexts of altered DUSP11 activity. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of host DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cell's sensitivity to 5'pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a context where DUSP11 activity has been co-opted by viruses to alter RNA metabolism and influence the outcome of infection.
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Affiliation(s)
- Kayla H Szymanik
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America
| | - Emily A Rex
- Department of Microbiology, UT. Southwestern Medical Center, Dallas, Texas, United States of America
| | - Vamshikrishna R Pothireddy
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America
| | - Don B Gammon
- Department of Microbiology, UT. Southwestern Medical Center, Dallas, Texas, United States of America
| | - Dustin C Hancks
- Department of Immunology, UT. Southwestern Medical Center, Dallas, Texas, United States of America
| | - Christopher S Sullivan
- Department of Molecular Biosciences, LaMontagne Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, United States of America
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2
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Ma Y, Mao Y, Luo S, Zuo W, Gao P, Ying B. Development and characterization of a miRNA-responsive circular RNA expression system with cell type specificity. MOLECULAR THERAPY. NUCLEIC ACIDS 2025; 36:102450. [PMID: 39967851 PMCID: PMC11834102 DOI: 10.1016/j.omtn.2025.102450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Accepted: 01/10/2025] [Indexed: 02/20/2025]
Abstract
Although microRNAs (miRNAs) binding to messenger RNAs (mRNAs) generally results in mRNA degradation and reduced protein expression, their interaction with the internal ribosome entry sites (IRES) of certain RNA viruses enhances viral amplification and expression. In this study, we utilized the natural hepatitis C cirus (HCV) 5' UTR region, which contains miR-122 binding sites, as the IRES of circular RNA (circRNAs) constructs. These circRNAs allowed inducible expression of downstream genes with high specificity in response to both exogenous and endogenous miR-122. Substituting the miR-122 binding sites with those for other miRNAs also resulted in the translational activation of circRNAs by their respective miRNAs in transfected cells. Furthermore, mouse models administered intravenously with lipid nanoparticle-formulated circRNAs containing miRNA binding sites (circRNA-LNP) exhibited higher expression in targeted tissues compared to those with mutated binding sites. Our research introduces a novel strategy for tissue-specific regulation of circRNA expression, potentially broadening the therapeutic applications of circRNAs and paving the way for more precise and effective treatments in gene therapy.
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Affiliation(s)
- Yu Ma
- Suzhou Abogen Biosciences Company, Suzhou 215123, China
| | - Yuqiao Mao
- College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
| | - Shirui Luo
- College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
| | - Wenjie Zuo
- Suzhou Abogen Biosciences Company, Suzhou 215123, China
| | - Peng Gao
- Suzhou Abogen Biosciences Company, Suzhou 215123, China
| | - Bo Ying
- Suzhou Abogen Biosciences Company, Suzhou 215123, China
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3
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Hanson WA, Romero Agosto GA, Rouskin S. Viral RNA Interactome: The Ultimate Researcher's Guide to RNA-Protein Interactions. Viruses 2024; 16:1702. [PMID: 39599817 PMCID: PMC11599142 DOI: 10.3390/v16111702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Revised: 10/18/2024] [Accepted: 10/25/2024] [Indexed: 11/29/2024] Open
Abstract
RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA-protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA-protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.
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Affiliation(s)
| | | | - Silvi Rouskin
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; (W.A.H.); (G.A.R.A.)
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4
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Szymanik KH, Hancks DC, Sullivan CS. Viral piracy of host RNA phosphatase DUSP11 by avipoxviruses. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.06.606876. [PMID: 39211142 PMCID: PMC11361023 DOI: 10.1101/2024.08.06.606876] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/04/2024]
Abstract
Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5' end (5'ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5'pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5'pppRNA generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how artificially altered DUSP11 can impact immune activation, prompting further exploration into natural contexts of altered DUSP11. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of endogenous DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cell's sensitivity to 5'pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a virus-relevant context where DUSP11 activity has been co-opted to alter RNA metabolism and influence the outcome of infection.
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5
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Frericks N, Brown RJP, Reinecke BM, Herrmann M, Brüggemann Y, Todt D, Miskey C, Vondran FWR, Steinmann E, Pietschmann T, Sheldon J. Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes. J Virol 2024; 98:e0192123. [PMID: 38319104 PMCID: PMC10949430 DOI: 10.1128/jvi.01921-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Accepted: 01/12/2024] [Indexed: 02/07/2024] Open
Abstract
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.
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Affiliation(s)
- Nicola Frericks
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Richard J. P. Brown
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
- Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
| | | | - Maike Herrmann
- Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
| | - Yannick Brüggemann
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Daniel Todt
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
- European Virus Bioinformatics Center (EVBC), Jena, Germany
| | - Csaba Miskey
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Florian W. R. Vondran
- Department for General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
- Clinic for General, Visceral and Transplant Surgery, University Hospital RWTH Aachen, Aachen, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
| | - Eike Steinmann
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Thomas Pietschmann
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Julie Sheldon
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
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6
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Scott S, Li Y, Bermek O, Griffith JD, Lemon SM, Choi K. Binding of microRNA-122 to the hepatitis C virus 5' untranslated region modifies interactions with poly(C) binding protein 2 and the NS5B viral polymerase. Nucleic Acids Res 2023; 51:12397-12413. [PMID: 37941151 PMCID: PMC10711565 DOI: 10.1093/nar/gkad1000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2023] [Revised: 10/12/2023] [Accepted: 10/18/2023] [Indexed: 11/10/2023] Open
Abstract
Hepatitis C virus (HCV) requires two cellular factors, microRNA-122 (miR-122) and poly(C) binding protein 2 (PCBP2), for optimal replication. These host factors compete for binding to the 5' end of the single-stranded RNA genome to regulate the viral replication cycle. To understand how they interact with the RNA, we measured binding affinities of both factors for an RNA probe representing the 5' 45 nucleotides of the HCV genome (HCV45). Isothermal titration calorimetry revealed two, unequal miR-122 binding sites in HCV45, high-affinity (S1) and low-affinity (S2), differing roughly 100-fold in binding affinity. PCBP2 binds a site overlapping S2 with affinity similar to miR-122 binding to S2. PCBP2 circularizes the genome by also binding to the 3' UTR, bridging the 5' and 3' ends of the genome. By competing with PCBP2 for binding at S2, miR-122 disrupts PCBP2-mediated genome circularization. We show that the viral RNA-dependent RNA polymerase, NS5B, also binds to HCV45, and that the binding affinity of NS5B is increased in the presence of miR-122, suggesting miR-122 promotes recruitment of the polymerase. We propose that competition between miR-122 and PCBP2 for HCV45 functions as a translation-to-replication switch, determining whether the RNA genome templates protein synthesis or RNA replication.
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Affiliation(s)
- Seth Scott
- Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - You Li
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
| | - Oya Bermek
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
| | - Jack D Griffith
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
| | - Stanley M Lemon
- Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
- Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27517, USA
| | - Kyung H Choi
- Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
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7
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Rheault M, Cousineau SE, Fox DR, Abram QH, Sagan S. Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly. Nucleic Acids Res 2023; 51:2447-2463. [PMID: 36807979 PMCID: PMC10018354 DOI: 10.1093/nar/gkad094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2022] [Revised: 01/20/2023] [Accepted: 02/18/2023] [Indexed: 02/23/2023] Open
Abstract
Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or 'riboswitch', allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5' untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.
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Affiliation(s)
- Marylin Rheault
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
| | - Sophie E Cousineau
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
| | - Danielle R Fox
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
- Department of Physiology, McGill University, Montréal, Canada
| | - Quinn H Abram
- Department of Biochemistry, McGill University, Montréal, Canada
| | - Selena M Sagan
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
- Department of Biochemistry, McGill University, Montréal, Canada
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8
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Abdel Halim AS, Rudayni HA, Chaudhary AA, Ali MAM. MicroRNAs: Small molecules with big impacts in liver injury. J Cell Physiol 2023; 238:32-69. [PMID: 36317692 DOI: 10.1002/jcp.30908] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 09/30/2022] [Accepted: 10/14/2022] [Indexed: 11/07/2022]
Abstract
A type of small noncoding RNAs known as microRNAs (miRNAs) fine-tune gene expression posttranscriptionally by binding to certain messenger RNA targets. Numerous physiological processes in the liver, such as differentiation, proliferation, and apoptosis, are regulated by miRNAs. Additionally, there is growing evidence that miRNAs contribute to liver pathology. Extracellular vesicles like exosomes, which contain secreted miRNAs, may facilitate paracrine and endocrine communication between various tissues by changing the gene expression and function of distal cells. The use of stable miRNAs as noninvasive biomarkers was made possible by the discovery of these molecules in body fluids. Circulating miRNAs reflect the conditions of the liver that are abnormal and may serve as new biomarkers for the early detection, prognosis, and evaluation of liver pathological states. miRNAs are appealing therapeutic targets for a range of liver disease states because altered miRNA expression is associated with deregulation of the liver's metabolism, liver damage, liver fibrosis, and tumor formation. This review provides a comprehensive review and update on miRNAs biogenesis pathways and mechanisms of miRNA-mediated gene silencing. It also outlines how miRNAs affect hepatic cell proliferation, death, and regeneration as well as hepatic detoxification. Additionally, it highlights the diverse functions that miRNAs play in the onset and progression of various liver diseases, including nonalcoholic fatty liver disease, alcoholic liver disease, fibrosis, hepatitis C virus infection, and hepatocellular carcinoma. Further, it summarizes the diverse liver-specific miRNAs, illustrating the potential merits and possible caveats of their utilization as noninvasive biomarkers and appealing therapeutic targets for liver illnesses.
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Affiliation(s)
- Alyaa S Abdel Halim
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - Hassan Ahmed Rudayni
- Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia
| | - Anis Ahmad Chaudhary
- Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia
| | - Mohamed A M Ali
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.,Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia
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9
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Panigrahi M, Palmer MA, Wilson JA. MicroRNA-122 Regulation of HCV Infections: Insights from Studies of miR-122-Independent Replication. Pathogens 2022; 11:1005. [PMID: 36145436 PMCID: PMC9504723 DOI: 10.3390/pathogens11091005] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 08/26/2022] [Accepted: 08/29/2022] [Indexed: 11/18/2022] Open
Abstract
Despite the advancement in antiviral therapy, Hepatitis C remains a global health challenge and one of the leading causes of hepatitis related deaths worldwide. Hepatitis C virus, the causative agent, is a positive strand RNA virus that requires a liver specific microRNA called miR-122 for its replication. Unconventional to the canonical role of miRNAs in translation suppression by binding to 3'Untranslated Region (UTR) of messenger RNAs, miR-122 binds to two sites on the 5'UTR of viral genome and promotes viral propagation. In this review, we describe the unique relationship between the liver specific microRNA and HCV, the current knowledge on the mechanisms by which the virus uses miR-122 to promote the virus life cycle, and how miR-122 impacts viral tropism and pathogenesis. We will also discuss the use of anti-miR-122 therapy and its impact on viral evolution of miR-122-independent replication. This review further provides insight into how viruses manipulate host factors at the initial stage of infection to establish a successful infection.
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Affiliation(s)
| | | | - Joyce A. Wilson
- Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
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10
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Lakhia R, Ramalingam H, Chang CM, Cobo-Stark P, Biggers L, Flaten A, Alvarez J, Valencia T, Wallace DP, Lee EC, Patel V. PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression. Nat Commun 2022; 13:4765. [PMID: 35965273 PMCID: PMC9376183 DOI: 10.1038/s41467-022-32543-2] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 08/04/2022] [Indexed: 12/22/2022] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation occurs when PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are repressed via their 3'-UTR miR-17 binding element. Eliminating this motif (Pkd1∆17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also inhibited via its 3'-UTR miR-17 motif, and Pkd2∆17-induced Polycystin-2 derepression retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, modeling PKD1∆17 or PKD2∆17 alleles in patient-derived primary ADPKD cultures leads to smaller cysts, reduced proliferation, lower pCreb1 expression, and improved mitochondrial membrane potential. Thus, evading 3'-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.
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Affiliation(s)
- Ronak Lakhia
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Harini Ramalingam
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Chun-Mien Chang
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Patricia Cobo-Stark
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Laurence Biggers
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Andrea Flaten
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Jesus Alvarez
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | | | - Darren P Wallace
- Department of Internal Medicine and the Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA
| | - Edmund C Lee
- Regulus Therapeutics Inc., San Diego, CA, 92121, USA
| | - Vishal Patel
- Department of Internal Medicine, Nephrology, UT Southwestern Medical Center, Dallas, TX, 75390, USA.
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11
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Wang Y, Lin S, Zhao Z, Xu P, Gao K, Qian H, Zhang Z, Guo X. Functional analysis of a putative Bombyx mori cypovirus miRNA BmCPV-miR-10 and its effect on virus replication. INSECT MOLECULAR BIOLOGY 2021; 30:552-565. [PMID: 34296485 DOI: 10.1111/imb.12725] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 07/11/2021] [Accepted: 07/13/2021] [Indexed: 06/13/2023]
Abstract
Bombyx mori cypovirus (BmCPV) is an important pathogen of silkworm (B. mori), the economically beneficial insect. The mechanism of its interaction with host immune defence system in the process of infection is still not yet completely clear. Researches have demonstrated that virus-encoded microRNAs (miRNA) play a crucial role in regulating host-pathogen interaction, but few reports are available so far on miRNAs encoded by insect viruses, especially the RNA viruses. In this study, a putative miRNA encoded by the 10th segment of BmCPV genomic RNA, BmCPV-miR-10, was identified and functionally analysed. The expression of the putative BmCPV-miR-10 could be detected via stem-loop RT-PCR (reverse transcription-Polymerase Chain Reaction) in the midgut of silkworm larvae infected with BmCPV. BmCSDE1 (B. mori cold shock domain E1 protein) gene was predicted to be a candidate target gene for BmCPV-miR-10 with the miRNA binding site located in 3' untranslated region of its mRNA. The regulation effect of the putative BmCPV-miR-10 on BmCSDE1 was verified in HEK293 cells by lentiviral expression system, in BmN cells by transfecting BmCPV-miR-10 mimics. The qRT-PCR (quantitative real-time PCR) results showed that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1. By injection of BmCPV-miR-10 mimics into the silkworm larvae infected with BmCPV, it was further proved that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1 in vivo, then inhibit the expression of BmApaf-1 (B. mori apoptotic protease activating factor 1), while enhance the replication of BmCPV genomic RNAs to a certain extent. These results implied that the putative BmCPV-miR-10 could down-regulate the expression of BmCSDE1, then suppress the expression of BmApaf-1, thereby created a favourable intracellular environment for virus replication and proliferation.
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Affiliation(s)
- Y Wang
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - S Lin
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - Z Zhao
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - P Xu
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - K Gao
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - H Qian
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericulture Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Z Zhang
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - X Guo
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericulture Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
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12
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A structured RNA motif locks Argonaute2:miR-122 onto the 5' end of the HCV genome. Nat Commun 2021; 12:6836. [PMID: 34824224 PMCID: PMC8616905 DOI: 10.1038/s41467-021-27177-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 10/27/2021] [Indexed: 12/03/2022] Open
Abstract
microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5’ end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex. The RNA genome of the Hepatitis C Virus binds to the liver-specific miR122. Here the authors report the crystal structure of the Ago2:miR122:HCV complex showing that the viral RNA’s structural element traps the Ago2:miR-122 complex on the 5’ end of the viral genome to protect it from degradation.
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13
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RNA Structure Protects the 5'-end of an Uncapped Tombusvirus RNA Genome from Xrn Digestion. J Virol 2021; 95:e0103421. [PMID: 34346764 DOI: 10.1128/jvi.01034-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5'-termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'UTR folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5'-access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5'-terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5'-access, we have termed this 5'-terminal structure an Xrn-evading RNA or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5'-terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5'-capped. Here we explored how a species in the type genus Tombusvirus protects its genomic 5'-end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a genome-encoded higher-order RNA structure independently conferring resistance to cellular 5'-end-attacking enzymes in an RNA plant virus.
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14
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Li HC, Yang CH, Lo SY. Cellular factors involved in the hepatitis C virus life cycle. World J Gastroenterol 2021; 27:4555-4581. [PMID: 34366623 PMCID: PMC8326260 DOI: 10.3748/wjg.v27.i28.4555] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/04/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 970, Taiwan
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan
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15
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Li HC, Yang CH, Lo SY. Hepatitis C Viral Replication Complex. Viruses 2021; 13:v13030520. [PMID: 33809897 PMCID: PMC8004249 DOI: 10.3390/v13030520] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 03/18/2021] [Accepted: 03/19/2021] [Indexed: 12/16/2022] Open
Abstract
The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 97004, Taiwan;
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 97004, Taiwan
- Correspondence: ; Tel.: +886-3-8565301 (ext. 2322)
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16
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Kunden RD, Ghezelbash S, Khan JQ, Wilson JA. Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5' UTR regulatory element with distinct impacts on virus translation and genome stability. Nucleic Acids Res 2020; 48:9235-9249. [PMID: 32810257 PMCID: PMC7498337 DOI: 10.1093/nar/gkaa664] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 07/21/2020] [Accepted: 07/28/2020] [Indexed: 12/24/2022] Open
Abstract
Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.
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Affiliation(s)
- Rasika D Kunden
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Sarah Ghezelbash
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Juveriya Q Khan
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Joyce A Wilson
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
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17
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Zhou Y, Ng DYE, Richards AM, Wang P. microRNA-221 Inhibits Latent TGF-β1 Activation through Targeting Thrombospondin-1 to Attenuate Kidney Failure-Induced Cardiac Fibrosis. MOLECULAR THERAPY. NUCLEIC ACIDS 2020; 22:803-814. [PMID: 33230477 PMCID: PMC7645417 DOI: 10.1016/j.omtn.2020.09.041] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Accepted: 09/30/2020] [Indexed: 12/21/2022]
Abstract
Kidney failure (KF) is associated with cardiac fibrosis and significantly increased mortality in heart failure. Thrombospondin-1 (TSP1), a key regulator of latent transforming growth factor-β1 (L-TGF-β1) activation, is a predicted target of miR-221. We hypothesized miR-221 attenuates severe KF-associated cardiac fibrosis via targeting of Thbs1 with subsequent inhibition of L-TGF-β1 activation. Rat cardiac fibroblasts (cFB) were isolated and transfected with microRNA-221 (miR-221) mimics or mimic control (miR-221 and MC) with or without exposure to L-TGF-β1. We demonstrate miR-221 downregulates Thbs1 via direct 3′ untranslated region (3′ UTR) targeting with consequent inhibition of L-TGF-β1 activation in cFB as proven by the significant reduction of myofibroblast activation, collagen secretion, TGF-β1 signaling, TSP1 secretion, and TGF-β1 bioactivity measured by Pai1 promoter reporter. The 5/6 nephrectomy (Nx) model of cardiac fibrosis was used to test the in vivo therapeutic efficacy of miR-221 (i.v. 1 mg/kg ×3). miR-221 significantly inhibited Nx-induced upregulation of TSP1 and p-SMAD3 in the heart at day-7 and reduced cardiac fibrosis (picro-sirius), improved cardiac function (±dP/dt), and improved 8-week survival rate (60% versus 36%; p = 0.038). miR-221 mimic treatment improved survival and reduced cardiac fibrosis in a model of severe KF. miR-221 is a therapeutic target to address cardiac fibrosis originating from renal disease and other causes.
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Affiliation(s)
- Yue Zhou
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599 Singapore.,Department of Medicine, National University Health System, Singapore 119228, Singapore
| | - Denise Yu En Ng
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599 Singapore.,Department of Medicine, National University Health System, Singapore 119228, Singapore
| | - Arthur Mark Richards
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599 Singapore.,Department of Medicine, National University Health System, Singapore 119228, Singapore.,Christchurch Heart Institute, Department of Medicine, University of Otago, Christchurch, New Zealand
| | - Peipei Wang
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599 Singapore.,Department of Medicine, National University Health System, Singapore 119228, Singapore
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18
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The Role of the Liver-Specific microRNA, miRNA-122 in the HCV Replication Cycle. Int J Mol Sci 2020; 21:ijms21165677. [PMID: 32784807 PMCID: PMC7460827 DOI: 10.3390/ijms21165677] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 08/04/2020] [Accepted: 08/05/2020] [Indexed: 02/08/2023] Open
Abstract
Hepatitis C virus (HCV) replication requires annealing of a liver specific microRNA, miR-122 to 2 sites on 5' untranslated region (UTR). While, microRNAs downregulate gene expression by binding to the 3' untranslated region of the target mRNA, in this case, the microRNA anneals to the 5'UTR of the viral genomes and upregulates the viral lifecycle. In this review, we explore the current understandings of the mechanisms by which miR-122 promotes the HCV lifecycle, and its contributions to pathogenesis. Annealing of miR-122 has been reported to (a) stimulate virus translation by promoting the formation of translationally active internal ribosome entry site (IRES) RNA structure, (b) stabilize the genome, and (c) induce viral genomic RNA replication. MiR-122 modulates lipid metabolism and suppresses tumor formation, and sequestration by HCV may influence virus pathogenesis. We also discuss the possible use of miR-122 as a biomarker for chronic hepatitis and as a therapeutic target. Finally, we discuss roles for miR-122 and other microRNAs in promoting other viruses.
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19
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Guo JY, Wang YS, Chen T, Jiang XX, Wu P, Geng T, Pan ZH, Shang MK, Hou CX, Gao K, Guo XJ. Functional analysis of a miRNA-like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus. INSECT SCIENCE 2020; 27:449-462. [PMID: 30869181 DOI: 10.1111/1744-7917.12671] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 01/14/2019] [Accepted: 01/29/2019] [Indexed: 06/09/2023]
Abstract
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5' untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.
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Affiliation(s)
- Jian-Yong Guo
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Yong-Sheng Wang
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Tian Chen
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - Xiao-Xu Jiang
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - Ping Wu
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Tao Geng
- Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikoou, China
| | - Zhong-Hua Pan
- School of Biology & Basic Medical Science, Soochow University, Suzhou, China
| | - Meng-Ke Shang
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
| | - Cheng-Xiang Hou
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Kun Gao
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
| | - Xi-Jie Guo
- School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
- Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
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20
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Amador-Cañizares Y, Panigrahi M, Huys A, Kunden RD, Adams HM, Schinold MJ, Wilson JA. miR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5' UTR RNA to stabilize and promote viral RNA accumulation. Nucleic Acids Res 2019; 46:9776-9792. [PMID: 30053137 PMCID: PMC6182169 DOI: 10.1093/nar/gky662] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Accepted: 07/11/2018] [Indexed: 01/01/2023] Open
Abstract
Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5′ UTR, in conjunction with any Ago isoform, modifies the 5′ UTR structure to stabilize the viral genome and promote HCV RNA accumulation.
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Affiliation(s)
- Yalena Amador-Cañizares
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Mamata Panigrahi
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Adam Huys
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Rasika D Kunden
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Halim M Adams
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Michael J Schinold
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
| | - Joyce A Wilson
- Department of Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
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21
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Chahal J, Gebert LF, Gan HH, Camacho E, Gunsalus KC, MacRae IJ, Sagan SM. miR-122 and Ago interactions with the HCV genome alter the structure of the viral 5' terminus. Nucleic Acids Res 2019; 47:5307-5324. [PMID: 30941417 PMCID: PMC6547439 DOI: 10.1093/nar/gkz194] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2019] [Revised: 03/11/2019] [Accepted: 03/20/2019] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5' UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1-117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5' terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5' UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5' terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES-40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5' terminus and promote formation of the HCV IRES.
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Affiliation(s)
- Jasmin Chahal
- Department of Microbiology & Immunology, McGill University, Montréal, QC H3G 1Y6, Canada
| | - Luca F R Gebert
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Hin Hark Gan
- Center for Genomics and Systems Biology, Department of Biology, New York University, 12 Waverly Place, New York, NY 10003, USA
| | - Edna Camacho
- Department of Biochemistry, McGill University, Montréal, QC H3G 1Y6, Canada
| | - Kristin C Gunsalus
- Center for Genomics and Systems Biology, Department of Biology, New York University, 12 Waverly Place, New York, NY 10003, USA
- Division of Biology, New York University Abu Dhabi, Abu Dhabi, UAE
| | - Ian J MacRae
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Selena M Sagan
- Department of Microbiology & Immunology, McGill University, Montréal, QC H3G 1Y6, Canada
- Department of Biochemistry, McGill University, Montréal, QC H3G 1Y6, Canada
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22
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Huang Y, Liu H, Sun X, Ding M, Tao G, Li X. Honeysuckle-derived microRNA2911 directly inhibits varicella-zoster virus replication by targeting IE62 gene. J Neurovirol 2019; 25:457-463. [PMID: 31140131 DOI: 10.1007/s13365-019-00741-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 02/17/2019] [Accepted: 02/26/2019] [Indexed: 12/16/2022]
Abstract
Varicella-zoster virus (VZV) leads to chicken pox on primary infection and herpes zoster on reactivation. Recent studies suggest that microRNA2911 (MIR2911), honeysuckle (HS)-encoded atypical microRNA, has potential as a therapeutic agent against influenza and EV71 virus infections. Here, we report that MIR2911 directly inhibits VZV replication by targeting the IE62 gene. The luciferase reporter assay and bioinformatics prediction revealed that MIR2911 could target the IE62 gene of VZV. The VZV-encoded IE62 protein expression was inhibited significantly by synthetic MIR2911, while the expression of the mutants, whose MIR2911-binding sites were modified, was not inhibited. The RNA extracted from HS decoction and synthetic MIR2911 considerably suppressed VZV infection. However, it did not influence viral replication of a mutant virus with alterations in the nucleotide sequences of IE62. At the same time, the RNA extracted from HS decoction treated with the anti-MIR2911 antagomir could not inhibit the VZV replication, demonstrating that VZV replication was specifically and sufficiently inhibited by MIR2911. These results indicated that, by targeting the IE62 gene, MIR2911 may effectively inhibit VZV replication. Our results also suggest a potential novel strategy for the treatment and prevention of diseases caused by VZV infection.
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Affiliation(s)
- Ying Huang
- Department of Pain, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China
| | - Huabo Liu
- Department of Pain, Zhejiang Province Zhoushan Hospital, Zhoushan, Zhejiang, China
| | - Xinlei Sun
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210046, Jiangsu, China
| | - Meng Ding
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210046, Jiangsu, China
| | - Gaojian Tao
- Department of Pain, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China.
| | - Xihan Li
- Department of Gastroenterology, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China.
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23
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Mata M, Neben S, Majzoub K, Carette J, Ramanathan M, Khavari PA, Sarnow P. Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site. PLoS Pathog 2019; 15:e1007467. [PMID: 31075158 PMCID: PMC6530871 DOI: 10.1371/journal.ppat.1007467] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2018] [Revised: 05/22/2019] [Accepted: 04/15/2019] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5’ end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5’ genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5’ end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5’ end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood. With the advent of potent direct-acting antivirals (DAA), hepatitis C virus (HCV) can now be eliminated from the majority of patients, using multidrug therapy with DAAs. However, such DAAs are not available for the treatment of most RNA virus infections. The main problem is the high error rate by which RNA-dependent RNA polymerases copy viral RNA genomes, allowing the selection of mutations that are resistant to DAAs. Thus, targeting host-encoded functions that are essential for growth of the virus but not for the host cell offer promising, novel approaches. HCV needs host-encoded microRNA miR-122 for its viral RNA replication in the liver, and depletion of miR-122 in HCV patients results in loss of viral RNA. This study shows that a single-nucleotide mutation in HCV allows viral RNA amplification when miR-122 abundances are low, concomitant with changes in its tropism.
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Affiliation(s)
- Miguel Mata
- Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States of America
| | - Steven Neben
- Regulus Therapeutics, San Diego, CA, United States of America
| | - Karim Majzoub
- Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States of America.,INSERM U1110, Institute of Viral and Liver Disease, University of Strasbourg, France
| | - Jan Carette
- Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States of America
| | - Muthukumar Ramanathan
- Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA, United States of America; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA, United States of America
| | - Paul A Khavari
- Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA, United States of America; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA, United States of America
| | - Peter Sarnow
- Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States of America
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24
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Adams RL, Huston NC, Tavares RCA, Pyle AM. Sensitive detection of structural features and rearrangements in long, structured RNA molecules. Methods Enzymol 2019; 623:249-289. [PMID: 31239050 DOI: 10.1016/bs.mie.2019.04.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suited for a given application. Here, using a long, highly structured viral RNA as an example (the positive strand genome of Hepatitis C Virus), we present a semi-comprehensive analysis and describe the major approaches for analyzing the architecture of RNA that is modified with structure-sensitive probes. Additionally, we present an updated method for generating in vitro transcribed and folded RNA that maintains native secondary structures in long RNA molecules. We anticipate that the methods described here will streamline the use of current approaches and help investigators who are unfamiliar with structure probing, obviating the need for time-consuming and expensive optimization.
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Affiliation(s)
- Rebecca L Adams
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States
| | - Nicholas C Huston
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States
| | - Rafael C A Tavares
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States; Department of Chemistry, Yale University, New Haven, CT, United States
| | - Anna M Pyle
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States; Department of Chemistry, Yale University, New Haven, CT, United States; Howard Hughes Medical Institute, Chevy Chase, MD, United States.
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25
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Abstract
Xrn1 is a major 5ʹ-3ʹ exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3ʹ untranslated region of several beny- and cucumovirus RNAs harbors a so-called ‘coremin’ motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5ʹ proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3ʹ proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.
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Affiliation(s)
- Ivar W Dilweg
- a Leiden Institute of Chemistry , Leiden University , Leiden , The Netherlands
| | - Alexander P Gultyaev
- b Leiden Institute of Advanced Computer Science , Leiden University , Leiden , The Netherlands.,c Department of Viroscience , Erasmus Medical Center , Rotterdam , The Netherlands
| | - René C Olsthoorn
- a Leiden Institute of Chemistry , Leiden University , Leiden , The Netherlands
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26
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Abstract
Hepatitis C virus represents a global pathogen of human health significance. In the space of less than three decades, we have witnessed the discovery of the virus, a growing understanding of the structure and biology of the viral-encoded proteins and their interaction with the host cell and the sequencing of the viral genome. Most importantly, we have moved from early therapeutic strategies aimed at crude boosting of host anti-viral immunity, limited by side effects and with poor response rates, to therapies that directly exploit our understanding of viral biology. In this review, we discuss the significance of the virus, its' discovery and outline the advances in the molecular characterisation of the virus, before setting these within the context of contemporary and emerging therapeutic strategies as well as viral resistance mechanisms.
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27
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Bentley K, Cook JP, Tuplin AK, Evans DJ. Structural and functional analysis of the roles of the HCV 5' NCR miR122-dependent long-range association and SLVI in genome translation and replication. PeerJ 2018; 6:e5870. [PMID: 30416884 PMCID: PMC6225842 DOI: 10.7717/peerj.5870] [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: 07/20/2018] [Accepted: 09/30/2018] [Indexed: 11/29/2022] Open
Abstract
The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA–RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5′ end of the core protein-coding region. This element forms an alternative RNA–RNA interaction with complementary sequences in the 5′ untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between ‘open’ and ‘closed’ structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the ‘closed’ conformation. In the current study, we have used selective 2′-hydroxyl acylation analysed by primer extension to validate this RNA–RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus.
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Affiliation(s)
- Kirsten Bentley
- BSRC and School of Biology, University of St Andrews, St Andrews, UK
| | - Jonathan P Cook
- School of Life Sciences, University of Warwick, Coventry, UK
| | - Andrew K Tuplin
- The Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - David J Evans
- BSRC and School of Biology, University of St Andrews, St Andrews, UK
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28
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Kanokudom S, Mahony TJ, Smith DR, Assavalapsakul W. Modulation of bovine herpesvirus 1 infection by virally encoded microRNAs. Virus Res 2018; 257:1-6. [PMID: 30193942 DOI: 10.1016/j.virusres.2018.08.022] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Revised: 08/24/2018] [Accepted: 08/31/2018] [Indexed: 12/27/2022]
Abstract
Bovine herpesvirus 1 (BoHV-1), is a member of the subfamily Alphaherpesvirinae in the order Herpesviridae and is a ubiquitous pathogen of cattle responsible for significant economic loss worldwide. The BoHV-1 genome encodes at least 10 BoHV-1 microRNA (miRNA) genes, whose functions remain poorly understood. This study sought to understand the role of three BoHV-1 miRNA genes, Bhv1-miR-B6, Bhv1-miR-B8 and Bhv1-miR-B9, which are located proximal to the BoHV-1 origins of replication (OriS). Therefore, plasmids expressing the precursor miRNA hairpins for the Bhv1-miR-B6, Bhv1-miR-B8, and Bhv1-miR-B9 genes were constructed and transfected into Madin-Darby bovine kidney cells prior to BoHV-1 infection. Interestingly, transient expression of either Bhv1-miR-B8 or Bhv1-miR-B9 in Madin-Darby bovine kidney cells prior to infection resulted in partial suppression of BoHV-1 replication, quantified through estimating levels of glycoprotein C mRNA and protein levels. Putative interactions between the mature miRNA bhv1-miR-B8-3p and bhv1-miR-B9 and BoHV-1 transcripts were identified providing plausible pathways for these molecules to affect virus replication. Therefore, these two miRNAs are implicated in the post-transcriptional regulation of BoHV-1 transcripts important for virus replication and could be used to limit BoHV-1 replication.
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Affiliation(s)
- Sitthichai Kanokudom
- Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Timothy J Mahony
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072, Australia
| | - Duncan R Smith
- Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand
| | - Wanchai Assavalapsakul
- Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
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29
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Kincaid RP, Lam VL, Chirayil RP, Randall G, Sullivan CS. RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus. Proc Natl Acad Sci U S A 2018; 115:8197-8202. [PMID: 30038017 PMCID: PMC6094126 DOI: 10.1073/pnas.1802326115] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Seventy percent of people infected with hepatitis C virus (HCV) will suffer chronic infection, putting them at risk for liver disease, including hepatocellular carcinoma. The full range of mechanisms that render some people more susceptible to chronic infection and liver disease is still being elucidated. XRN exonucleases can restrict HCV replication and may help to resolve HCV infections. However, it is unknown how 5' triphosphorylated HCV transcripts, primary products of the viral polymerase, become susceptible to attack by 5' monophosphate-specific XRNs. Here, we show that the 5' RNA triphosphatase DUSP11 acts on HCV transcripts, rendering them susceptible to XRN-mediated attack. Cells lacking DUSP11 show substantially enhanced HCV replication, and this effect is diminished when XRN expression is reduced. MicroRNA-122 (miR-122), a target of current phase II anti-HCV drugs, is known to protect HCV transcripts against XRNs. We show that HCV replication is less dependent on miR-122 in cells lacking DUSP11. Combined, these results implicate DUSP11 as an important component of XRN-mediated restriction of HCV.
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Affiliation(s)
- Rodney P Kincaid
- Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
- LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
| | - Victor L Lam
- Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
- LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
| | - Rachel P Chirayil
- Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
- LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
| | - Glenn Randall
- Department of Microbiology, The University of Chicago, Chicago, IL 60637
| | - Christopher S Sullivan
- Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162;
- LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712-0162
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30
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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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31
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Schult P, Roth H, Adams RL, Mas C, Imbert L, Orlik C, Ruggieri A, Pyle AM, Lohmann V. microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site. Nat Commun 2018; 9:2613. [PMID: 29973597 PMCID: PMC6031695 DOI: 10.1038/s41467-018-05053-3] [Citation(s) in RCA: 89] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2018] [Accepted: 06/11/2018] [Indexed: 12/24/2022] Open
Abstract
The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5′ terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures. The liver-specific microRNA-122 is an essential proviral host factor of Hepatitis C virus replication. Here the authors show that microRNA-122 functions as an RNA chaperone that guides the formation of a functional internal ribosome entry site by preventing energetically more favorable secondary structures within the HCV RNA genome.
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Affiliation(s)
- Philipp Schult
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany
| | - Hanna Roth
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany
| | - Rebecca L Adams
- Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect St, New Haven, CT, 06511, USA
| | - Caroline Mas
- University Grenoble Alpes, CNRS, CEA, IBS, 71 Avenue des Martyrs, CS 10090, 38044, Grenoble CEDEX 9, France
| | - Lionel Imbert
- University Grenoble Alpes, CNRS, CEA, IBS, 71 Avenue des Martyrs, CS 10090, 38044, Grenoble CEDEX 9, France
| | - Christian Orlik
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.,Department of Immunology, Molecular Immunology, University of Heidelberg, 69120, Heidelberg, Germany
| | - Alessia Ruggieri
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany
| | - Anna M Pyle
- Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect St, New Haven, CT, 06511, USA.,Howard Hughes Medical Institute, 219 Prospect St, New Haven, CT, 06511, USA
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.
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32
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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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33
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Yamane D, Selitsky SR, Shimakami T, Li Y, Zhou M, Honda M, Sethupathy P, Lemon SM. Differential hepatitis C virus RNA target site selection and host factor activities of naturally occurring miR-122 3΄ variants. Nucleic Acids Res 2017; 45:4743-4755. [PMID: 28082397 PMCID: PMC5416874 DOI: 10.1093/nar/gkw1332] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Accepted: 12/22/2016] [Indexed: 01/07/2023] Open
Abstract
In addition to suppressing cellular gene expression, certain miRNAs potently facilitate replication of specific positive-strand RNA viruses. miR-122, a pro-viral hepatitis C virus (HCV) host factor, binds and recruits Ago2 to tandem sites (S1 and S2) near the 5΄ end of the HCV genome, stabilizing it and promoting its synthesis. HCV target site selection follows canonical miRNA rules, but how non-templated 3΄ miR-122 modifications impact this unconventional miRNA action is unknown. High-throughput sequencing revealed that a 22 nt miRNA with 3΄G (‘22–3΄G’) comprised <63% of total miR-122 in human liver, whereas other variants (23–3΄A, 23–3΄U, 21–3΄U) represented 11–17%. All loaded equivalently into Ago2, and when tested individually functioned comparably in suppressing gene expression. In contrast, 23–3΄A and 23–3΄U were more active than 22–3΄G in stabilizing HCV RNA and promoting its replication, whereas 21–3΄U was almost completely inactive. This lack of 21–3΄U HCV host factor activity correlated with reduced recruitment of Ago2 to the HCV S1 site. Additional experiments demonstrated strong preference for guanosine at nt 22 of miR-122. Our findings reveal the importance of non-templated 3΄ miR-122 modifications to its HCV host factor activity, and identify unexpected differences in miRNA requirements for host gene suppression versus RNA virus replication.
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Affiliation(s)
- Daisuke Yamane
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Departments of Medicine and Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA
| | - Sara R Selitsky
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Bioinformatics and Computational Biology Curriculum, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Tetsuro Shimakami
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8641, Japan
| | - You Li
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Departments of Medicine and Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA
| | - Mi Zhou
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Bioinformatics and Computational Biology Curriculum, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Masao Honda
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8641, Japan
| | - Praveen Sethupathy
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Bioinformatics and Computational Biology Curriculum, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Stanley M Lemon
- Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Departments of Medicine and Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA
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34
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Jackowiak P, Hojka-Osinska A, Philips A, Zmienko A, Budzko L, Maillard P, Budkowska A, Figlerowicz M. Small RNA fragments derived from multiple RNA classes - the missing element of multi-omics characteristics of the hepatitis C virus cell culture model. BMC Genomics 2017; 18:502. [PMID: 28666407 PMCID: PMC5493846 DOI: 10.1186/s12864-017-3891-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Accepted: 06/21/2017] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND A pool of small RNA fragments (RFs) derived from diverse cellular RNAs has recently emerged as a rich source of functionally relevant molecules. Although their formation and accumulation has been connected to various stress conditions, the knowledge on RFs produced upon viral infections is very limited. Here, we applied the next generation sequencing (NGS) to characterize RFs generated in the hepatitis C virus (HCV) cell culture model (HCV-permissive Huh-7.5 cell line). RESULTS We found that both infected and non-infected cells contained a wide spectrum of RFs derived from virtually all RNA classes. A significant fraction of identified RFs accumulated to similar levels as miRNAs. Our analysis, focused on RFs originating from constitutively expressed non-coding RNAs, revealed three major patterns of parental RNA cleavage. We found that HCV infection induced significant changes in the accumulation of low copy number RFs, while subtly altered the levels of high copy number ones. Finally, the candidate RFs potentially relevant for host-virus interactions were identified. CONCLUSIONS Our results indicate that RFs should be considered an important component of the Huh-7.5 transcriptome and suggest that the main factors influencing the RF biogenesis are the RNA structure and RNA protection by interacting proteins. The data presented here significantly complement the existing transcriptomic, miRnomic, proteomic and metabolomic characteristics of the HCV cell culture model.
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Affiliation(s)
- Paulina Jackowiak
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Anna Hojka-Osinska
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Anna Philips
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Agnieszka Zmienko
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.,Institute of Computing Science, Poznan University of Technology, Piotrowo 3A, 60-965, Poznan, Poland
| | - Lucyna Budzko
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland
| | - Patrick Maillard
- Institut Pasteur, Hepacivirus and Innate Immunity, CNRS, UMR3569, 75724, Paris, France
| | - Agata Budkowska
- Institut Pasteur, Hepacivirus and Innate Immunity, CNRS, UMR3569, 75724, Paris, France.,Scientific Advisor for the Department of International Affairs, Institut Pasteur, 75724, Paris, France
| | - Marek Figlerowicz
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland. .,Institute of Computing Science, Poznan University of Technology, Piotrowo 3A, 60-965, Poznan, Poland.
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35
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Functional RNA structures throughout the Hepatitis C Virus genome. Curr Opin Virol 2017; 24:79-86. [PMID: 28511116 DOI: 10.1016/j.coviro.2017.04.007] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Accepted: 04/21/2017] [Indexed: 12/18/2022]
Abstract
The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics.
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Ono C, Fukuhara T, Motooka D, Nakamura S, Okuzaki D, Yamamoto S, Tamura T, Mori H, Sato A, Uemura K, Fauzyah Y, Kurihara T, Suda T, Nishio A, Hmwe SS, Okamoto T, Tatsumi T, Takehara T, Chayama K, Wakita T, Koike K, Matsuura Y. Characterization of miR-122-independent propagation of HCV. PLoS Pathog 2017; 13:e1006374. [PMID: 28494029 PMCID: PMC5441651 DOI: 10.1371/journal.ppat.1006374] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2017] [Revised: 05/23/2017] [Accepted: 04/24/2017] [Indexed: 12/12/2022] Open
Abstract
miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. A liver-specific microRNA, miR-122, is one of the key determinants of hepatitis C virus (HCV) hepatotropism and is required for efficient propagation of HCV. On the other hand, chronic infection with HCV is often associated with extrahepatic manifestations (EHMs), and a low level of HCV-RNA replication has been detected in some non-hepatic cells. Nonetheless, the detailed mechanisms underlying these phenomena remain unknown. Here, we show that miR-122 is dispensable for low-level replication or infectious particle formation, and a mutant virus adapted to miR-122-knockout cells exhibited efficient but miR-122-independent propagation. The adaptive virus of HCV genotype 2a possessed a G28A substitution in the 5’UTR and facilitated efficient replication complex formation under an miR-122-deficient condition, while it propagated at a level comparable to the wild type HCV in the presence of miR-122. Moreover, various adaptive mutations including C30U were introduced into genotype 1b, which originally had an adenine at the nucleotide position 29. These observations suggest that substitutions that yield miR-122-independent propagation are not induced during propagation in hepatocytes; however, treatment with an miR-122 inhibitor or persistent infection of HCV in non-hepatic cells may induce the emergence of mutant viruses, as evidenced by clinical samples.
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Affiliation(s)
- Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Motooka
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Shota Nakamura
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Okuzaki
- DNA-Chip Developmental Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomokazu Tamura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Asuka Sato
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Kentaro Uemura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yuzy Fauzyah
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takeshi Kurihara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takahiro Suda
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Akira Nishio
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Su Su Hmwe
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomohide Tatsumi
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Kazuaki Chayama
- Department of Medicine and Molecular Science, Hiroshima University School of Medicine, Hiroshima, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuhiko Koike
- Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- * E-mail:
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Elberry MH, Darwish NHE, Mousa SA. Hepatitis C virus management: potential impact of nanotechnology. Virol J 2017; 14:88. [PMID: 28464951 PMCID: PMC5414367 DOI: 10.1186/s12985-017-0753-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2017] [Accepted: 04/19/2017] [Indexed: 12/21/2022] Open
Abstract
Around 170–200 million individuals have hepatitis C virus (HCV), which represents ~ 3% of the world population, including ~ 3–5 million people in the USA. According to the WHO regional office in the Middle East, Egypt has the highest prevalence in the world, with 7% prevalence in adults. There had been no effective vaccine for HCV; a combination of PEG-Interferon and ribavirin for at least 48 weeks was the standard therapy, but it failed in more than 40% of the patients and has a high cost and serious side effects. The recent introduction of direct-acting antivirals (DAA) resulted in major advances toward the cure of HCV. However, relapse and reduced antiviral efficacy in fibrotic, cirrhotic HCV patients in addition to some undesired effects restrain the full potential of these combinations. There is a need for new approaches for the combinations of different DAA and their targeted delivery using novel nanotechnology approaches. In this review, the role of nanoparticles as a carrier for HCV vaccines, anti-HCV combinations, and their targeted delivery are discussed.
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Affiliation(s)
- Mostafa H Elberry
- The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, 12144, USA.,National Cancer Institute, Cairo University, Cairo, Egypt
| | - Noureldien H E Darwish
- The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, 12144, USA.,Faculty of Medicine, Mansoura University, Mansoura, Egypt
| | - Shaker A Mousa
- The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, 12144, USA.
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Biegel JM, Henderson E, Cox EM, Bonenfant G, Netzband R, Kahn S, Eager R, Pager CT. Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5' untranslated region of hepatitis C virus RNA. Virology 2017; 507:231-241. [PMID: 28456022 DOI: 10.1016/j.virol.2017.04.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2016] [Revised: 04/13/2017] [Accepted: 04/14/2017] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication.
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Affiliation(s)
- Jason M Biegel
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Eric Henderson
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Erica M Cox
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Gaston Bonenfant
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Rachel Netzband
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Samantha Kahn
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Rachel Eager
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA
| | - Cara T Pager
- Department of Biological Sciences, The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA.
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Identification and characterization of two putative microRNAs encoded by Bombyx mori cypovirus. Virus Res 2017; 233:86-94. [DOI: 10.1016/j.virusres.2017.03.009] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2017] [Revised: 03/03/2017] [Accepted: 03/04/2017] [Indexed: 01/23/2023]
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40
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Regulation of hepatitis C virus genome replication by microRNA-122. Uirusu 2017; 65:277-286. [PMID: 27760927 DOI: 10.2222/jsv.65.277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
microRNA-122 (miR-122) is an abundant, liver-specific miRNA that regulates gene expression post-transcriptionally, typically by binding to the 3' untranslated region (UTR) of mRNAs, repressing their translation and mediating their degradation. Hepatitis C virus (HCV) is uniquely dependent on miR-122. Similar to conventional miRNA action, miR-122 recruits Argonaute-2 (AGO2) protein to the 5' UTR of the viral genome. However, in contrast to typical miRNA function, this stabilizes HCV RNA and slows its decay in infected cells. We found that HCV RNA is degraded primarily by the cytoplasmic 5' exonuclease XRN1 and that miR-122 acts to protect the viral RNA from XRN1-mediated 5' exonucleolytic decay. However, HCV replication still requires miR-122 in XRN1-depleted cells, suggesting additional functions. We also showed that miR-122 enhances HCV RNA synthesis by reducing viral genomes engaged in translation while increasing the fraction available for RNA synthesis. In this review, we summarize the recent progress on the regulatory mechanisms of HCV genome replication by miR-122.
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41
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Kanokudom S, Vilaivan T, Wikan N, Thepparit C, Smith DR, Assavalapsakul W. miR-21 promotes dengue virus serotype 2 replication in HepG2 cells. Antiviral Res 2017; 142:169-177. [PMID: 28365456 DOI: 10.1016/j.antiviral.2017.03.020] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Revised: 02/09/2017] [Accepted: 03/27/2017] [Indexed: 11/19/2022]
Abstract
Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR-21, was differentially expressed at all time points examined. Interestingly, miR-21 was also differentially regulated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR-21 during DENV infection was further examined by treating HepG2 cells with an anti-miR-21 (AMO-21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR-21 plays a key role in DENV 2 replication. To further confirm the role of miR-21 in DENV infection, a peptide nucleic acid-21 (PNA-21) construct with a nucleotide sequence complementary to AMO-21, was co-administered with AMO-21 as an AMO-21/PNA-21 complex followed by DENV 2 infection. The results showed that AMO-21 significantly reduced DENV 2 titer, PNA-21 significantly increased DENV 2 titer and the combined AMO-21/PNA-21 showed no difference from non-treated infection controls. Taken together, the results show that miR-21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point.
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Affiliation(s)
- Sitthichai Kanokudom
- Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Tirayut Vilaivan
- Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand
| | - Nitwara Wikan
- Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand
| | - Chutima Thepparit
- Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand
| | - Duncan R Smith
- Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand
| | - Wanchai Assavalapsakul
- Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
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42
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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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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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Lafreniere MA, Powdrill MH, Singaravelu R, Pezacki JP. 6-Hydroxydopamine Inhibits the Hepatitis C Virus through Alkylation of Host and Viral Proteins and the Induction of Oxidative Stress. ACS Infect Dis 2016; 2:863-871. [PMID: 27682680 DOI: 10.1021/acsinfecdis.6b00098] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Many viruses, including the hepatitis C virus (HCV), are dependent on the host RNA silencing pathway for replication. In this study, we screened small molecule probes, previously reported to disrupt loading of the RNA-induced silencing complex (RISC), including 6-hydroxydopamine (6-OHDA), suramin (SUR), and aurintricarboxylic acid (ATA), to examine their effects on viral replication. We found that 6-OHDA inhibited HCV replication; however, 6-OHDA was a less potent inhibitor of RISC than either SUR or ATA. By generating a novel chemical probe (6-OHDA-yne), we determined that 6-OHDA covalently modifies host and virus proteins. Moreover, 6-OHDA was shown to be an alkylating agent that is capable of generating adducts with a number of enzymes involved in the oxidative stress response. Furthermore, modification of viral enzymes with 6-OHDA and 6-OHDA-yne was found to inhibit their enzymatic activity. Our findings suggest that 6-OHDA is a probe for oxidative stress as well as protein alkylation, and these properties together contribute to the antiviral effects of this compound.
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Affiliation(s)
- Matthew A. Lafreniere
- Department of Chemistry
and Biomolecular Sciences, University of Ottawa, 10 Marie Curie
Private, Ottawa, ON, Canada K1N 6N5
| | - Megan H. Powdrill
- Department of Chemistry
and Biomolecular Sciences, University of Ottawa, 10 Marie Curie
Private, Ottawa, ON, Canada K1N 6N5
| | - Ragunath Singaravelu
- Department
of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H 8M5
| | - John Paul Pezacki
- Department of Chemistry
and Biomolecular Sciences, University of Ottawa, 10 Marie Curie
Private, Ottawa, ON, Canada K1N 6N5
- Department
of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H 8M5
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Miyamura T, Lemon SM, Walker CM, Wakita T. The HCV Replicase Complex and Viral RNA Synthesis. HEPATITIS C VIRUS I 2016. [PMCID: PMC7122888 DOI: 10.1007/978-4-431-56098-2_8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Replication of hepatitis C virus (HCV) is tightly linked to membrane alterations designated the membranous web, harboring the viral replicase complex. In this chapter we describe the morphology and 3D architecture of the HCV-induced replication organelles, mainly consisting of double membrane vesicles, which are generated by a concerted action of the nonstructural proteins NS3 to NS5B. Recent studies have furthermore identified a number of host cell proteins and lipids contributing to the biogenesis of the membranous web, which are discussed in this chapter. Viral RNA synthesis is tightly associated with these membrane alterations and mainly driven by the viral RNA dependent RNA polymerase NS5B. We summarize our current knowledge of the structure and function of NS5B, the role of cis-acting replication elements at the termini of the genome in regulating RNA synthesis and the contribution of additional viral and host factors to viral RNA synthesis, which is still ill defined.
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Affiliation(s)
- Tatsuo Miyamura
- National Institute of Infectious Diseases, Tokyo, Tokyo Japan
| | - Stanley M. Lemon
- Departments of Medicine and Microbiology & Immunology , The University of North Carolina, Chapel Hill, North Carolina USA
| | - Christopher M. Walker
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio USA
| | - Takaji Wakita
- National Institute of Infectious Diseases, Tokyo, Tokyo Japan
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Abstract
The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition.
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Sarnow P, Sagan SM. Unraveling the Mysterious Interactions Between Hepatitis C Virus RNA and Liver-Specific MicroRNA-122. Annu Rev Virol 2016; 3:309-332. [PMID: 27578438 DOI: 10.1146/annurev-virology-110615-042409] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Many viruses encode or subvert cellular microRNAs (miRNAs) to aid in their gene expression, amplification strategies, or pathogenic signatures. miRNAs typically downregulate gene expression by binding to the 3' untranslated region of their mRNA targets. As a result, target mRNAs are translationally repressed and subsequently deadenylated and degraded. Curiously, hepatitis C virus (HCV), a member of the Flaviviridae family, recruits two molecules of liver-specific microRNA-122 (miR-122) to the 5' end of its genome. In contrast to the canonical activity of miRNAs, the interactions of miR-122 with the viral genome promote viral RNA accumulation in cultured cells and in animal models of HCV infection. Sequestration of miR-122 results in loss of viral RNA both in cell culture and in the livers of chronic HCV-infected patients. This review discusses the mechanisms by which miR-122 is thought to enhance viral RNA abundance and the consequences of miR-122-HCV interactions. We also describe preliminary findings from phase II clinical trials in patients treated with miR-122 antisense oligonucleotides.
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Affiliation(s)
- Peter Sarnow
- Department of Microbiology and Immunology, Stanford University, Stanford, California 94305
| | - Selena M Sagan
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada;
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Ganesan M, Natarajan SK, Zhang J, Mott JL, Poluektova LI, McVicker BL, Kharbanda KK, Tuma DJ, Osna NA. Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde. Am J Physiol Gastrointest Liver Physiol 2016; 310:G930-G940. [PMID: 27056722 PMCID: PMC6842882 DOI: 10.1152/ajpgi.00021.2016] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Accepted: 03/31/2016] [Indexed: 02/08/2023]
Abstract
Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.
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Affiliation(s)
- Murali Ganesan
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Sathish Kumar Natarajan
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Jinjin Zhang
- School of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska; and
| | - Justin L Mott
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska
| | | | - Benita L McVicker
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Kusum K Kharbanda
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Dean J Tuma
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Natalia A Osna
- Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska;
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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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50
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RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA. Proc Natl Acad Sci U S A 2015; 112:14036-41. [PMID: 26504242 DOI: 10.1073/pnas.1509251112] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
RNAIII, the effector of the agr quorum-sensing system, plays a key role in virulence gene regulation in Staphylococcus aureus, but how RNAIII transcriptionally regulates its downstream genes is not completely understood. Here, we show that RNAIII stabilizes mgrA mRNA, thereby increasing the production of MgrA, a global transcriptional regulator that affects the expression of many genes. The mgrA gene is transcribed from two promoters, P1 and P2, to produce two mRNA transcripts with long 5' UTR. Two adjacent regions of the mgrA mRNA UTR transcribed from the upstream P2 promoter, but not the P1 promoter, form a stable complex with two regions of RNAIII near the 5' and 3' ends. We further demonstrate that the interaction has several biological effects. We propose that MgrA can serve as an intermediary regulator through which agr exerts its regulatory function.
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