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1
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Sun M, Manson ML, Guo T, de Lange ECM. CNS Viral Infections-What to Consider for Improving Drug Treatment: A Plea for Using Mathematical Modeling Approaches. CNS Drugs 2024; 38:349-373. [PMID: 38580795 PMCID: PMC11026214 DOI: 10.1007/s40263-024-01082-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/10/2024] [Indexed: 04/07/2024]
Abstract
Neurotropic viruses may cause meningitis, myelitis, encephalitis, or meningoencephalitis. These inflammatory conditions of the central nervous system (CNS) may have serious and devastating consequences if not treated adequately. In this review, we first summarize how neurotropic viruses can enter the CNS by (1) crossing the blood-brain barrier or blood-cerebrospinal fluid barrier; (2) invading the nose via the olfactory route; or (3) invading the peripheral nervous system. Neurotropic viruses may then enter the intracellular space of brain cells via endocytosis and/or membrane fusion. Antiviral drugs are currently used for different viral CNS infections, even though their use and dosing regimens within the CNS, with the exception of acyclovir, are minimally supported by clinical evidence. We therefore provide considerations to optimize drug treatment(s) for these neurotropic viruses. Antiviral drugs should cross the blood-brain barrier/blood cerebrospinal fluid barrier and pass the brain cellular membrane to inhibit these viruses inside the brain cells. Some antiviral drugs may also require intracellular conversion into their active metabolite(s). This illustrates the need to better understand these mechanisms because these processes dictate drug exposure within the CNS that ultimately determine the success of antiviral drugs for CNS infections. Finally, we discuss mathematical model-based approaches for optimizing antiviral treatments. Thereby emphasizing the potential of CNS physiologically based pharmacokinetic models because direct measurement of brain intracellular exposure in living humans faces ethical restrictions. Existing physiologically based pharmacokinetic models combined with in vitro pharmacokinetic/pharmacodynamic information can be used to predict drug exposure and evaluate efficacy of antiviral drugs within the CNS, to ultimately optimize the treatments of CNS viral infections.
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Affiliation(s)
- Ming Sun
- Division of Systems Pharmacology and Pharmacy, Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
| | - Martijn L Manson
- Division of Systems Pharmacology and Pharmacy, Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
| | - Tingjie Guo
- Division of Systems Pharmacology and Pharmacy, Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
| | - Elizabeth C M de Lange
- Division of Systems Pharmacology and Pharmacy, Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
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2
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RNA-Binding Proteins as Regulators of Internal Initiation of Viral mRNA Translation. Viruses 2022; 14:v14020188. [PMID: 35215780 PMCID: PMC8879377 DOI: 10.3390/v14020188] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/03/2022] [Accepted: 01/14/2022] [Indexed: 12/17/2022] Open
Abstract
Viruses are obligate intracellular parasites that depend on the host’s protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. Many viruses utilize non-canonical strategies such as targeting host eIFs and RNA elements known as internal ribosome entry sites (IRESs) to reprogram cellular gene expression, ensuring preferential translation of vRNAs. In this review, we discuss vRNA IRES-mediated translation initiation, highlighting the role of RNA-binding proteins (RBPs), other than the canonical translation initiation factors, in regulating their activity.
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3
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Arhab Y, Bulakhov AG, Pestova TV, Hellen CU. Dissemination of Internal Ribosomal Entry Sites (IRES) Between Viruses by Horizontal Gene Transfer. Viruses 2020; 12:E612. [PMID: 32512856 PMCID: PMC7354566 DOI: 10.3390/v12060612] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Revised: 06/01/2020] [Accepted: 06/02/2020] [Indexed: 12/19/2022] Open
Abstract
Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5'-untranslated region (5'UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5'-end-independent initiation of translation by a different mechanism. Picornavirus 5'UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution.
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Affiliation(s)
| | | | | | - Christopher U.T. Hellen
- Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, USA; (Y.A.); (A.G.B.); (T.V.P.)
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Godet AC, David F, Hantelys F, Tatin F, Lacazette E, Garmy-Susini B, Prats AC. IRES Trans-Acting Factors, Key Actors of the Stress Response. Int J Mol Sci 2019; 20:ijms20040924. [PMID: 30791615 PMCID: PMC6412753 DOI: 10.3390/ijms20040924] [Citation(s) in RCA: 119] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2018] [Revised: 02/12/2019] [Accepted: 02/14/2019] [Indexed: 12/16/2022] Open
Abstract
The cellular stress response corresponds to the molecular changes that a cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression at transcriptional and posttranscriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support the translation of specific mRNAs. A major mechanism involved in stress-activated translation is the internal ribosome entry site (IRES)-driven initiation. IRESs, first discovered in viral mRNAs, are present in cellular mRNAs coding for master regulators of cell responses, whose expression must be tightly controlled. IRESs allow the translation of these mRNAs in response to different stresses, including DNA damage, amino-acid starvation, hypoxia or endoplasmic reticulum stress, as well as to physiological stimuli such as cell differentiation or synapse network formation. Most IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents the history of viral and cellular IRES discovery as well as an update of the reported ITAFs regulating cellular mRNA translation and of their different mechanisms of action. The impact of ITAFs on the coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted.
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Affiliation(s)
- Anne-Claire Godet
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Florian David
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Fransky Hantelys
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Florence Tatin
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Eric Lacazette
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Barbara Garmy-Susini
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
| | - Anne-Catherine Prats
- UMR 1048-I2MC, Inserm, Université de Toulouse, UT3, 31432 Toulouse cedex 4, France.
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5
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Attal J, Theron MC, Puissant C, Houdebine LM. Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA. Gene Expr 2018; 8:299-309. [PMID: 10947079 PMCID: PMC6157382] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
Abstract
Specific structures found in the mRNA of picornavirus are known to allow a cap-independent translation. These structures, named internal ribosome entry sites (IRES), are also able to favor translation of the second cistron in bicistronic mRNAs. Their mechanism of action is not well understood. In the present study, two IRESs have been used: the IRES from poliovirus and a newly discovered IRES (SUR) composed of the 5' P untranslated sequence from SV40 early genes, the R structure, and a small part of the U5 region from the human leukemia virus-1 (HTLV-1). The bicistronic constructs containing the firefly luciferase gene as the first cistron and the chloramphenicol acetyltransferase (CAT) as the second cistron were driven by the Rous sarcoma virus (RSV) promoter and contained the early gene SV40 terminator. All the resulting plasmids were tested by transfection in HeLa and CHO cells. In the bicistronic mRNAs without IRES, the expression of the CAT gene was dependent on the distance between the two cistrons. The maximum efficiency in the expression of the second cistron was obtained when the intercalating RNA was composed of 30 to 90 nucleotides. This expression was deeply reduced when the intercalating fragment contained 8 or 300 nucleotides and was undetectable with 500 nucleotides. Unexpectedly, the luciferase mRNA was almost not expressed when the intercalating RNA was of 8 or 30 nucleotides. Expression of the luciferase gene occurred when the intercistronic RNA fragment was of 80 nucleotides and it became lower at 300 and 500 nucleotides. The same observations were done when the poliovirus or the SUR IRESs were added after the intercistronic spacers. However, expression of the CAT gene was amplified by both IRESs. When the CAT cistron preceded by the poliovirus or SUR IRES was introduced within luciferase cistron, 316 nucleotides before its termination codon, the IRESs were able to initiate translation of the following CAT gene irrespectively of the mRNA luciferase reading frame. Moreover, with all these constructs the highest expression level of the CAT cistron did not exceed 10% of that obtained with the same vector carrying only the CAT cistron. To identify a possible relation between the IRESs and the cap site, the CAT cistron preceded or not with an IRES was introduced 210 nucleotides downstream of the AUG codon of the luciferase gene (i.e., 258 nucleotides from the cap site) and 100 nucleotides after an added UAG termination codon. Expression of the CAT gene was not modified by the addition of the poliovirus IRES but it was strongly stimulated by the SUR IRES (the level of expression corresponded to 65% of that obtained with the same vector carrying only the CAT cistron). These results suggest that there is a cooperation between the cap and the SUR IRES and not the poliovirus IRES to stimulate translation. These data indicate that IRESs must be introduced in precise position to allow an efficient expression of the second cistron in bicistronic mRNAs.
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Affiliation(s)
- Joé Attal
- Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, CedexFrance
| | - Marie-Claire Theron
- Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, CedexFrance
| | - Claudine Puissant
- Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, CedexFrance
| | - Louis Marie Houdebine
- Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, CedexFrance
- Address correspondence to Louis Marie Houdebine, Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, Cedex France. Tel: 33 1 34 65 25 40; Fax: 33 1 34 65 22 41; E-mail:
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6
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Abstract
Reproduction of RNA viruses is typically error-prone due to the infidelity of their replicative machinery and the usual lack of proofreading mechanisms. The error rates may be close to those that kill the virus. Consequently, populations of RNA viruses are represented by heterogeneous sets of genomes with various levels of fitness. This is especially consequential when viruses encounter various bottlenecks and new infections are initiated by a single or few deviating genomes. Nevertheless, RNA viruses are able to maintain their identity by conservation of major functional elements. This conservatism stems from genetic robustness or mutational tolerance, which is largely due to the functional degeneracy of many protein and RNA elements as well as to negative selection. Another relevant mechanism is the capacity to restore fitness after genetic damages, also based on replicative infidelity. Conversely, error-prone replication is a major tool that ensures viral evolvability. The potential for changes in debilitated genomes is much higher in small populations, because in the absence of stronger competitors low-fit genomes have a choice of various trajectories to wander along fitness landscapes. Thus, low-fit populations are inherently unstable, and it may be said that to run ahead it is useful to stumble. In this report, focusing on picornaviruses and also considering data from other RNA viruses, we review the biological relevance and mechanisms of various alterations of viral RNA genomes as well as pathways and mechanisms of rehabilitation after loss of fitness. The relationships among mutational robustness, resilience, and evolvability of viral RNA genomes are discussed.
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Willcocks MM, Zaini S, Chamond N, Ulryck N, Allouche D, Rajagopalan N, Davids NA, Fahnøe U, Hadsbjerg J, Rasmussen TB, Roberts LO, Sargueil B, Belsham GJ, Locker N. Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites. Nucleic Acids Res 2018; 45:13016-13028. [PMID: 29069411 PMCID: PMC5727462 DOI: 10.1093/nar/gkx991] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2017] [Accepted: 10/12/2017] [Indexed: 01/23/2023] Open
Abstract
Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles.
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Affiliation(s)
- Margaret M Willcocks
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Salmah Zaini
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Nathalie Chamond
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Nathalie Ulryck
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Delphine Allouche
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Noemie Rajagopalan
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Nana A Davids
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Ulrik Fahnøe
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Johanne Hadsbjerg
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Thomas Bruun Rasmussen
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Lisa O Roberts
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK.,School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Bruno Sargueil
- Faculté des Sciences Pharmaceutiques et Biologiques, UMR8015, Université Paris Descartes, Paris, France
| | - Graham J Belsham
- DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
| | - Nicolas Locker
- Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
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8
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Rivas-Aravena A, Muñoz P, Jorquera P, Diaz A, Reinoso C, González-Catrilelbún S, Sandino AM. Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus. Virus Res 2017; 240:121-129. [PMID: 28743463 DOI: 10.1016/j.virusres.2017.07.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2017] [Revised: 06/08/2017] [Accepted: 07/12/2017] [Indexed: 01/24/2023]
Abstract
The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly.
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Affiliation(s)
- Andrea Rivas-Aravena
- Comisión Chilena de Energía Nuclear, Departamento de Aplicaciones Nucleares, Laboratorio de Radiobiología Celular y Molecular. Nueva Bilbao 12501, Las Condes, Santiago, Chile; Universidad San Sebastián, Facultad de Ciencias, Lota 2465, Providencia, Santiago, Chile.
| | - Patricio Muñoz
- Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile
| | - Patricia Jorquera
- Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile
| | - Alvaro Diaz
- Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile
| | - Claudia Reinoso
- Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile
| | - Sebastián González-Catrilelbún
- Comisión Chilena de Energía Nuclear, Departamento de Aplicaciones Nucleares, Laboratorio de Radiobiología Celular y Molecular. Nueva Bilbao 12501, Las Condes, Santiago, Chile; Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile
| | - Ana María Sandino
- Universidad de Santiago de Chile, Centro de Biotecnología Acuícola, Laboratorio de Virología,Av. Bernardo O'Higgins 3303, Estación Central, Santiago, Chile.
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9
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Abstract
Hepatitis C virus (HCV) is the major cause of transfusion-associated hepatitis and accounts for a significant proportion of hepatitis cases worldwide. Most, if not all, infections become persistent and about 60% of cases develop chronic liver disease with various outcomes ranging from an asymptomatic carrier state to chronic active hepatitis and liver cirrhosis, which is strongly associated with the development of hepatocellular carcinoma. Since the initial cloning of the viral genome in 1989, our knowledge of the molecular biology of HCV has increased rapidly and led to the identification of several potential targets for antiviral intervention. In contrast, the low replication of the virus in cell culture, the lack of convenient animal models and the high genome variability present major challenges for drug development. This review will describe candidate drug targets and summarize ‘classical’ and ‘novel’ approaches currently being pursued to develop efficient HCV-specific therapies.
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Affiliation(s)
- R Bartenschlager
- Institute for Virology, Johannes-Gutenberg University of Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany
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10
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Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation. Proc Natl Acad Sci U S A 2015; 112:3505-10. [PMID: 25737543 DOI: 10.1073/pnas.1420477112] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.
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11
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Asnani M, Kumar P, Hellen CUT. Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae. Virology 2015; 478:61-74. [PMID: 25726971 DOI: 10.1016/j.virol.2015.02.016] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 02/05/2015] [Accepted: 02/09/2015] [Indexed: 01/13/2023]
Abstract
Picornavirus genomes contain internal ribosomal entry sites (IRESs) that promote end-independent translation initiation. Five structural classes of picornavirus IRES have been identified, but numerous IRESs remain unclassified. Here, previously unrecognized Type IV IRESs were identified in members of three proposed picornavirus genera (Limnipivirus, Pasivirus, Rafivirus) and four recognized genera (Kobuvirus, Megrivirus, Sapelovirus, Parechovirus). These IRESs are ~230-420 nucleotides long, reflecting heterogeneity outside a common structural core. Closer analysis yielded insights into evolutionary processes that have shaped contemporary IRESs. The presence of related IRESs in diverse genera supports the hypothesis that they are heritable genetic elements that spread by horizontal gene transfer. Recombination likely also accounts for the exchange of some peripheral subdomains, suggesting that IRES evolution involves incremental addition of elements to a pre-existing core. Nucleotide conservation is concentrated in ribosome-binding sites, and at the junction of helical domains, likely to ensure orientation of subdomains in an active conformation.
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Affiliation(s)
- Mukta Asnani
- Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA
| | - Parimal Kumar
- Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA
| | - Christopher U T Hellen
- Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA.
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12
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Analysis of genotype 1b hepatitis C virus IRES in serum and peripheral blood mononuclear cells in patients treated with interferon and ribavirin. BIOMED RESEARCH INTERNATIONAL 2014; 2014:175405. [PMID: 25136559 PMCID: PMC4106116 DOI: 10.1155/2014/175405] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Accepted: 06/16/2014] [Indexed: 02/05/2023]
Abstract
Hepatitis C virus (HCV) highly conserved IRES (internal ribosome entry site) sequence, localized within the 5(')-untranslated region (5(')UTR), may determine viral properties like replication efficiency and cell tropism. The aim of the present study was to characterize newly emerging 5(')UTR variants in serum and peripheral blood mononuclear cells (PBMC) in chronic hepatitis C patients treated with interferon (IFN) and ribavirin and to identify their effect on IRES secondary structures. The study group consisted of 87 patients infected with genotype 1b from whom serum and PBMC samples were collected at 9 time points (before, during, and after treatment). New 5(')UTR variants developed in 9 patients. Out of the overall 14 new variants, 9 (64%) were found in PBMC. HCV variants with decreased thermodynamic stability were identified only in PBMC and C183U mutation was the most common one in this compartment. In conclusion, antiviral treatment may favor emergence of new 5(')UTR variants both in blood and in PBMC compartments. However, variants developing in the latter compartment were predicted to have lower thermodynamic stability of the IRES secondary structures compared to serum strains. C-U change in position 183, which has not been described previously, might indicate viral adaptation to lymphoid cells.
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13
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Inhibition of hepatitis C virus in chimeric mice by short synthetic hairpin RNAs: sequence analysis of surviving virus shows added selective pressure of combination therapy. J Virol 2014; 88:4647-56. [PMID: 24478422 DOI: 10.1128/jvi.00105-14] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
UNLABELLED We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63-66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action. IMPORTANCE This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle, if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.
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Ali AK, Lin J, Han J, Ibrahim KM, Jarjees MM, Qu F. The 5' untranslated region of Bean pod mottle virus RNA2 tolerates unusually large deletions or insertions. Virus Res 2014; 179:247-50. [PMID: 24211666 DOI: 10.1016/j.virusres.2013.10.024] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2013] [Revised: 10/24/2013] [Accepted: 10/25/2013] [Indexed: 10/26/2022]
Abstract
Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA virus of Secoviridae. We recently reported that a 137 nucleotide (nt) stretch (#263-399) of the 466 nt 5' untranslated region (5' UTR) of BPMV RNA2 can be deleted without compromising BPMV propagation in host plants [Lin et al., J. Gen. Virol. 94 (2013) 1415-1420]. Here we demonstrate that nonviral insertions of up to 625 nt is tolerated by the same region. Furthermore, one insertion mutant underwent recombination in infected plants, leading to the truncation of nt #250-361, thus extending the dispensable sequence to 150 nt (nt #250-399). We are unaware of any other (+) RNA virus that tolerates insertion/deletion of these sizes (625 nt/150 nt) within its 5' UTR. Importantly, tolerance of large insertions within the RNA2 5' UTR offers a novel, more convenient site for incorporating host gene fragments, making BPMV a more versatile vector of virus-induced gene silencing.
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Affiliation(s)
- Ahmed Khamis Ali
- Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA; Department of Biology, College of Science, The University of Mustansiriyah, Iraq
| | - Junyan Lin
- Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA
| | - Junping Han
- Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA
| | | | - Mysire Majeed Jarjees
- Plant Protection Department, College of Agriculture, University of Baghdad, Abu-Ghraib, Iraq
| | - Feng Qu
- Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA.
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15
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Huang M, Deshpande M. Hepatitis C drug discovery: in vitro and in vivo systems and drugs in the pipeline. Expert Rev Anti Infect Ther 2014; 2:375-88. [PMID: 15482203 DOI: 10.1586/14787210.2.3.375] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The combination therapy of ribavirin and pegylated interferon-alpha for hepatitis C has significant side effects, is often poorly tolerated and is ineffective in many patients, despite causing impressive improvement in the sustained virological response. Discovery and development of more effective and well-tolerated antihepatitis C virus drugs are clearly in great demand. During the past few years, remarkable advances have been made in the establishment of in vitro and in vivo systems. Armed with these systems, a wave of specific antihepatitis C virus compounds have been discovered and are moving into the clinical phase. More effective combination therapies with specific antivirals are predicted to emerge in the near future for the treatment of hepatitis C.
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Affiliation(s)
- Mingjun Huang
- Antiviral Drug Discovery, Achillion Pharmaceuticals, New Haven, CT 06511, USA.
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16
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Jahan N, Wimmer E, Mueller S. Polypyrimidine tract binding protein-1 (PTB1) is a determinant of the tissue and host tropism of a human rhinovirus/poliovirus chimera PV1(RIPO). PLoS One 2013; 8:e60791. [PMID: 23593313 PMCID: PMC3617181 DOI: 10.1371/journal.pone.0060791] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2012] [Accepted: 03/03/2013] [Indexed: 01/08/2023] Open
Abstract
The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)'s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the functional IRES structure.
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Affiliation(s)
- Nusrat Jahan
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, New York, United States of America
| | - Eckard Wimmer
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, New York, United States of America
| | - Steffen Mueller
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, New York, United States of America
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Seago J, Juleff N, Moffat K, Berryman S, Christie JM, Charleston B, Jackson T. An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein. J Gen Virol 2013; 94:1517-1527. [PMID: 23559477 PMCID: PMC3709630 DOI: 10.1099/vir.0.052308-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed.
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Affiliation(s)
- Julian Seago
- The Pirbright Institute, Woking, Surrey GU24 0NF, UK
| | | | - Katy Moffat
- The Pirbright Institute, Woking, Surrey GU24 0NF, UK
| | | | - John M Christie
- Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
| | | | - Terry Jackson
- The Pirbright Institute, Woking, Surrey GU24 0NF, UK
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18
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Inhibition of the interaction between NS3 protease and HCV IRES with a small peptide: a novel therapeutic strategy. Mol Ther 2012; 21:57-67. [PMID: 22910295 DOI: 10.1038/mt.2012.151] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.
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Toward genetics-based virus taxonomy: comparative analysis of a genetics-based classification and the taxonomy of picornaviruses. J Virol 2012; 86:3905-15. [PMID: 22278238 DOI: 10.1128/jvi.07174-11] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Virus taxonomy has received little attention from the research community despite its broad relevance. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3890-3904, 2012), we have introduced a quantitative approach to hierarchically classify viruses of a family using pairwise evolutionary distances (PEDs) as a measure of genetic divergence. When applied to the six most conserved proteins of the Picornaviridae, it clustered 1,234 genome sequences in groups at three hierarchical levels (to which we refer as the "GENETIC classification"). In this study, we compare the GENETIC classification with the expert-based picornavirus taxonomy and outline differences in the underlying frameworks regarding the relation of virus groups and genetic diversity that represent, respectively, the structure and content of a classification. To facilitate the analysis, we introduce two novel diagrams. The first connects the genetic diversity of taxa to both the PED distribution and the phylogeny of picornaviruses. The second depicts a classification and the accommodated genetic diversity in a standardized manner. Generally, we found striking agreement between the two classifications on species and genus taxa. A few disagreements concern the species Human rhinovirus A and Human rhinovirus C and the genus Aphthovirus, which were split in the GENETIC classification. Furthermore, we propose a new supergenus level and universal, level-specific PED thresholds, not reached yet by many taxa. Since the species threshold is approached mostly by taxa with large sampling sizes and those infecting multiple hosts, it may represent an upper limit on divergence, beyond which homologous recombination in the six most conserved genes between two picornaviruses might not give viable progeny.
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20
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Yao L, Dong H, Zhu H, Nelson D, Liu C, Lambiase L, Li X. Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line. J Viral Hepat 2011; 18:e523-9. [PMID: 21914072 PMCID: PMC3736357 DOI: 10.1111/j.1365-2893.2011.01452.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)-mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES-mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES-mediated translation.
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Affiliation(s)
- L. Yao
- Division of Gastroenterology, Department of Medicine, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL
| | - H. Dong
- Department of Pathology, College of Medicine, University of Florida, Gainesville, FL, USA
| | - H. Zhu
- Department of Molecular Medicine, College of Biology Hunan University, Changsha, Hunan Province, China
| | - D. Nelson
- Department of Medicine, College of Medicine, University of Florida, Gainesville, FL
| | - C. Liu
- Department of Pathology, College of Medicine, University of Florida, Gainesville, FL, USA
| | - L. Lambiase
- Division of Gastroenterology, Department of Medicine, University of Tennessee College of Medicine, Chattanooga, TN, USA
| | - X. Li
- Division of Gastroenterology, Department of Medicine, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL
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21
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A host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, PV1(RIPO). J Virol 2011; 85:7225-35. [PMID: 21561914 DOI: 10.1128/jvi.01804-09] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5'-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.
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22
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Liu G, Yángüez E, Chen Z, Li C. The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences. Virol J 2011; 8:147. [PMID: 21450110 PMCID: PMC3072930 DOI: 10.1186/1743-422x-8-147] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2010] [Accepted: 03/31/2011] [Indexed: 02/05/2023] Open
Abstract
Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.
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Affiliation(s)
- Guangqing Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, PR China.
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23
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Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis. PLoS Pathog 2010; 6:e1001066. [PMID: 20865167 PMCID: PMC2928791 DOI: 10.1371/journal.ppat.1001066] [Citation(s) in RCA: 107] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2010] [Accepted: 07/26/2010] [Indexed: 12/27/2022] Open
Abstract
In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase) in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase) of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase) and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C(ATPase) coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C(ATPase) is an essential component of the replication complex, and (iv) 2C(ATPase) has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.
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Liu Y, Wimmer E, Paul AV. Cis-acting RNA elements in human and animal plus-strand RNA viruses. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2009; 1789:495-517. [PMID: 19781674 PMCID: PMC2783963 DOI: 10.1016/j.bbagrm.2009.09.007] [Citation(s) in RCA: 134] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/04/2009] [Revised: 09/09/2009] [Accepted: 09/13/2009] [Indexed: 02/08/2023]
Abstract
The RNA genomes of plus-strand RNA viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. Those structures that are functionally important are called cis-acting RNA elements because their functions cannot be complemented in trans. They can be involved not only in RNA/RNA interactions but also in binding of viral and cellular proteins during the complex processes of translation, RNA replication and encapsidation. Most viral cis-acting RNA elements are located in the highly structured 5'- and 3'-nontranslated regions of the genomes but sometimes they also extend into the adjacent coding sequences. In addition, some cis-acting RNA elements are embedded within the coding sequences far away from the genomic ends. Although the functional importance of many of these structures has been confirmed by genetic and biochemical analyses, their precise roles are not yet fully understood. In this review we have summarized what is known about cis-acting RNA elements in nine families of human and animal plus-strand RNA viruses with an emphasis on the most thoroughly characterized virus families, the Picornaviridae and Flaviviridae.
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Affiliation(s)
- Ying Liu
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11790, USA
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25
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Inhibition of hepatitis C virus IRES-mediated translation by oligonucleotides. Virus Res 2009; 146:29-35. [PMID: 19720092 DOI: 10.1016/j.virusres.2009.08.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2009] [Revised: 08/11/2009] [Accepted: 08/21/2009] [Indexed: 02/08/2023]
Abstract
Two oligodeoxynucleotides (ODNs) were found to have a strong inhibition on the hepatitis C virus (HCV) internal ribosomal entry sites (IRES)-mediated translation but not the rabbit globin mRNA translation. Specific inhibition of those ODNs on HCV IRES-mediated translation was confirmed with heat treatment of ODNs in formic acid and dosage-dependent manners. Heat treatment of ODNs presented a decreasing inhibitory effect on HCV IRES-mediated translation. A dosage-dependent decrease of HCV IRES-mediated translation was observed with increasing amount of these ODNs in HeLa cell extracts. The minimal sequences of ODNs (A11) were identified as 5'-CGCGTTACG-3' with the strongest inhibition of the HCV IRES-mediated translation. In a search for cellular factors, two cellular factors (p68 and p70) were identified to interact with ODNs A1 and A11, but not A5 (CT-oligo). This data showed new kinds of cellular proteins involved in HCV IRES-mediated translation. Further study of ODNs and these cellular proteins will provide important information for understanding the mechanistic basis and molecular regulation of HCV IRES-mediated translation.
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26
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Savolainen-Kopra C, Samoilovich E, Kahelin H, Hiekka AK, Hovi T, Roivainen M. Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages. J Gen Virol 2009; 90:1859-1868. [PMID: 19403755 DOI: 10.1099/vir.0.010942-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The roles of recombination and accumulation of point mutations in the origin of new poliovirus (PV) characteristics have been hypothesized, but it is not known which are essential to evolution. We studied phenotypic differences between recombinant PV strains isolated from successive stool specimens of an oral PV vaccine recipient. The studied strains included three PV2/PV1 recombinants with increasing numbers of mutations in the VP1 gene, two of the three with an amino acid change I-->T in the DE-loop of VP1, their putative PV1 parent and strains Sabin 1 and 2. Growth of these viruses was examined in three cell lines: colorectal adenocarcinoma, neuroblastoma and HeLa. The main observation was a higher growth rate between 4 and 6 h post-infection of the two recombinants with the I-->T substitution. All recombinants grew at a higher rate than parental strains in the exponential phase of the replication cycle. In a temperature sensitivity test, the I-->T-substituted recombinants replicated equally well at an elevated temperature. Complete genome sequencing of the three recombinants revealed 12 (3), 19 (3) and 27 (3) nucleotide (amino acid) differences from Sabin. Mutations were located in regions defining attenuation, temperature sensitivity, antigenicity and the cis-acting replicating element. The recombination site was in the 5' end of 3D. In a competition assay, the most mutated recombinant beat parental Sabin in all three cell lines, strongly suggesting that this virus has an advantage. Two independent intertypic recombinants, PV3/PV1 and PV3/PV2, also showed similar growth advantages, but they also contained several point mutations. Thus, our data defend the hypothesis that accumulation of certain advantageous mutations plays a key role in gaining increased fitness.
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Affiliation(s)
| | - Elena Samoilovich
- Immunoprofylaxis Laboratory, Research Institute of Epidemiology and Microbiology, Minsk, Belarus
| | - Heidi Kahelin
- National Institute for Health and Welfare, Helsinki, Finland
| | | | - Tapani Hovi
- National Institute for Health and Welfare, Helsinki, Finland
| | - Merja Roivainen
- National Institute for Health and Welfare, Helsinki, Finland
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27
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Steil BP, Barton DJ. Cis-active RNA elements (CREs) and picornavirus RNA replication. Virus Res 2009; 139:240-52. [PMID: 18773930 PMCID: PMC2692539 DOI: 10.1016/j.virusres.2008.07.027] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2008] [Revised: 07/25/2008] [Accepted: 07/29/2008] [Indexed: 10/21/2022]
Abstract
Our understanding of picornavirus RNA replication has improved over the past 10 years, due in large part to the discovery of cis-active RNA elements (CREs) within picornavirus RNA genomes. CREs function as templates for the conversion of VPg, the Viral Protein of the genome, into VPgpUpU(OH). These so called CREs are different from the previously recognized cis-active RNA sequences and structures within the 5' and 3' NTRs of picornavirus genomes. Two adenosine residues in the loop of the CRE RNA structures allow the viral RNA-dependent RNA polymerase 3D(Pol) to add two uridine residues to the tyrosine residue of VPg. Because VPg and/or VPgpUpU(OH) prime the initiation of viral RNA replication, the asymmetric replication of viral RNA could not be explained without an understanding of the viral RNA template involved in the conversion of VPg into VPgpUpU(OH) primers. We review the growing body of knowledge regarding picornavirus CREs and discuss how CRE RNAs work coordinately with viral replication proteins and other cis-active RNAs in the 5' and 3' NTRs during RNA replication.
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Affiliation(s)
- Benjamin P Steil
- Department of Microbiology and Program in Molecular Biology, University of Colorado Denver, School of Medicine, United States
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Moes L, Wirth M. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon. Virol J 2007; 4:124. [PMID: 18034871 PMCID: PMC2212637 DOI: 10.1186/1743-422x-4-124] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2007] [Accepted: 11/22/2007] [Indexed: 01/14/2023] Open
Abstract
Background Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA [1]. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein [2]. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the hepatitis C viruses, another genus of the Flaviviridae. Conclusion The IRES of the non-cytopathic BVDV SD-1 strain displays features known from other pestivirus IRESes. The predicted pseudoknot in the 5'UTR of BVDV SD-1 virus represents an important structural element in BVDV translation.
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29
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Suzuki T, Ishii K, Aizaki H, Wakita T. Hepatitis C viral life cycle. Adv Drug Deliv Rev 2007; 59:1200-12. [PMID: 17825945 DOI: 10.1016/j.addr.2007.04.014] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2007] [Accepted: 04/11/2007] [Indexed: 12/16/2022]
Abstract
Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. Molecular studies of the virus became possible with the successful cloning of its genome in 1989. Although much work remains to be done regarding early and late stages of the HCV life cycle, significant progress has been made with respect to the molecular biology of HCV, especially the viral protein processing and the genome replication. This review summarizes our current understanding of genomic organization of HCV, features of the viral protein characteristics, and the viral life cycle.
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Affiliation(s)
- Tetsuro Suzuki
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
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Bradrick SS, Dobrikova EY, Kaiser C, Shveygert M, Gromeier M. Poly(A)-binding protein is differentially required for translation mediated by viral internal ribosome entry sites. RNA (NEW YORK, N.Y.) 2007; 13:1582-93. [PMID: 17652408 PMCID: PMC1950770 DOI: 10.1261/rna.556107] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2023]
Abstract
The 3' poly(A) tail present on the majority of mature eukaryotic mRNAs is an important regulator of protein synthesis and mRNA stability. The poly(A) tail improves the efficiency of translation initiation through recruitment of PABP, enabling its interaction with eIF4F located at the mRNA 5'-end. Recent evidence has also implicated a possible role for PABP and the poly(A) tract in translation control at steps beyond the initiation phase. Similar to conventional mRNAs, plus-strand RNA virus genomes that utilize internal ribosome entry sites (IRESes) to promote cap-independent translation are influenced by PABP and poly(A) status. However, the relative contribution of these factors to translation initiation mediated by distinct IRESes is unclear. We have investigated cis- and trans-acting effects of poly(A) and PABP, respectively, on RNAs harboring IRESes from three diverse viruses: encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), and coxsackievirus B3 (CBV3). A 3' poly(A) tract enhanced translation of both capped and IRES-containing reporter RNAs. However, only CBV3 and capped transcripts were stabilized as a result of polyadenylation. Correspondingly, translation of polyadenylated CBV3 and capped RNAs displayed heightened sensitivity to the PABP inhibitor Paip2 compared with EMCV and HCV. Sucrose density gradient analyses suggested a stimulatory role for PABP and 3' poly(A) in the CBV3 initiation phase, while assembly of HCV and EMCV RNAs into ribosomal complexes was little affected by either factor. Collectively, the observed differential effects of PABP and poly(A) on translation imply mechanistic differences between viral IRES elements and suggest modulating roles for PABP and the poly(A) tail at multiple phases of translation.
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Affiliation(s)
- Shelton S Bradrick
- Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
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31
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Suzuki T, Aizaki H, Murakami K, Shoji I, Wakita T. Molecular biology of hepatitis C virus. J Gastroenterol 2007; 42:411-23. [PMID: 17671755 DOI: 10.1007/s00535-007-2030-3] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2007] [Accepted: 02/10/2007] [Indexed: 02/04/2023]
Abstract
Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes persistent, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. For many years, the characterization of the HCV genome and its products has been done by heterologous expression systems because of the lack of a productive cell culture system. The development of the HCV replicon system is a highlight of HCV research and has allowed examination of the viral RNA replication in cell culture. Recently, a robust system for production of recombinant infectious HCV has been established, and classical virological techniques are now able to be applied to HCV. This development of reverse genetics-based experimental tools in HCV research can bring a greater understanding of the viral life cycle and pathogenesis of HCV-induced diseases. This review summarizes the current knowledge of cell culture systems for HCV research and recent advances in the investigation of the molecular virology of HCV.
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Affiliation(s)
- Tetsuro Suzuki
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Tokyo, Japan
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32
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Hellen CUT, de Breyne S. A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination. J Virol 2007; 81:5850-63. [PMID: 17392358 PMCID: PMC1900287 DOI: 10.1128/jvi.02403-06] [Citation(s) in RCA: 114] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2006] [Accepted: 03/20/2007] [Indexed: 01/17/2023] Open
Abstract
The 5' untranslated regions (UTRs) of the RNA genomes of Flaviviridae of the Hepacivirus and Pestivirus genera contain internal ribosomal entry sites (IRESs) that are unrelated to the two principal classes of IRESs of Picornaviridae. The mechanism of translation initiation on hepacivirus/pestivirus (HP) IRESs, which involves factor-independent binding to ribosomal 40S subunits, also differs fundamentally from initiation on these picornavirus IRESs. Ribosomal binding to HP IRESs requires conserved sequences that form a pseudoknot and the adjacent IIId and IIIe domains; analogous elements do not occur in the two principal groups of picornavirus IRESs. Here, comparative sequence analysis was used to identify a subset of picornaviruses from multiple genera that contain 5' UTR sequences with significant similarities to HP IRESs. They are avian encephalomyelitis virus, duck hepatitis virus 1, duck picornavirus, porcine teschovirus, porcine enterovirus 8, Seneca Valley virus, and simian picornavirus. Their 5' UTRs are predicted to form several structures, in some of which the peripheral elements differ from the corresponding HP IRES elements but in which the core pseudoknot, domain IIId, and domain IIIe elements are all closely related. These findings suggest that HP-like IRESs have been exchanged between unrelated virus families by recombination and support the hypothesis that RNA viruses consist of modular coding and noncoding elements that can exchange and evolve independently.
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Affiliation(s)
- Christopher U T Hellen
- Department of Microbiology and Immunology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203, USA.
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33
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Bailey JM, Tapprich WE. Structure of the 5' nontranslated region of the coxsackievirus b3 genome: Chemical modification and comparative sequence analysis. J Virol 2006; 81:650-68. [PMID: 17079314 PMCID: PMC1797431 DOI: 10.1128/jvi.01327-06] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.
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Affiliation(s)
- Jennifer M Bailey
- Department of Biology, University of Nebraska at Omaha, 6001 Dodge St, Omaha, NE 68182, USA
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34
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Dobrikova EY, Grisham RN, Kaiser C, Lin J, Gromeier M. Competitive translation efficiency at the picornavirus type 1 internal ribosome entry site facilitated by viral cis and trans factors. J Virol 2006; 80:3310-21. [PMID: 16537598 PMCID: PMC1440366 DOI: 10.1128/jvi.80.7.3310-3321.2006] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.
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Affiliation(s)
- Elena Y Dobrikova
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27710, USA
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35
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Racaniello VR. One hundred years of poliovirus pathogenesis. Virology 2006; 344:9-16. [PMID: 16364730 DOI: 10.1016/j.virol.2005.09.015] [Citation(s) in RCA: 187] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2005] [Accepted: 09/10/2005] [Indexed: 10/25/2022]
Abstract
Poliovirus was first isolated nearly 100 years ago in a landmark experiment that established the viral etiology of poliomyelitis. This discovery stimulated investigation of the pathogenesis of poliomyelitis in many laboratories. Nearly 50 years later, when two effective poliovirus vaccines were developed, the impetus to study poliovirus pathogenesis waned. The identification of the cell receptor for poliovirus, CD155, and its use in the development of transgenic mice susceptible to poliovirus revived interest in understanding how the virus causes disease. Experiments in CD155 transgenic mice have provided new information on the initial sites of virus replication in the host, how the virus spreads to the central nervous system through the blood and by axonal transport, the determinants of viral tropism, and the basis for the attenuation phenotype of the Sabin vaccine strains. Despite these advances, our understanding of poliovirus pathogenesis is still incomplete. The dilemma is not how to answer the remaining questions, but whether there will be sufficient time to do so before global eradication of poliomyelitis leads to cessation of research on the disease.
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Affiliation(s)
- Vincent R Racaniello
- Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032, USA.
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Abstract
Replication of poliovirus RNA is accomplished by the error-prone viral RNA-dependent RNA polymerase and hence is accompanied by numerous mutations. In addition, genetic errors may be introduced by nonreplicative mechanisms. Resulting variability is manifested by point mutations and genomic rearrangements (e.g., deletions, insertions and recombination). After description of basic mechanisms underlying this variability, the review focuses on regularities of poliovirus evolution (mutation fixation) in tissue cultures, human organisms and populations.
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Affiliation(s)
- V I Agol
- M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, 142782, Russia.
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37
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Shimoike T, Koyama C, Murakami K, Suzuki R, Matsuura Y, Miyamura T, Suzuki T. Down-regulation of the internal ribosome entry site (IRES)-mediated translation of the hepatitis C virus: Critical role of binding of the stem-loop IIId domain of IRES and the viral core protein. Virology 2006; 345:434-45. [PMID: 16297950 DOI: 10.1016/j.virol.2005.10.013] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2005] [Revised: 07/01/2005] [Accepted: 10/07/2005] [Indexed: 01/06/2023]
Abstract
In a previous study, we observed that hepatitis C virus (HCV) core protein specifically inhibits translation initiated by an HCV internal ribosome entry site (IRES). To investigate the mechanism by which down-regulation of HCV translation occurs, a series of mutations were introduced into the IRES element, as well as the core protein, and their effect on IRES activity examined in this study. We found that expression of the core protein inhibits HCV translation possibly by binding to a stem-loop IIId domain, particularly a GGG triplet within the hairpin loop structure of the domain, within the IRES. Basic-residue clusters located at the N-terminus of the core protein have an inhibitory effect on HCV translation, and at least one of three known clusters is required for inhibition. We propose a model in which competitive binding of the core protein for the IRES and 40S ribosomal subunit regulates HCV translation.
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Affiliation(s)
- Takashi Shimoike
- Department of Virology II, National Institute of Infectious Diseases, Musashi-murayama, Tokyo 208-0011, Japan. ,jp
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38
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Jang SK. Internal initiation: IRES elements of picornaviruses and hepatitis c virus. Virus Res 2005; 119:2-15. [PMID: 16377015 DOI: 10.1016/j.virusres.2005.11.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2005] [Revised: 08/29/2005] [Accepted: 11/02/2005] [Indexed: 02/08/2023]
Abstract
The scanning hypothesis provides an explanation for events preceding the first peptide bond formation during the translation of the vast majority of eukaryotic mRNAs. However, this hypothesis does not explain the translation of eukaryotic mRNAs lacking the cap structure required for scanning. The existence of a group of positive sense RNA viruses lacking cap structures (e.g. picornaviruses) indicates that host cells also contain a 5' cap-independent translation mechanism. This review discusses the translation mechanisms of atypical viral mRNAs such as picornaviruses and hepatitis c virus, and uses these mechanisms to propose a general theme for all translation, including that of both eukaryotic and prokaryotic mRNAs.
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Affiliation(s)
- Sung Key Jang
- NRL, PBC, Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
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39
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Teterina NL, Gorbalenya AE, Egger D, Bienz K, Rinaudo MS, Ehrenfeld E. Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein. Virology 2005; 344:453-67. [PMID: 16226781 PMCID: PMC7111807 DOI: 10.1016/j.virol.2005.08.044] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2005] [Revised: 08/04/2005] [Accepted: 08/24/2005] [Indexed: 01/06/2023]
Abstract
The N-terminal region of the picornaviral 2C protein is predicted to fold into an amphipathic α-helix that is responsible for the protein's association with membranes in the viral RNA replication complex. We have identified a similar sequence in the N-terminal region of NS5A of hepaciviruses that was recently shown to form an amphipathic α-helix. The conservation of the N-terminal region in two apparently unrelated proteins of two different RNA virus families suggested that this helix might represent an independent module. To test this hypothesis, we constructed chimeric poliovirus (PV) genomes in which the sequence encoding the N-terminal 2C amphipathic helix was replaced by orthologous sequences from other picornaviral genomes or a similar sequence from NS5A of HCV. Effects of the mutations were assessed by measuring the accumulation of viable virus and viral RNA in HeLa cells after transfection, examining membrane morphology in cells expressing chimeric proteins and by in vitro analysis of RNA translation, protein processing and negative strand RNA synthesis in HeLa cell extracts. The chimeras manifested a wide range of growth and RNA synthesis phenotypes. The results are compatible with our hypothesis, although they demonstrate that helix exchangeability may be restricted due to requirements for interactions with other viral components involved in virus replication.
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Affiliation(s)
- Natalya L Teterina
- Laboratory of Infectious Diseases, LID, NIAID, NIH, Bldg. 50, Room 6122, 50 South Drive, Bethesda, MD 20892-8011, USA.
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40
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Nagashima S, Sasaki J, Taniguchi K. The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis. J Virol 2005; 79:6918-31. [PMID: 15890931 PMCID: PMC1112095 DOI: 10.1128/jvi.79.11.6918-6931.2005] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.
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Affiliation(s)
- Shigeo Nagashima
- Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
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Grassmann CW, Yu H, Isken O, Behrens SE. Hepatitis C virus and the related bovine viral diarrhea virus considerably differ in the functional organization of the 5' non-translated region: implications for the viral life cycle. Virology 2005; 333:349-66. [PMID: 15721367 DOI: 10.1016/j.virol.2005.01.007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2004] [Revised: 12/04/2004] [Accepted: 01/07/2005] [Indexed: 01/26/2023]
Abstract
The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.
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Affiliation(s)
- Claus Wilhelm Grassmann
- Institute for Virology, Justus-Liebig-Universität Giessen, Frankfurter Street 107, 35392 Giessen, Germany
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42
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Liang C, Rieder E, Hahm B, Jang SK, Paul A, Wimmer E. Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells. Virology 2005; 333:41-53. [PMID: 15708591 DOI: 10.1016/j.virol.2004.12.031] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2004] [Revised: 10/17/2004] [Accepted: 12/23/2004] [Indexed: 11/21/2022]
Abstract
Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either "conditioned" Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naive Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.
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Affiliation(s)
- Chengyu Liang
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11790, USA
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Honda M, Shimazaki T, Kaneko S. La protein is a potent regulator of replication of hepatitis C virus in patients with chronic hepatitis C through internal ribosomal entry site-directed translation. Gastroenterology 2005; 128:449-62. [PMID: 15685555 DOI: 10.1053/j.gastro.2004.11.064] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND AND AIMS Translation of hepatitis C virus is an essential step of viral replication and is mediated by an internal ribosome entry site. We previously reported that the hepatitis C virus internal ribosome entry site is most active during the synthetic (S) or mitotic (M) phases and lowest during quiescent (G 0 ) phase. Here, we investigated host factors responsible for the regulation of the hepatitis C virus internal ribosome entry site. METHODS We synchronized the cell-cycle progression and evaluated gene-expression dynamics of host factors and kinetics of hepatitis C virus internal ribosome entry site activity in cells at various points during the cell cycle by using a complementary DNA microarray. We also validated the significance of identified host factors on hepatitis C virus replication in vivo. RESULTS Hepatitis C virus internal ribosome entry site activity correlated with a gene cluster induced in the S and G 2 /M phases. It is interesting to note that most initiation factors known to bind or interact with the hepatitis C virus internal ribosome entry site [poly(rC)-binding protein 2, polypyrimidine tract binding protein, eukaryotic initiation factor 3, eukaryotic initiation factor 2gamma, eukaryotic initiation factor 2beta, La protein, and heterogenous nuclear ribonucleoprotein L] were induced during the S and G 2 /M phases. Expression of La protein, polypyrimidine tract binding protein, and eukaryotic initiation factor 3 (p116, p170) were predominantly repressed in G 0 phase and induced in S and G 2 /M phases. Suppression or overexpression of La protein and polypyrimidine tract binding protein in RCF-26 significantly changed hepatitis C virus internal ribosome entry site activity. In the livers of patients with chronic hepatitis C, expression of La protein was significantly increased and correlated with the amount of hepatitis C virus RNA. CONCLUSIONS Hepatitis C virus uses host factors induced during cell division but not during quiescence for replication. Of these, La protein is a potent regulator and enhances hepatitis C virus replication in regenerating hepatocytes in patients with chronic hepatitis C.
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Affiliation(s)
- Masao Honda
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
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Bartenschlager R, Frese M, Pietschmann T. Novel insights into hepatitis C virus replication and persistence. Adv Virus Res 2005; 63:71-180. [PMID: 15530561 DOI: 10.1016/s0065-3527(04)63002-8] [Citation(s) in RCA: 212] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the family Flaviviridae. A hallmark of HCV is its high propensity to establish a persistent infection that in many cases leads to chronic liver disease. Molecular studies of the virus became possible with the first successful cloning of its genome in 1989. Since then, the genomic organization has been delineated, and viral proteins have been studied in some detail. In 1999, an efficient cell culture system became available that recapitulates the intracellular part of the HCV life cycle, thereby allowing detailed molecular studies of various aspects of viral RNA replication and persistence. This chapter attempts to summarize the current state of knowledge in these most actively worked on fields of HCV research.
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Affiliation(s)
- Ralf Bartenschlager
- Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
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Li D, Lott WB, Martyn J, Haqshenas G, Gowans EJ. Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B12 and the HCV core protein. J Virol 2004; 78:12075-81. [PMID: 15479850 PMCID: PMC523236 DOI: 10.1128/jvi.78.21.12075-12081.2004] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B(12), was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B(12) and the core protein differ, and they target different regions of the IRES.
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Affiliation(s)
- Dongsheng Li
- Macfarlane Burnet Institute for Medical Research and Public Health, GPO Box 2284, Melbourne, VIC 3001, Australia
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Kim JH, Paek KY, Ha SH, Cho S, Choi K, Kim CS, Ryu SH, Jang SK. A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon. Mol Cell Biol 2004; 24:7878-90. [PMID: 15340051 PMCID: PMC515056 DOI: 10.1128/mcb.24.18.7878-7890.2004] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2004] [Revised: 05/17/2004] [Accepted: 06/17/2004] [Indexed: 02/07/2023] Open
Abstract
Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5' nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.
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Affiliation(s)
- Jong Heon Kim
- NRL, PBC, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Hyoja-Dong San 31, Pohang, Kyungbuk 790-784, South Korea
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Costa-Mattioli M, Svitkin Y, Sonenberg N. La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro. Mol Cell Biol 2004; 24:6861-70. [PMID: 15254251 PMCID: PMC444852 DOI: 10.1128/mcb.24.15.6861-6870.2004] [Citation(s) in RCA: 129] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2004] [Revised: 04/02/2004] [Accepted: 04/30/2004] [Indexed: 12/12/2022] Open
Abstract
Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.
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Affiliation(s)
- Mauro Costa-Mattioli
- Department of Biochemistry and McGill Cancer Center, McGill University, McIntyre Medical Building, Montreal, Quebec, Canada H3G 1Y6
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Pudi R, Srinivasan P, Das S. La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation. J Biol Chem 2004; 279:29879-88. [PMID: 15138264 DOI: 10.1074/jbc.m403417200] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S. (2003) J. Biol. Chem. 278, 12231-12240). Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA. Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex. Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes. These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation.
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Affiliation(s)
- Renuka Pudi
- Department of Microbiology and Cell Biology, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
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Kauder SE, Racaniello VR. Poliovirus tropism and attenuation are determined after internal ribosome entry. J Clin Invest 2004; 113:1743-53. [PMID: 15199409 PMCID: PMC420511 DOI: 10.1172/jci21323] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2004] [Accepted: 04/02/2004] [Indexed: 11/17/2022] Open
Abstract
Poliovirus replication is limited to a few organs, including the brain and spinal cord. This restricted tropism may be a consequence of organ-specific differences in translation initiation by the poliovirus internal ribosome entry site (IRES). A C-to-U mutation at base 472 in the IRES of the Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence, may further restrict tropism by eliminating viral replication in the CNS. To determine the relationship between IRES-mediated translation and poliovirus tropism, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The IRESs of poliovirus, the cardiotropic coxsackievirus B3 (CVB3), and the hepatotropic hepatitis C virus (HCV) mediate translation in many organs, including those that do not support viral replication. A translation defect associated with the Sabin type 3 IRES was observed in all organs examined. Poliovirus type 1 and recombinant polioviruses dependent on the IRES of CVB3 or HCV replicate in the CNS of mice and cause paralysis. Although the type 3 Sabin strain is an effective vaccine, polioviruses with a U at base 472 of the IRES cause paralysis in newborn mice. Tropism of wild-type and vaccine strains of poliovirus is therefore determined after internal ribosome entry.
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Affiliation(s)
- Steven E Kauder
- Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA
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