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van den Akker GGH, Zacchini F, Housmans BAC, van der Vloet L, Caron MMJ, Montanaro L, Welting TJM. Current Practice in Bicistronic IRES Reporter Use: A Systematic Review. Int J Mol Sci 2021; 22:5193. [PMID: 34068921 PMCID: PMC8156625 DOI: 10.3390/ijms22105193] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Revised: 05/05/2021] [Accepted: 05/12/2021] [Indexed: 12/26/2022] Open
Abstract
Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994-2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication.
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Affiliation(s)
- Guus Gijsbertus Hubert van den Akker
- Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands; (G.G.H.v.d.A.); (B.A.C.H.); (L.v.d.V.); (M.M.J.C.)
| | - Federico Zacchini
- Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, I-40138 Bologna, Italy; (F.Z.); (L.M.)
- Centro di Ricerca Biomedica Applicata—CRBA, Bologna University, Policlinico di Sant’Orsola, I-40138 Bologna, Italy
| | - Bas Adrianus Catharina Housmans
- Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands; (G.G.H.v.d.A.); (B.A.C.H.); (L.v.d.V.); (M.M.J.C.)
| | - Laura van der Vloet
- Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands; (G.G.H.v.d.A.); (B.A.C.H.); (L.v.d.V.); (M.M.J.C.)
| | - Marjolein Maria Johanna Caron
- Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands; (G.G.H.v.d.A.); (B.A.C.H.); (L.v.d.V.); (M.M.J.C.)
| | - Lorenzo Montanaro
- Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, I-40138 Bologna, Italy; (F.Z.); (L.M.)
- Centro di Ricerca Biomedica Applicata—CRBA, Bologna University, Policlinico di Sant’Orsola, I-40138 Bologna, Italy
- Programma Dipartimentale in Medicina di Laboratorio, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Via Albertoni 15, I-40138 Bologna, Italy
| | - Tim Johannes Maria Welting
- Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands; (G.G.H.v.d.A.); (B.A.C.H.); (L.v.d.V.); (M.M.J.C.)
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Moreira-Soto A, Arroyo-Murillo F, Sander AL, Rasche A, Corman V, Tegtmeyer B, Steinmann E, Corrales-Aguilar E, Wieseke N, Avey-Arroyo J, Drexler JF. Cross-order host switches of hepatitis C-related viruses illustrated by a novel hepacivirus from sloths. Virus Evol 2020; 6:veaa033. [PMID: 32704383 PMCID: PMC7368370 DOI: 10.1093/ve/veaa033] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The genealogy of the hepatitis C virus (HCV) and the genus Hepacivirus remains elusive despite numerous recently discovered animal hepaciviruses (HVs). Viruses from evolutionarily ancient mammals might elucidate the HV macro-evolutionary patterns. Here, we investigated sixty-seven two-toed and nine three-toed sloths from Costa Rica for HVs using molecular and serological tools. A novel sloth HV was detected by reverse transcription polymerase chain reaction (RT-PCR) in three-toed sloths (2/9, 22.2%; 95% confidence interval (CI), 5.3-55.7). Genomic characterization revealed typical HV features including overall polyprotein gene structure, a type 4 internal ribosomal entry site in the viral 5'-genome terminus, an A-U-rich region and X-tail structure in the viral 3'-genome terminus. Different from other animal HVs, HV seropositivity in two-toed sloths was low at 4.5 per cent (3/67; CI, 1.0-12.9), whereas the RT-PCR-positive three-toed sloths were seronegative. Limited cross-reactivity of the serological assay implied exposure of seropositive two-toed sloths to HVs of unknown origin and recent infections in RT-PCR-positive animals preceding seroconversion. Recent infections were consistent with only 9 nucleotide exchanges between the two sloth HVs, located predominantly within the E1/E2 encoding regions. Translated sequence distances of NS3 and NS5 proteins and host comparisons suggested that the sloth HV represents a novel HV species. Event- and sequence distance-based reconciliations of phylogenies of HVs and of their hosts revealed complex macro-evolutionary patterns, including both long-term evolutionary associations and host switches, most strikingly from rodents into sloths. Ancestral state reconstructions corroborated rodents as predominant sources of HV host switches during the genealogy of extant HVs. Sequence distance comparisons, partial conservation of critical amino acid residues associated with HV entry and selection pressure signatures of host genes encoding entry and antiviral protein orthologs were consistent with HV host switches between genetically divergent mammals, including the projected host switch from rodents into sloths. Structural comparison of HCV and sloth HV E2 proteins suggested conserved modes of hepaciviral entry. Our data corroborate complex macro-evolutionary patterns shaping the genus Hepacivirus, highlight that host switches are possible across highly diverse host taxa, and elucidate a prominent role of rodent hosts during the Hepacivirus genealogy.
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Affiliation(s)
- Andres Moreira-Soto
- Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany.,Virology-CIET, Faculty of Microbiology, University of Costa Rica, San José, Costa Rica
| | | | - Anna-Lena Sander
- Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany
| | - Andrea Rasche
- Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany
| | - Victor Corman
- Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany
| | - Birthe Tegtmeyer
- Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover 30625, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Faculty of Medicine, Ruhr-University Bochum, Bochum 44801, Germany
| | | | - Nicolas Wieseke
- Swarm Intelligence and Complex Systems Group, Department of Computer Science, Leipzig University, Leipzig, Germany
| | | | - Jan Felix Drexler
- Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin 10117, Germany.,German Centre for Infection Research (DZIF), Germany
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3
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Hepatitis C Virus Translation Regulation. Int J Mol Sci 2020; 21:ijms21072328. [PMID: 32230899 PMCID: PMC7178104 DOI: 10.3390/ijms21072328] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 03/18/2020] [Accepted: 03/25/2020] [Indexed: 12/12/2022] Open
Abstract
Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.
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Dutkiewicz M, Ciesiołka J. Form confers function: Case of the 3’X region of the hepatitis C virus genome. World J Gastroenterol 2018; 24:3374-3383. [PMID: 30122877 PMCID: PMC6092582 DOI: 10.3748/wjg.v24.i30.3374] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Revised: 06/25/2018] [Accepted: 06/30/2018] [Indexed: 02/06/2023] Open
Abstract
At the 3’ end of genomic hepatitis C virus (HCV) RNA there is a highly conserved untranslated region, the 3’X-tail, which forms part of the 3’UTR. This region plays key functions in regulation of critical processes of the viral life cycle. The 3’X region is essential for viral replication and infectivity. It is also responsible for regulation of switching between translation and transcription of the viral RNA. There is some evidence indicating the contribution of the 3’X region to the translation efficiency of the viral polyprotein and to the encapsidation process. Several different secondary structure models of the 3’X region, based on computer predictions and experimental structure probing, have been proposed. It is likely that the 3’X region adopts more than one structural form in infected cells and that a specific equilibrium between the various forms regulates several aspects of the viral life cycle. The most intriguing explanations of the structural heterogeneity problem of the 3’X region came with the discovery of its involvement in long-range RNA-RNA interactions and the potential for homodimer formation. This article summarizes current knowledge on the structure and function of the 3’X region of hepatitis C genomic RNA, reviews previous opinions, presents new hypotheses and summarizes the questions that still remain unanswered.
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Affiliation(s)
- Mariola Dutkiewicz
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan 61-704, Poland
| | - Jerzy Ciesiołka
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan 61-704, Poland
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Liang R, Li C, Jin H, Meng C, Chen Z, Zhu J, Miao Q, Ding C, Liu G. Duck hepatitis A virus serotype 1 minigenome: a model for studying the viral 3'UTR effect on viral translation. Virus Genes 2015; 51:367-74. [PMID: 26578153 DOI: 10.1007/s11262-015-1255-0] [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: 06/16/2015] [Accepted: 10/09/2015] [Indexed: 10/22/2022]
Abstract
To date, the genetic replication and translation mechanisms as well as the pathogenesis of duck hepatitis A virus type 1 (DHAV-1) have not been adequately characterized due to the lack of a reliable and efficient cell culture system. Although the full-length infections clone system is the best platform to manipulate the virus, it is relatively difficult to assemble this system due to the lack of a suitable cell line. It has been proven that the minigenome system an efficient reverse genetics system for the study of RNA viruses. In some cases, it can be used to displace the infectious clone of RNA viruses. Here, we generated a minigenome for DHAV-1 with two luciferase reporter genes, firefly luciferase (Fluc) and Renilla luciferase (Rluc). The Rluc gene was used as a reference gene for the normalization of the Fluc gene expression in transfected cells, which provided a platform for studying the regulatory mechanisms of DHAV-1. Furthermore, to investigate the role of DHAV-3'UTR in the regulation of viral protein translation, deletions in the 3'UTR were introduced into the DHAV-1 minigenome. Luciferase activity, an indicator of virus translation, was then determined. These results showed that a minigenome system for DHAV-1 was successfully constructed for the first time and that the complete or partial deletion of the DHAV-3'UTR did not affect the expression level of the reporter gene, indicating that DHAV-1 translation may not be modulated by the viral genomic 3'UTR sequence.
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Affiliation(s)
- Ruiying Liang
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
- College of Veterinary Medicine, China Agricultural University, Beijing, 100094, China
| | - Chuanfeng Li
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Hongyan Jin
- Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Chunchun Meng
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Zongyan Chen
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Jie Zhu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Qiuhong Miao
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Chan Ding
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China
| | - Guangqing Liu
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
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Hoffman B, Li Z, Liu Q. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR. J Gen Virol 2015; 96:2114-2121. [PMID: 25862017 DOI: 10.1099/vir.0.000141] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.
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Affiliation(s)
- Brett Hoffman
- VIDO-InterVac, Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Zhubing Li
- VIDO-InterVac, Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Qiang Liu
- VIDO-InterVac, Vaccinology and Immunotherapeutics, Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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Abstract
Hepatitis C viral protein translation occurs in a cap-independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5'-untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap-dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis-acting viral factors, as well as trans-acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.
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Affiliation(s)
- Brett Hoffman
- Vaccine and Infectious Disease Organization/International Vaccine Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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Romero-López C, Berzal-Herranz A. A long-range RNA-RNA interaction between the 5' and 3' ends of the HCV genome. RNA (NEW YORK, N.Y.) 2009; 15:1740-1752. [PMID: 19605533 PMCID: PMC2743058 DOI: 10.1261/rna.1680809] [Citation(s) in RCA: 101] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/07/2009] [Accepted: 06/04/2009] [Indexed: 02/05/2023]
Abstract
The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5' UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3' UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3' end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3' end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA-RNA interaction between the 5' and 3' ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5'-3' end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.
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Affiliation(s)
- Cristina Romero-López
- Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Armilla, 18100 Granada, Spain
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Lourenço S, Costa F, Débarges B, Andrieu T, Cahour A. Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors. FEBS J 2008; 275:4179-97. [DOI: 10.1111/j.1742-4658.2008.06566.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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Wolf M, Dimitrova M, Baumert TF, Schuster C. The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression. Nucleic Acids Res 2008; 36:3054-64. [PMID: 18400784 PMCID: PMC2396417 DOI: 10.1093/nar/gkn111] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an ‘alternate reading frame’ (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named 86/88ARFP. Interestingly, 86/88ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle.
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Affiliation(s)
- Marie Wolf
- Inserm, U748, Université Louis Pasteur, Strasbourg and Service d'Hépatogastroentérologie, Hôpitaux Universitaires de Strasbourg, F-67000, France
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3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 2007; 82:184-95. [PMID: 17942554 DOI: 10.1128/jvi.01796-07] [Citation(s) in RCA: 99] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3' nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3' X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3' NTR, 3' SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3' SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3' X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.
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Tellinghuisen TL, Evans MJ, von Hahn T, You S, Rice CM. Studying hepatitis C virus: making the best of a bad virus. J Virol 2007; 81:8853-67. [PMID: 17522203 PMCID: PMC1951464 DOI: 10.1128/jvi.00753-07] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
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Pawlotsky JM, Chevaliez S, McHutchison JG. The hepatitis C virus life cycle as a target for new antiviral therapies. Gastroenterology 2007; 132:1979-98. [PMID: 17484890 DOI: 10.1053/j.gastro.2007.03.116] [Citation(s) in RCA: 223] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/22/2007] [Accepted: 03/23/2007] [Indexed: 12/11/2022]
Abstract
The burden of disease consequent to hepatitis C virus (HCV) infection has been well described and is expected to increase dramatically over the next decade. Current approved antiviral therapies are effective in eradicating the virus in approximately 50% of infected patients. However, pegylated interferon and ribavirin-based therapy is costly, prolonged, associated with significant adverse effects, and not deemed suitable for many HCV-infected patients. As such, there is a clear and pressing need for the development of additional agents that act through alternate or different mechanisms, in the hope that such regimens could lead to enhanced response rates more broadly applicable to patients with hepatitis C infection. Recent basic science enhancements in HCV cell culture systems and replication assays have led to a broadening of our understanding of many of the mechanisms of HCV replication and, therefore, potential novel antiviral targets. In this article, we have attempted to highlight important new information as it relates to our understanding of the HCV life cycle. These steps broadly encompass viral attachment, entry, and fusion; viral RNA translation; posttranslational processing; HCV replication; and viral assembly and release. In each of these areas, we present up-to-date knowledge of the relevant aspects of that component of the viral life cycle and then describe the preclinical and clinical development targets and pathways being explored in the translational and clinical settings.
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Affiliation(s)
- Jean-Michel Pawlotsky
- French National Reference Center for Viral Hepatitis B, C, and delta, Department of Virology, Hôpital Henri Mondor, Université Paris 12, Créteil, France.
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14
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Song Y, Friebe P, Tzima E, Jünemann C, Bartenschlager R, Niepmann M. The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 2006; 80:11579-88. [PMID: 16971433 PMCID: PMC1642618 DOI: 10.1128/jvi.00675-06] [Citation(s) in RCA: 108] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.
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Affiliation(s)
- Yutong Song
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany
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Bradrick SS, Walters RW, Gromeier M. The hepatitis C virus 3'-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res 2006; 34:1293-303. [PMID: 16510853 PMCID: PMC1388098 DOI: 10.1093/nar/gkl019] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Enhancement of eukaryotic messenger RNA (mRNA) translation initiation by the 3′ poly(A) tail is mediated through interaction of poly(A)-binding protein with eukaryotic initiation factor (eIF) 4G, bridging the 5′ terminal cap structure. In contrast to cellular mRNA, translation of the uncapped, non-polyadenylated hepatitis C virus (HCV) genome occurs independently of eIF4G and a role for 3′-untranslated sequences in modifying HCV gene expression is controversial. Utilizing cell-based and in vitro translation assays, we show that the HCV 3′-untranslated region (UTR) or a 3′ poly(A) tract of sufficient length interchangeably stimulate translation dependent upon the HCV internal ribosomal entry site (IRES). However, in contrast to cap-dependent translation, the rate of initiation at the HCV IRES was unaffected by 3′-untranslated sequences. Analysis of post-initiation events revealed that the 3′ poly(A) tract and HCV 3′-UTR improve translation efficiency by enabling termination and possibly ribosome recycling for successive rounds of translation.
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Affiliation(s)
| | | | - Matthias Gromeier
- To whom correspondence should be addressed. Tel: +1 919 668 6205; Fax: +1 919 684 8735;
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Morikawa K, Ito T, Nozawa H, Inokuchi M, Uchikoshi M, Saito T, Mitamura K, Imawari M. Translational enhancement of HCV RNA genotype 1b by 3'-untranslated and envelope 2 protein-coding sequences. Virology 2005; 345:404-15. [PMID: 16289655 DOI: 10.1016/j.virol.2005.10.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2005] [Revised: 08/10/2005] [Accepted: 10/04/2005] [Indexed: 11/20/2022]
Abstract
HCV RNA has a unique regulatory mechanism for translation. The X region of 3'-UTR and core-coding sequence regulate HCV translation. In this study, we clarified that the entire 3'-UTR also enhances HCV translation, and the envelope-coding sequence of HCV genotype 1b increases degree of this enhancement. In the luciferase reporter assay using rabbit reticulocyte lysates, translational enhancement by 3'-UTR with core to E2 regions was 25-fold higher when compared with control RNA lacking the 3'-UTR. Presence of the entire E2 sequence was important for this enhancement. This phenomenon was not due to transcript stability, and envelope protein alone did not affect translation. E2-coding sequence of genotype 1a had no effect on translation. We observed the same results in animal cell culture systems using bicistronic RNA. Structural protein-coding sequences and 3'-UTR of HCV RNA regulate viral translation, and a target for antiviral agents may be present in these regions.
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Affiliation(s)
- Kenichi Morikawa
- The Second Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
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17
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Pappas CL, Tzeng WP, Frey TK. Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation. Arch Virol 2005; 151:327-46. [PMID: 16172842 DOI: 10.1007/s00705-005-0614-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2005] [Accepted: 07/04/2005] [Indexed: 10/25/2022]
Abstract
The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5' untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5'UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5'UTR was necessary for translation; interestingly, when plasmids were used the 5'UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SLL) that has a structure and stability similar to SL's found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SLL had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SLL or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5'UTR of the SG RNA is required. Further investigation of the differing effects of the SLL on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SLLs could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SLL only functions in a "cytopathic environment" in which cell translation has been altered.
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Affiliation(s)
- C L Pappas
- Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA
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Kalliampakou KI, Kalamvoki M, Mavromara P. Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. J Gen Virol 2005; 86:1015-1025. [PMID: 15784895 DOI: 10.1099/vir.0.80728-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5' non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.
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Affiliation(s)
- Katerina I Kalliampakou
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens 115 21, Greece
| | - Maria Kalamvoki
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens 115 21, Greece
| | - Penelope Mavromara
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens 115 21, Greece
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Topliff CL, Chon SK, Donis RO, Eskridge KM, Kelling CL. In vitro and in vivo translational efficiencies of the 5' untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates. Virology 2005; 331:349-56. [PMID: 15629777 DOI: 10.1016/j.virol.2004.09.044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2004] [Accepted: 09/28/2004] [Indexed: 11/30/2022]
Abstract
We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of sequence identity in the 5' untranslated region (UTR), subtle differences in the primary and secondary structures, as well as differences in cell lines, influence translational efficiencies.
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Affiliation(s)
- Christina L Topliff
- Department of Veterinary and Biomedical Sciences, Veterinary Basic Science, University of Nebraska, East Campus Loop and Fair Street, VBS 210, Lincoln, NE 68583-0905, USA
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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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Abstract
Bicistronic reporter assay systems have become a mainstay of molecular biology. While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties. In general, a primary dataset measures the paired expression of two internally controlled reporter genes. The expression ratio of these two genes is then normalized to an external control reporter. The end result is a ‘ratio of ratios’ that is inherently sensitive to propagation of the error contributed by each of the respective numerical components. The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects. A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems. In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems. Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed −1 frameshift signals. These frameshift signals originate from the L-A virus, the SARS-associated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes. Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed −1 frameshifting are statistically significant. A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website (http://dinmanlab.umd.edu/statistics). These spreadsheets could be easily adapted to any bicistronic reporter assay system.
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Affiliation(s)
- Jonathan L Jacobs
- Department of Cell Biology and Molecular Genetics, 2135 Microbiology Building, University of Maryland, College Park, MD 20742, USA
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