|
1
|
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.
Collapse
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.)
| |
Collapse
|
|
2
|
Romero-López C, Berzal-Herranz A. The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle. Int J Mol Sci 2020; 21:1479. [PMID: 32098260 PMCID: PMC7073135 DOI: 10.3390/ijms21041479] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 02/05/2023] Open
Abstract
RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.
Collapse
Affiliation(s)
- Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
| |
Collapse
|
|
3
|
Khawaja A, Vopalensky V, Pospisek M. Understanding the potential of hepatitis C virus internal ribosome entry site domains to modulate translation initiation via their structure and function. WILEY INTERDISCIPLINARY REVIEWS-RNA 2014; 6:211-24. [PMID: 25352252 PMCID: PMC4361049 DOI: 10.1002/wrna.1268] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/14/2014] [Revised: 08/31/2014] [Accepted: 09/02/2014] [Indexed: 12/16/2022]
Abstract
Translation initiation in the hepatitis C virus (HCV) occurs through a cap-independent mechanism that involves an internal ribosome entry site (IRES) capable of interacting with and utilizing the eukaryotic translational machinery. In this review, we focus on the structural configuration of the different HCV IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. In some cases, multiple mutations, even those scattered across different domains, led to restoration of the translational activity of the HCV IRES, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long-range inter- and/or intra-domain functional interactions. The precise functioning of the HCV IRES requires the specific interaction of its domains with ribosomal subunits and a subset of eukaryotic translation initiation factors (eIFs). The structural conformation, sequence preservation and variability, and translational machinery association with the HCV IRES regions are also thoroughly discussed, along with other factors that can affect and influence the formation of translation initiation complexes. WIREs RNA 2015, 6:211–224. doi: 10.1002/wrna.1268
Collapse
Affiliation(s)
- Anas Khawaja
- Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic
| | | | | |
Collapse
|
|
4
|
Chan SW. Establishment of chronic hepatitis C virus infection: Translational evasion of oxidative defence. World J Gastroenterol 2014; 20:2785-2800. [PMID: 24659872 PMCID: PMC3961964 DOI: 10.3748/wjg.v20.i11.2785] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Revised: 12/03/2013] [Accepted: 01/15/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) causes a clinically important disease affecting 3% of the world population. HCV is a single-stranded, positive-sense RNA virus belonging to the genus Hepacivirus within the Flaviviridae family. The virus establishes a chronic infection in the face of an active host oxidative defence, thus adaptation to oxidative stress is key to virus survival. Being a small RNA virus with a limited genomic capacity, we speculate that HCV deploys a different strategy to evade host oxidative defence. Instead of counteracting oxidative stress, it utilizes oxidative stress to facilitate its own survival. Translation is the first step in the replication of a plus strand RNA virus so it would make sense if the virus can exploit the host oxidative defence in facilitating this very first step. This is particularly true when HCV utilizes an internal ribosome entry site element in translation, which is distinctive from that of cap-dependent translation of the vast majority of cellular genes, thus allowing selective translation of genes under conditions when global protein synthesis is compromised. Indeed, we were the first to show that HCV translation was stimulated by an important pro-oxidant-hydrogen peroxide in hepatocytes, suggesting that HCV is able to adapt to and utilize the host anti-viral response to facilitate its own translation thus allowing the virus to thrive under oxidative stress condition to establish chronicity. Understanding how HCV translation is regulated under oxidative stress condition will advance our knowledge on how HCV establishes chronicity. As chronicity is the initiator step in disease progression this will eventually lead to a better understanding of pathogenicity, which is particularly relevant to the development of anti-virals and improved treatments of HCV patients using anti-oxidants.
Collapse
|
|
5
|
Li YP, Ramirez S, Gottwein JM, Bukh J. Non-genotype-specific role of the hepatitis C virus 5' untranslated region in virus production and in inhibition by interferon. Virology 2011; 421:222-34. [PMID: 22029937 DOI: 10.1016/j.virol.2011.10.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2011] [Revised: 06/15/2011] [Accepted: 10/03/2011] [Indexed: 12/18/2022]
Abstract
The 5' untranslated region (5'UTR) of hepatitis C virus (HCV) is structured into four domains (I-IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5'UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1-7 recombinants with highly diverse 5'UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5'UTR of genotypes 1-6 or with heterotypic chimeric (1a/3a and 3a/1a) 5'UTR domains I, II or III, and 1a recombinants with 5'UTR domain I of genotypes 1-6. All were fully functional in Huh7.5 cells; therefore, the 5'UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5'-terminus was required. We demonstrated that J6/JFH1 with 5'UTR of genotypes 1-6 responded similarly to interferon-α2b. This study provides novel insight into the role of the 5'UTR in the HCV life cycle and facilitates HCV basic research and testing of 5'UTR-targeting antivirals.
Collapse
Affiliation(s)
- Yi-Ping Li
- Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark
| | | | | | | |
Collapse
|
|
6
|
Burks JM, Zwieb C, Müller F, Wower IK, Wower J. Comparative structural studies of bovine viral diarrhea virus IRES RNA. Virus Res 2011; 160:136-42. [DOI: 10.1016/j.virusres.2011.06.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2011] [Revised: 05/28/2011] [Accepted: 06/01/2011] [Indexed: 02/03/2023]
|
|
7
|
López-Lastra M, Ramdohr P, Letelier A, Vallejos M, Vera-Otarola J, Valiente-Echeverría F. Translation initiation of viral mRNAs. Rev Med Virol 2010; 20:177-95. [PMID: 20440748 PMCID: PMC7169124 DOI: 10.1002/rmv.649] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell‐type‐specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs. Copyright © 2010 John Wiley & Sons, Ltd.
Collapse
Affiliation(s)
- Marcelo López-Lastra
- Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile.
| | | | | | | | | | | |
Collapse
|
|
8
|
Sequence variability at the internal ribosome entry site of the HCV genome in relation to therapy outcome. ARCH BIOL SCI 2009. [DOI: 10.2298/abs0902205j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
Different types of interferon are widely used to treat hepatitis C virus (HCV) infection. Results obtained in vitro suggest that interferon inhibits internal ribosome entry site (IRES)-mediated translation of the HCV genome. To elucidate the possible effect of the nucleotide sequence of IRES on therapy outcome, we compared HCV isolates from patients with sustained response and non-response to interferon/ribavirin combination therapy. In 56 analyzed HCV isolates, nucleotide changes appeared strictly in the stem-loop IIIb region, the stem part from 243 nt to 248 nt, and the polypyrimidine-II region. The natural sequence variability of IRES in isolates of genotype 3a was significantly higher than in isolates of genotype 1b (p < 0.05). The average number of nucleotide changes in genotype 3a correlated with response to therapy (p < 0.05).
Collapse
|
|
9
|
Barría MI, González A, Vera-Otarola J, León U, Vollrath V, Marsac D, Monasterio O, Pérez-Acle T, Soza A, López-Lastra M. Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation. Nucleic Acids Res 2008; 37:957-71. [PMID: 19106142 PMCID: PMC2647302 DOI: 10.1093/nar/gkn1022] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The HCV internal ribosome entry site (IRES) spans a region of ∼340 nt that encompasses most of the 5′ untranslated region (5′UTR) of the viral mRNA and the first 24–40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5′UTR on IRES activity, naturally occurring variants of the 5′UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.
Collapse
Affiliation(s)
- María Inés Barría
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Guerniou V, Gillet R, Berrée F, Carboni B, Felden B. Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates. Nucleic Acids Res 2007; 35:6778-87. [PMID: 17921501 PMCID: PMC2175329 DOI: 10.1093/nar/gkm770] [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] [Indexed: 01/23/2023] Open
Abstract
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the ‘cap-independent’ translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5′ or 3′-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37°C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the ‘IRES-dependent’ translation in vitro. The 5′-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.
Collapse
Affiliation(s)
- Valérie Guerniou
- Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France
| | | | | | | | | |
Collapse
|
|
11
|
Motazakker M, Preikschat P, Elliott J, Smith CA, Mills PR, Oien K, Spence E, Elliott RM, McCruden EAB. Translation efficiencies of the 5'-untranslated region of genotypes 1a and 3a in hepatitis C infected patients. J Med Virol 2007; 79:259-69. [PMID: 17245719 DOI: 10.1002/jmv.20794] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Differences between the translation efficiencies mediated by the 5'-untranslated regions (5'-UTR) of genotypes (gt) 1 and 3 of hepatitis C virus (HCV) have been reported but it is unknown if such differences are biologically significant. The 5'-UTR was sequenced from paired serum and liver samples from 26 patients with chronic HCV hepatitis (11 gt 1a, 15 gt 3a). To determine whether there is a consistent difference between gts 1a and 3a translation efficiency, 5'-UTR (nt 1-356) and 5'-UTR plus core (nt 1-914) sequences were cloned into bicistronic, luciferase-encoding constructs and relative translation efficiencies (RTE) measured in Huh7 cells and BHK cells. The relationships between viral load, liver biopsy Ishak scores, degree of steatosis and translational activity of the patient-derived nucleotide sequence were examined. There were no differences in 5'-UTR sequence between serum and corresponding liver samples. The mean RTE of 5'-UTR sequences from gt 3a isolates was not significantly different from gt 1a whether or not the core encoding sequence was included, although inclusion of core led to a reduction in RTE by 93-97% for both genotypes. No correlation was found between RTE and serum HCV RNA levels, liver steatosis, inflammation, or fibrosis. However, a significant correlation was found between the presence of steatosis and infection with HCV gt 3a. It is concluded that there was no difference in translation efficiencies of 5'-UTRs from patients infected with gts 1a and 3a, and translation activity measured in vitro does not correlate with viral load or severity of liver disease.
Collapse
Affiliation(s)
- M Motazakker
- Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK
| | | | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Gallegos-Orozco JF, Arenas JI, Vargas HE, Kibler KV, Wilkinson JK, Nowicki M, Radkowski M, Nasseri J, Rakela J, Laskus T. Selection of different 5' untranslated region hepatitis C virus variants during post-transfusion and post-transplantation infection. J Viral Hepat 2006; 13:489-98. [PMID: 16792543 DOI: 10.1111/j.1365-2893.2006.00724.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND Hepatitis C virus (HCV) translation is initiated in a cap-independent manner by an internal ribosome entry site (IRES) located within the 5' untranslated region (5'UTR). Sequence changes in this region could affect translation efficiency and presumably viral replication. AIM To determine translation efficiency of 5'UTR variants developing during post-transfusion hepatitis C in two immunocompetent subjects and in two immunosuppressed liver recipients with recurrent HCV. METHODS Sequential samples were screened for 5'UTR changes by single-strand conformation polymorphism followed by cloning and sequencing whenever band pattern suggested sequence changes. 5'UTR variants were tested for IRES activity using a bicistronic dual luciferase expression plasmid transfected into HepG2 and Huh7 cell-lines. RESULTS In the transfused patients, translation efficiency of 5'UTR variants from early post-transfusion samples was 5.1- to 13.7-fold higher than that of predominant variants found in late follow-up samples. Post-transplant variants in the other two patients had 2.6- to 5.9-fold higher translation efficiency than those present only in pretransplant samples. CONCLUSION In the immunocompetent host there may be selection of low translation efficiency HCV variants over the course of infection. However, in immunosuppressed subjects the opposite seems to be true as low translation efficiency variants are superseded by high translation efficiency variants.
Collapse
Affiliation(s)
- J F Gallegos-Orozco
- Division of Transplantation Medicine, Mayo Clinic, Scottsdale, AZ 85259, USA.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Yasmeen A, Hamid S, Granath FN, Lindström H, Elliott RM, Siddiqui AA, Persson MAA. Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3. J Viral Hepat 2006; 13:87-95. [PMID: 16436126 DOI: 10.1111/j.1365-2893.2005.00660.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Combination therapy with interferon-alpha (IFN-alpha) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20-30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1-407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 +/- 13 vs 69.4 +/- 22; P < 0.01). Subsequently, the effect of IFN-alpha plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection.
Collapse
Affiliation(s)
- A Yasmeen
- Department of Medicine, Center for Molecular Medicine, and Clinical Epidemiology Unit, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden
| | | | | | | | | | | | | |
Collapse
|
|
14
|
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.
Collapse
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
| | | | | | | | | |
Collapse
|
|
15
|
Rijnbrand R, Thiviyanathan V, Kaluarachchi K, Lemon SM, Gorenstein DG. Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites. J Mol Biol 2004; 343:805-17. [PMID: 15476802 DOI: 10.1016/j.jmb.2004.08.095] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2004] [Revised: 08/29/2004] [Accepted: 08/30/2004] [Indexed: 02/05/2023]
Abstract
Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.
Collapse
Affiliation(s)
- Rene Rijnbrand
- Department of Microbiology, University of Texas Medical Branch, Galveston, TX 77555, USA.
| | | | | | | | | |
Collapse
|
|
16
|
|
|
17
|
Ji H, Fraser CS, Yu Y, Leary J, Doudna JA. Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc Natl Acad Sci U S A 2004; 101:16990-5. [PMID: 15563596 PMCID: PMC534415 DOI: 10.1073/pnas.0407402101] [Citation(s) in RCA: 140] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site).
Collapse
Affiliation(s)
- Hong Ji
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | | | | | | | | |
Collapse
|
|
18
|
Thelu MA, Drouet E, Hilleret MN, Zarski JP. Lack of clinical significance of variability in the internal ribosome entry site of hepatitis C virus. J Med Virol 2004; 72:396-405. [PMID: 14748063 DOI: 10.1002/jmv.20021] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The extreme 5'-proximal sequence of the hepatitis C virus (HCV) genome including the 5' non-coding region (5'NCR) of 341 nucleotide long and the first 30 nucleotides of the core region is highly conserved among different HCV genotypes. It contains a segment termed Internal Ribosome Entry Site (IRES) that regulates the cap-independent translation of HCV-RNA to polyprotein. Sequence variability in this region has important implications for structural organisation and function of the IRES element and could correlate with HCV RNA concentration or response to antiviral therapy. Fourteen patients (seven women, seven men) with chronic hepatitis C were separated into two groups according to their response to antiviral therapy. Seven of these were sustained responders to treatment by Interferon alpha 2b and Ribavirin and seven were non-responders. After cloning-sequencing, the IRES (nt 21 to 374) appears to be structurally highly conserved. However some variability was found between the different isolates obtained: 209 substitutions with a median of four variants/patients. Comparison of the number of variants present in the viral population of the sustained responders and non-responders patients do not show any difference. Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. The translation initiator AUG-4 codon, located in the stem-loop IV, is never modified. Variations observed in the IRES are not a parameter of response to antiviral therapy, but the integrity of this region is a necessary condition to maintain its activity.
Collapse
Affiliation(s)
- Marie-Ange Thelu
- Département d'Hépato-Gastroentérologie, Hôpital Albert Michallon, Grenoble, France.
| | | | | | | |
Collapse
|
|
19
|
Kalliampakou KI, Psaridi-Linardaki L, Mavromara P. Mutational analysis of the apical region of domain II of the HCV IRES. FEBS Lett 2002; 511:79-84. [PMID: 11821053 DOI: 10.1016/s0014-5793(01)03300-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The hepatitis C virus internal ribosome entry site (IRES) binds directly to the 40S ribosomal subunit via domains III/IV while domain II induces conformational changes on the ribosome which have been implicated in the decoding process. Here, we performed an extensive mutational study within the apical portion of domain II in order to address the functional role of this region on translation. Our results showed that the conservation of most nucleotides in this region was only partially related to the IRES function. Notwithstanding, however, selected single point mutations within the apical loop had a deleterious effect on IRES activity.
Collapse
Affiliation(s)
- Katerina I Kalliampakou
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vassilisis Sofias Avenue, Athens 11521, Greece
| | | | | |
Collapse
|
|
20
|
Odreman-Macchioli F, Baralle FE, Buratti E. Mutational analysis of the different bulge regions of hepatitis C virus domain II and their influence on internal ribosome entry site translational ability. J Biol Chem 2001; 276:41648-55. [PMID: 11498532 DOI: 10.1074/jbc.m104128200] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The hepatitis C virus (HCV) 5'-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.
Collapse
Affiliation(s)
- F Odreman-Macchioli
- International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy
| | | | | |
Collapse
|
|
21
|
Lott WB, Takyar SS, Tuppen J, Crawford DH, Harrison M, Sloots TP, Gowans EJ. Vitamin B12 and hepatitis C: molecular biology and human pathology. Proc Natl Acad Sci U S A 2001; 98:4916-21. [PMID: 11296247 PMCID: PMC33138 DOI: 10.1073/pnas.081072798] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.
Collapse
Affiliation(s)
- W B Lott
- Clinical Medical Virology Research Centre, University of Queensland, St. Lucia, Queensland 4067, Australia.
| | | | | | | | | | | | | |
Collapse
|
|
22
|
Le SY, Liu WM, Maizel JV. Phylogenetic evidence for the improved RNA higher-order structure in internal ribosome entry sequences of HCV and pestiviruses. Virus Genes 2001; 17:279-95. [PMID: 9926403 DOI: 10.1023/a:1008073905920] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The strong requirement for a small segment of the 5'-proximal coding sequence of hepatitis C virus (HCV) is one of the most remarkable features in the internal initiation of HCV mRNA translation. Phylogenetic analysis and RNA folding indicate a common RNA structure of the 5' untranslated region (UTR) of HCV and the animal pestiviruses, including HCV types 1-11, bovine viral diarrhea (BVDV), border disease virus (BDV) and hog cholera (HoCV). Although the common RNA structure shares similar features to that proposed for the internal ribosome entry sequence (IRES) of picornavirus, phylogenetic evidence suggests four new tertiary interactions between conserved terminal hairpin loops and between the terminal hairpin loop of F2b and the short coding sequence for HCV and pestiviruses. We suggest that the higher-order structures of IRES cis-acting elements for HCV and animal pestivirus are composed of stem-loop structures B-C, domains E-H, stem-loop structure J and four additional tertiary interactions. The common structure of IRES elements for these viruses forms a compact structure by these tertiary interactions and stem stacking. The active structural core is centered in the junction domain of E-H that is also conserved in all members of picornaviruses. Our model suggests that the requirement for a small segment of the 5' coding sequence is to form the distinct tertiary structure that facilitates the cis-acting function of the HCV IRES in the internal initiation of the translational control.
Collapse
Affiliation(s)
- S Y Le
- Laboratory of Experimental and Computational Biology, National Cancer Institute, NIH, Frederick, Maryland 21702, USA.
| | | | | |
Collapse
|
|
23
|
Laporte J, Malet I, Andrieu T, Thibault V, Toulme JJ, Wychowski C, Pawlotsky JM, Huraux JM, Agut H, Cahour A. Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type. J Virol 2000; 74:10827-33. [PMID: 11044132 PMCID: PMC110962 DOI: 10.1128/jvi.74.22.10827-10833.2000] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.
Collapse
Affiliation(s)
- J Laporte
- Laboratoire de virologie, C.E.R.V.I., UPRES EA 2387, Hôpital Pitié-Salpêtrière, 75651 Paris Cedex 13, France
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
24
|
Abstract
As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell.
Collapse
Affiliation(s)
- M Gale
- University of Texas Southwestern Medical Center, Dallas, Texas, USA.
| | | | | |
Collapse
|
|
25
|
Odreman-Macchioli FE, Tisminetzky SG, Zotti M, Baralle FE, Buratti E. Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res 2000; 28:875-85. [PMID: 10648778 PMCID: PMC102586 DOI: 10.1093/nar/28.4.875] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/1999] [Revised: 12/22/1999] [Accepted: 12/22/1999] [Indexed: 01/15/2023] Open
Abstract
Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).
Collapse
Affiliation(s)
- F E Odreman-Macchioli
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy
| | | | | | | | | |
Collapse
|
|
26
|
Rijnbrand RC, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 1999; 242:85-116. [PMID: 10592657 DOI: 10.1007/978-3-642-59605-6_5] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- R C Rijnbrand
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston 77555-1019, USA
| | | |
Collapse
|
|
27
|
Féray C, Shouval D, Samuel D. Will transplantation of an hepatitis C-infected graft improve the outcome of liver transplantation in HCV patients? Gastroenterology 1999; 117:263-5. [PMID: 10381937 DOI: 10.1016/s0016-5085(99)70577-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
|
|
28
|
Tang S, Collier AJ, Elliott RM. Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus 1b 5' untranslated region (5'UTR) lead to mutants severely defective in translation which cannot be complemented in trans by the wild-type 5'UTR sequence. J Virol 1999; 73:2359-64. [PMID: 9971819 PMCID: PMC104481 DOI: 10.1128/jvi.73.3.2359-2364.1999] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/1998] [Accepted: 12/08/1998] [Indexed: 11/20/2022] Open
Abstract
Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5' untranslated region (5'UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5'UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5'UTR sequence were identified at various locations throughout the 5'UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.
Collapse
Affiliation(s)
- S Tang
- Institute of Virology, University of Glasgow, Glasgow G11 5JR, Scotland, United Kingdom
| | | | | |
Collapse
|
|
29
|
Abstract
The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.
Collapse
Affiliation(s)
- C U Hellen
- Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York Health Science Center at Brooklyn, Brooklyn, NY 11203, USA
| | | |
Collapse
|
|
30
|
Negro F, Krawczynski K, Quadri R, Rubbia-Brandt L, Mondelli M, Zarski JP, Hadengue A. Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reverse-transcriptase polymerase chain reaction. Hepatology 1999; 29:536-42. [PMID: 9918932 DOI: 10.1002/hep.510290223] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic- and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-alpha) treatment. Genomic- and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The HCV-RNA level in the serum correlated with the genomic-strand, but not with the minus-strand, HCV-RNA titer in the liver. No correlations were found between either strand of the intrahepatic HCV RNA and the level of expression of HCV antigens in the liver, or with the grading/staging of the underlying liver disease. The response to IFN-alpha treatment could be predicted by the serum HCV-RNA level and genotype, but not by the intrahepatic level of genomic- or minus-strand HCV RNA. These results suggest that, although the detection of the minus-strand HCV RNA reliably identifies the presence of replicating HCV in its target organ, the quantitative measurement of viremia remains the clinically meaningful "golden standard" for assessing the level of HCV replication.
Collapse
Affiliation(s)
- F Negro
- Divisions of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland.
| | | | | | | | | | | | | |
Collapse
|
|
31
|
Martell M, Gómez J, Esteban JI, Sauleda S, Quer J, Cabot B, Esteban R, Guardia J. High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA. J Clin Microbiol 1999; 37:327-32. [PMID: 9889212 PMCID: PMC84298 DOI: 10.1128/jcm.37.2.327-332.1999] [Citation(s) in RCA: 188] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.
Collapse
Affiliation(s)
- M Martell
- Liver Unit, Department of Medicine, Hospital General Universitari Vall d'Hebron, Barcelona, Spain
| | | | | | | | | | | | | | | |
Collapse
|
|
32
|
Buratti E, Tisminetzky S, Zotti M, Baralle FE. Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3. Nucleic Acids Res 1998; 26:3179-87. [PMID: 9628916 PMCID: PMC147697 DOI: 10.1093/nar/26.13.3179] [Citation(s) in RCA: 105] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Translation initiation in Hepatitis C Virus is controlled by the presence of an internal ribosome entry site element (IRES) principally located in its 5' untranslated region (UTR). Mutation/deletion analyses have shown that the integrity of this structure is essential for initiation of cap-independent protein synthesis. We have developed a strategy to swap the position of the two major domains (II and III) on the 5'UTR sequence. The aim was to further characterize this mechanism by preserving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the different domains on the 5'UTR is essential for efficient IRES functioning. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain III makes direct contact with two proteins (p170/p120) which can be associated with efficient IRES activity. In particular, we have mapped the binding sites of these proteins and shown that p120 binds to the apical loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryotic initiation factor eIF3: p170 and p116/p110.
Collapse
Affiliation(s)
- E Buratti
- International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
| | | | | | | |
Collapse
|