RESUMEN
The genome of RNA viruses folds into 3D structures that include long-range RNARNA interactions relevant to control critical steps of the viral cycle. In particular, initiation of translation driven by the IRES element of foot-and-mouth disease virus is stimulated by the 3ÎUTR. Here we sought to investigate the RNA local flexibility of the IRES element and the 3ÎUTR in living cells. The SHAPE reactivity observed in vivo showed statistically significant differences compared to the free RNA, revealing protected or exposed positions within the IRES and the 3ÎUTR. Importantly, the IRES local flexibility was modified in the presence of the 3ÎUTR, showing significant protections at residues upstream from the functional start codon. Conversely, presence of the IRES element in cis altered the 3ÎUTR local flexibility leading to an overall enhanced reactivity. Unlike the reactivity changes observed in the IRES element, the SHAPE differences of the 3ÎUTR were large but not statistically significant, suggesting multiple dynamic RNA interactions. These results were supported by covariation analysis, which predicted IRES-3ÎUTR conserved helices in agreement with the protections observed by SHAPE probing. Mutational analysis suggested that disruption of one of these interactions could be compensated by alternative base pairings, providing direct evidences for dynamic long-range interactions between these distant elements of the viral genome.
Asunto(s)
Regiones no Traducidas 3' , Virus de la Fiebre Aftosa/genética , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , Animales , Emparejamiento Base , Secuencia de Bases , Línea Celular , Codón Iniciador , Simulación por Computador , Virus de la Fiebre Aftosa/metabolismo , Genoma Viral , Sitios Internos de Entrada al Ribosoma , Modelos Moleculares , Mutagénesis , Estabilidad del ARN/genéticaRESUMEN
The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.
Asunto(s)
Antivirales/farmacología , Gentamicinas/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , ARN Viral/química , Línea Celular Tumoral/virología , Codón Iniciador , Hepacivirus/patogenicidad , Humanos , Cinética , Conformación de Ácido Nucleico , ARN Viral/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismo , Ribonucleasa P/química , Ribonucleasa P/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA-protein interactions on IRES activity.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Cromatografía de Afinidad/métodos , Electroforesis Capilar , Espectrometría de Masas , Conformación de Ácido Nucleico , Virus ARN/genética , Virus ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200 nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself.
Asunto(s)
Hepacivirus/metabolismo , Interferón-alfa/metabolismo , Hígado/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/genética , Emparejamiento Base , Secuencia de Bases , Cartilla de ADN/genética , Hepacivirus/genética , Humanos , Interferón-alfa/genética , Análisis por Micromatrices , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa P/genética , Ribonucleasa P/aislamiento & purificación , Ribonucleasa P/metabolismo , Análisis de Secuencia de ARN , Especificidad por Sustrato , Synechocystis/enzimología , Synechocystis/genéticaRESUMEN
A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C(79) and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U(78) and CSFV U(44) in others. Among other reaction products, the majority of biochemically active products detected contained 3'-phosphate and 5'-phosphate-end groups at the newly generated termini, along with a much lower amount of 3'-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (~33 nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures.
Asunto(s)
ARN Catalítico/química , ARN Catalítico/efectos de la radiación , ARN Viral/química , ARN Viral/efectos de la radiación , Rayos Ultravioleta , Antioxidantes/farmacología , Virus de la Fiebre Porcina Clásica/genética , Hepacivirus/genética , Radical Hidroxilo/química , Mutación , Oxidación-Reducción , ARN Catalítico/metabolismo , ARN Viral/metabolismoRESUMEN
It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5' untranslated region could ultimately represent a new mechanism of action of micro-RNAs.
Asunto(s)
Regiones no Traducidas 5' , Hepacivirus/genética , MicroARNs/química , ARN Viral/química , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Cinética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , ARN Viral/metabolismo , Ribonucleasa III/metabolismo , Ribonucleasa T1/metabolismo , Análisis de Secuencia de ARNRESUMEN
Internal ribosome entry site (IRES) elements were discovered in picornaviruses. These elements are cis-acting RNA sequences that adopt diverse three-dimensional structures and recruit the translation machinery using a 5' end-independent mechanism assisted by a subset of translation initiation factors and various RNA binding proteins termed IRES transacting factors (ITAFs). Many of these factors suffer important modifications during infection including cleavage by picornavirus proteases, changes in the phosphorylation level and/or redistribution of the protein from the nuclear to the cytoplasm compartment. Picornavirus IRES are amongst the most potent elements described so far. However, given their large diversity and complexity, the mechanistic basis of its mode of action is not yet fully understood. This review is focused to describe recent advances on the studies of RNA structure and RNA-protein interactions modulating picornavirus IRES activity.