RESUMEN
Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.
Asunto(s)
Regiones no Traducidas 3' , Virus Chikungunya , Genoma Viral , ARN Viral , Recombinación Genética , Replicación Viral , Virus Chikungunya/genética , Regiones no Traducidas 3'/genética , ARN Viral/genética , ARN Viral/metabolismo , Animales , Replicación Viral/genética , Fiebre Chikungunya/virología , Fiebre Chikungunya/genética , Fiebre Chikungunya/transmisión , Humanos , Aedes/virología , Aedes/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Línea CelularRESUMEN
Background: A coronavirus identified in 2019, SARS- CoV- 2, has caused a pandemic of respiratory illness, called COVID- 19. Most people with COVID-19 experience mild to moderate symptoms and recover without the need for special treatments. The SARSCoV2 RNAdependent RNA polymerase (RdRp) plays a crucial role in the viral life cycle. The active site of the RdRp is a very accessible region, so targeting this region to study the inhibition of viral replication may be an effective therapeutic approach. For this reason, this study has selected and analysed a series of ligands used as SARS-CoV-2 virus inhibitors, namely: the Zidovudine, Tromantadine, Pyramidine, Oseltamivir, Hydroxychoroquine, Cobicistat, Doravirine (Pifeltro), Dolutegravir, Boceprevir, Indinavir, Truvada, Trizivir, Trifluridine, Sofosbuvir and Zalcitabine. Methods: These ligands were analyzed using molecular docking, Receptor-Based Pharmacophore Modelling. On the other hand, these outcomes were supported with chemical reactivity indices defined within a conceptual density functional theory framework. Results: The results show the conformations with the highest root-mean-square deviation (RMSD), have π-π stacking interaction with residue LEU141, GLN189, GLU166 and GLY143, HIE41, among others. Also was development an electrostatic potential comparison using the global and local reactivity indices. Conclusions: These studies allow the identification of the main stabilizing interactions using the crystal structure of SARSCoV2 RNAdependent RNA polymerase. In this order of ideas, this study provides new insights into these ligands that can be used in the design of new COVID-19 treatments. The studies allowed us to find an explanation supported in the Density Functional Theory about the chemical reactivity and the stabilization in the active site of the ligands.
Asunto(s)
Antivirales , Simulación del Acoplamiento Molecular , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Ligandos , Antivirales/farmacología , Antivirales/química , Humanos , COVID-19/virología , Tratamiento Farmacológico de COVID-19 , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Pandemias , Betacoronavirus/efectos de los fármacos , FarmacóforoRESUMEN
The Zika virus (ZIKV) is a neurotropic arbovirus considered a global threat to public health. Although there have been several efforts in drug discovery projects for ZIKV in recent years, there are still no antiviral drugs approved to date. Here, we describe the results of a global collaborative crowdsourced open science project, the OpenZika project, from IBM's World Community Grid (WCG), which integrates different computational and experimental strategies for advancing a drug candidate for ZIKV. Initially, molecular docking protocols were developed to identify potential inhibitors of ZIKV NS5 RNA-dependent RNA polymerase (NS5 RdRp), NS3 protease (NS2B-NS3pro), and NS3 helicase (NS3hel). Then, a machine learning (ML) model was built to distinguish active vs inactive compounds for the cytoprotective effect against ZIKV infection. We performed three independent target-based virtual screening campaigns (NS5 RdRp, NS2B-NS3pro, and NS3hel), followed by predictions by the ML model and other filters, and prioritized a total of 61 compounds for further testing in enzymatic and phenotypic assays. This yielded five non-nucleoside compounds which showed inhibitory activity against ZIKV NS5 RdRp in enzymatic assays (IC50 range from 0.61 to 17 µM). Two compounds thermally destabilized NS3hel and showed binding affinity in the micromolar range (Kd range from 9 to 35 µM). Moreover, the compounds LabMol-301 inhibited both NS5 RdRp and NS2B-NS3pro (IC50 of 0.8 and 7.4 µM, respectively) and LabMol-212 thermally destabilized the ZIKV NS3hel (Kd of 35 µM). Both also protected cells from death induced by ZIKV infection in in vitro cell-based assays. However, while eight compounds (including LabMol-301 and LabMol-212) showed a cytoprotective effect and prevented ZIKV-induced cell death, agreeing with our ML model for prediction of this cytoprotective effect, no compound showed a direct antiviral effect against ZIKV. Thus, the new scaffolds discovered here are promising hits for future structural optimization and for advancing the discovery of further drug candidates for ZIKV. Furthermore, this work has demonstrated the importance of the integration of computational and experimental approaches, as well as the potential of large-scale collaborative networks to advance drug discovery projects for neglected diseases and emerging viruses, despite the lack of available direct antiviral activity and cytoprotective effect data, that reflects on the assertiveness of the computational predictions. The importance of these efforts rests with the need to be prepared for future viral epidemic and pandemic outbreaks.
Asunto(s)
Antivirales , Inhibidores de Proteasas , Virus Zika , Humanos , Antivirales/farmacología , Antivirales/química , Simulación del Acoplamiento Molecular , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/química , Virus Zika/efectos de los fármacos , Virus Zika/enzimología , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
Over recent years, many outbreaks caused by (re)emerging RNA viruses have been reported worldwide, including life-threatening Flaviviruses, such as Dengue (DENV) and Zika (ZIKV). Currently, there is only one licensed vaccine against Dengue, Dengvaxia®. However, its administration is not recommended for children under nine years. Still, there are no specific inhibitors available to treat these infectious diseases. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) is a metalloenzyme essential for viral replication, suggesting that it is a promising macromolecular target since it has no human homolog. Nowadays, several NS5 RdRp inhibitors have been reported, while none inhibitors are currently in clinical development. In this context, this review constitutes a comprehensive work focused on RdRp inhibitors from natural, synthetic, and even repurposing sources. Furthermore, their main aspects associated with the structure-activity relationship (SAR), proposed mechanisms of action, computational studies, and other topics will be discussed in detail.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Virus Zika/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Virus del Dengue/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-Actividad , Virus Zika/enzimologíaRESUMEN
We report here high rates (75.38%, 49/65) of detection of genogroup I (GI) PBVs in diarrheic pigs on the Caribbean island of St. Kitts. High quality gene segment-2 sequences encoding a significant region (~350 amino acid (aa) residues) of the putative RNA-dependent RNA polymerase (RdRp) were obtained for 23 PBV strains. The porcine PBV strains from St. Kitts exhibited high genetic diversity among themselves (deduced aa identities of 56-100%) and with other PBVs (maximum deduced aa identities of 64-97%), and retained the three domains that are conserved in putative RdRps of PBVs. The nearly complete gene segment-2 sequence (full-length minus partial 3'- untranslated region) of a porcine PBV strain (strain PO36 from St. Kitts) that is closely related (deduced aa identities of 96-97%) to simian and human GI PBVs was determined using a combination of the non-specific primer-based amplification method and conventional RT-PCR. The complete putative RdRp sequence of strain PO36 preserved the various features that are maintained in PBVs from various species. For the first time, several co-circulating PBV strains from pigs were characterized for a significant region (~350 aa) of the putative RdRp, providing important insights into the genetic diversity of PBVs in a porcine population. Taken together, these observations corroborated growing evidence that PBVs can be highly prevalent and show limited correlation globally with host species or geography. This is the first report on detection of PBVs in pigs from the Caribbean region.
Asunto(s)
Variación Genética , Picobirnavirus/aislamiento & purificación , Infecciones por Virus ARN/veterinaria , Enfermedades de los Porcinos/virología , Secuencia de Aminoácidos , Animales , Diarrea/epidemiología , Diarrea/veterinaria , Diarrea/virología , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Picobirnavirus/genética , Infecciones por Virus ARN/epidemiología , Infecciones por Virus ARN/virología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , San Kitts y Nevis/epidemiología , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2' carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti-hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide-range antiviral drug.
Asunto(s)
Antivirales/metabolismo , Antivirales/farmacología , Nucleótidos/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Zika/efectos de los fármacos , Virus Zika/metabolismo , Antivirales/química , Sitios de Unión , Brasil , Guanosina/antagonistas & inhibidores , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Virus Zika/enzimologíaRESUMEN
Viruses are lifeless particles designed for setting virus-host interactome assuring a new generation of virions for dissemination. This interactome generates a pressure on host organisms evolving mechanisms to neutralize viral infection, which places the pressure back onto virus, a process known as virus-host cell co-evolution. Positive-single stranded RNA (+sRNA) viruses are an important group of viral agents illustrating this interesting phenomenon. During replication, their genomic +sRNA is employed as template for translation of viral proteins; among them the RNA-dependent RNA polymerase (RdRp) is responsible of viral genome replication originating double-strand RNA molecules (dsRNA) as intermediates, which accumulate representing a potent threat for cellular dsRNA receptors to initiate an antiviral response. A common feature shared by these viruses is their ability to rearrange cellular membranes to serve as platforms for genome replication and assembly of new virions, supporting replication efficiency increase by concentrating critical factors and protecting the viral genome from host anti-viral systems. This review summarizes current knowledge regarding cellular dsRNA receptors and describes prototype viruses developing replication niches inside rearranged membranes. However, for several viral agents it's been observed both, a complex rearrangement of cellular membranes and a strong innate immune antiviral response induction. So, we have included recent data explaining the mechanism by, even though viruses have evolved elegant hideouts, host cells are still able to develop dsRNA receptors-dependent antiviral response.
Asunto(s)
Inmunidad Innata , Virus ARN/enzimología , Virus ARN/inmunología , ARN Mensajero/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Bicatenario/metabolismoRESUMEN
The RNA-dependent RNA polymerase (RdRP) of the Hepatitis C virus (HCV), named NS5B, is phosphorylated by the cellular protein kinase C-related kinase 2 (PRK2) at two serine residues (Ser29 and Ser42) of the finger subdomain (genotype 1b). Herein, using bioinformatics, we selected four potential phosphorylation residues (Ser46, Ser76, Ser96 and Ser112) of NS5B (genotype 2a) for study. Whereas the NS5B Ser46D and Ser76D substitutions seemed to improve polymerase activity, the Ser96D mutation decreased colony formation efficiency. Active WT NS5B was utilized in in vitro kinase assays, and phosphopeptides were analyzed by mass spectrometry. Interestingly, the data indicated that both the NS5B Ser29 and Ser76 residues resulted phosphorylated. Thus, as Ser76 is absolutely conserved across HCV genotypes, our results confirmed the relevance of these sites for both genotypes and suggested that Ser76 becomes phosphorylated by a cellular kinase different from PRK2. By molecular dynamic simulations, we show that new interactions between space-adjacent amino acid chains could be established by the presence of a di-anionic phosphate group on the analyzed serines to possibly modify RNA polymerase activity. Together, our data present novel evidence on the complex regulation at the finger subdomain of HCV NS5B via phosphorylation.
Asunto(s)
Hepacivirus/enzimología , Hepatitis C/virología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Simulación de Dinámica Molecular , Fosforilación , Mutación Puntual , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Epigénesis Genética/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Gametogénesis en la Planta/genética , Gametogénesis en la Planta/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
The microRNA (miRNA) miR396 regulates GROWTH-REGULATING FACTORs (GRFs), a plant specific family of transcription factors. Overexpression of miR396 causes a decrease in the GRFs that has been shown to affect cell proliferation in the meristem and developing leaves. To bring further insights into the function of the miR396 regulatory network we performed a mutant enhancer screen of a stable Arabidopsis transgenic line expressing 35S:miR396b, which has a reduction in leaf size. From this screen we recovered several mutants enhancing this phenotype and displaying organs with lotus- or needle-like shape. Analysis of these plants revealed mutations in as2 and rdr6. While 35S:miR396b in an as2 context generated organs with lotus-like shape, the overexpression of the miRNA in an rdr6 mutant background caused more important developmental defects, including pin-like organs and lobed leaves. Combination of miR396 overexpressors, and rdr6 and as2 mutants show additional organ defects, suggesting that the three pathways act in concert. Genetic interactions during leaf development were observed in a similar way between miR396 overexpression and mutants in RDR6, SGS3 or AGO7, which are known to participate in trans-acting siRNA (ta-siRNA) biogenesis. Furthermore, we found that miR396 can cause lotus- and pin-like organs per se, once a certain expression threshold is overcome. In good agreement, mutants accumulating high levels of TCP4, which induces miR396, interacted with the AS1/AS2 pathway to generate lotus-like organs. The results indicate that the miR396 regulatory network and the ta-siRNA biogenesis pathway synergistically interact during leaf development and morphogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , MicroARNs , Hojas de la Planta , ARN Polimerasa Dependiente del ARN , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Meristema/genética , Meristema/metabolismo , MicroARNs/genética , Morfogénesis , Mutación , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5' untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.
Asunto(s)
Secuencias de Aminoácidos/genética , Virus del Dengue/enzimología , Regiones Promotoras Genéticas/genética , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/química , Animales , Línea Celular , Cricetinae , Virus del Dengue/genética , Virus del Dengue/metabolismo , Modelos Moleculares , Mutación , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Groundnut ringspot virus is a negative-sense single-stranded RNA virus that belongs to the genus Tospovirus and is the prevalent member of this genus in Brazil. This work presents the nucleotide sequence of the L RNA, with a single open reading frame of 2873 amino acids in the complementary strand corresponding to the RNA-dependent RNA polymerase (L protein), as well as the characterization of conserved domains of the L protein by in silico analysis. Phylogenetic analysis of different L protein domains confirmed that GRSV is a member of the American clade, and comparison with a N-protein indicates that phylogeny based on L protein sequences may be more reliable than that based on the N protein.
Asunto(s)
ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Tospovirus/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica/fisiología , Filogenia , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).
Asunto(s)
Antivirales/farmacología , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Indanos/farmacología , ARN Viral/biosíntesis , Tiosemicarbazonas/farmacología , Replicación Viral/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Antivirales/química , Línea Celular , Virus de la Diarrea Viral Bovina/fisiología , Farmacorresistencia Viral , Humanos , Indanos/química , Modelos Moleculares , Mutación Missense , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Ribavirina/farmacología , Tiosemicarbazonas/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
miR-122 is an abundant, liver-specific microRNA that is required for efficient amplification of hepatitis C virus (HCV) RNA. Recent studies with a miR-122-specific locked nucleic acid antagomir have shown it to be an important host target for therapeutic intervention. However, considerable controversy exists concerning the mechanisms underlying the dependence of HCV replication on miR-122. We studied the impact of miR-122 on the rate of [(32)P]-incorporation into positive-strand viral RNA by membrane-bound replicase complexes isolated from cells containing HCV RNA replicons. [(32)P]-incorporation in this cell-free system represents primarily the elongation phase of RNA synthesis, with little or no de novo initiation, and was not affected by the addition of either excess miR-122 or a miR-122-specific antisense oligonucleotide that suppresses replication in vivo. We also found no evidence that detectable quantities of miR-122 are specifically associated with replicase complexes in vivo. These results are consistent with miR-122 acting at an alternative step in the viral life cycle, promoting cap-independent viral translation, enhancing viral RNA stability, or facilitating de novo initiation of viral RNA synthesis.
Asunto(s)
Hepacivirus/genética , Hepacivirus/metabolismo , MicroARNs/metabolismo , ARN Viral/biosíntesis , Replicación Viral , Antivirales/farmacología , Línea Celular Tumoral , Sistema Libre de Células , Células Cultivadas , Regulación Viral de la Expresión Génica , Silenciador del Gen , Hepatocitos , Humanos , MicroARNs/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Mutación Puntual , Estabilidad del ARN , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón , Transcripción Genética , Proteínas Virales/biosíntesis , Replicación Viral/genéticaRESUMEN
In many eukaryotes, the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs through a post-transcriptional gene-silencing mechanism called RNA interference or RNAi. In the present study, we found that endogenous long-dsRNA was substantially more effective at producing interference than endogenous, or exogenous, short-dsRNA expression in Giardia lamblia . The effects of this interference were not evident in the highly expressed protein tubulin or the stage-specific cyst wall protein 2. However, long-dsRNA caused potent and specific interference in the medium subunits of adaptins, the RNA-dependent RNA polymerase, and the exogenous green fluorescence protein. Our results suggest that the ability of dsRNA antisense to inhibit the expression of these specific types of proteins is indicative of a gene-specific mechanism.
Asunto(s)
Regulación hacia Abajo/genética , Giardia lamblia/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN/fisiología , ARN Bicatenario/fisiología , Subunidades del Complejo de Proteínas Adaptadoras/genética , Subunidades del Complejo de Proteínas Adaptadoras/metabolismo , Chaperón BiP del Retículo Endoplásmico , Técnica del Anticuerpo Fluorescente Directa , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica/genética , Giardia lamblia/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Microscopía Confocal , Proteínas Protozoarias/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.
Asunto(s)
Virus del Dengue/genética , Genoma Viral , ARN Viral/biosíntesis , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Replicación Viral/fisiología , Virus del Dengue/fisiología , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , ARN Viral/química , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.