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1.
Int J Mol Sci ; 21(18)2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32971876

RESUMO

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3'-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.


Assuntos
Códon , Genoma Viral , Hepacivirus/fisiologia , Fases de Leitura Aberta , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/biossíntese , Ribossomos/metabolismo , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Humanos , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Ribossomos/genética
2.
Antiviral Res ; 165: 55-64, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30885750

RESUMO

Influenza viruses are major respiratory pathogens responsible for both seasonal epidemics and occasional pandemics worldwide. The current available treatment options have limited efficacy and thus the development of new antivirals is highly needed. We previously reported the identification of a series of cycloheptathiophene-3-carboxamide compounds as influenza A virus inhibitors that act by targeting the protein-protein interactions between the PA-PB1 subunits of the viral polymerase. In this study, we characterized the antiviral properties of the most promising compounds as well as investigated their propensity to induce drug resistance. Our results show that some of the selected compounds possess potent, broad-spectrum anti-influenza activity as they efficiently inhibited the replication of several strains of influenza A and B viruses, including an oseltamivir-resistant clinical isolate, with nanomolar or low-micromolar potency. The most promising compounds specifically inhibited the PA-PB1 binding in vitro and interfered with the influenza A virus polymerase activity in a cellular context, without showing cytotoxicity. The most active PA-PB1 inhibitors showed to possess a drug resistance barrier higher than that of oseltamivir. Indeed, no viral variants with reduced susceptibility to the selected compounds emerged after serial passages of influenza A virus under drug selective pressure. Overall, our studies identified potent PA-PB1 inhibitors as promising candidates for the development of new anti-influenza drugs.


Assuntos
Antivirais/farmacologia , Orthomyxoviridae/efeitos dos fármacos , RNA Polimerase Dependente de RNA/efeitos dos fármacos , Animais , Farmacorresistência Viral , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Oseltamivir/farmacologia , RNA Polimerase Dependente de RNA/biossíntese , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
3.
J Virol ; 91(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28148800

RESUMO

Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3' untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3' to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed.IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3' proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/biossíntese , Tombusviridae/genética , Cucumis/virologia , Análise Mutacional de DNA , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Viral/química , RNA Polimerase Dependente de RNA/genética , Tombusviridae/enzimologia
4.
BMC Plant Biol ; 16: 15, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26757721

RESUMO

BACKGROUND: Host RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1. RESULTS: In MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level. CONCLUSIONS: MtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.


Assuntos
Medicago truncatula/enzimologia , Nicotiana/virologia , Doenças das Plantas/virologia , RNA Polimerase Dependente de RNA/biossíntese , Ácido Salicílico/farmacologia , Vírus do Mosaico do Tabaco/fisiologia , Indução Enzimática , Expressão Gênica , Medicago truncatula/genética , Meristema/virologia , Plantas Geneticamente Modificadas , RNA Polimerase Dependente de RNA/metabolismo , Nicotiana/genética , Vírus do Mosaico do Tabaco/efeitos dos fármacos
5.
Virus Genes ; 51(3): 423-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26560706

RESUMO

The RNA-dependent RNA polymerase (RdRp) of rice stripe virus (RSV) is critical for both the transcription and replication of the viral genome. Despite its importance, little is known about how it functions in cells. In the present study, RSV RdRp was split into three pieces, since expression of the full protein could not be achieved. Then, the intracellular localization of these three RdRp fragments and their interactions with nucleocapsid protein (NP) were investigated, which is another viral protein required for viral RNA synthesis. The data showed that all three RdRp fragments displayed punctuate staining patterns in the cytoplasm, and the C-terminal fragment co-localized with NP in the perinuclear region. Both bimolecular fluorescence complementation and co-immunoprecipitation experiments demonstrated that of the three RdRp fragments, only the C-terminal fragment could interact with NP. Further analysis using a series of truncated NPs identified the N-terminal 50-amino-acid region within NP as the determinant for its interaction with the C-terminus of RdRp.


Assuntos
Proteínas do Nucleocapsídeo/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Tenuivirus/metabolismo , Animais , Coinfecção/virologia , Imunoprecipitação/métodos , Mutação , Sinais de Localização Nuclear , Proteínas do Nucleocapsídeo/biossíntese , Proteínas do Nucleocapsídeo/genética , Oryza/virologia , Folhas de Planta/virologia , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Viral/biossíntese , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/genética , Células Sf9/virologia , Tenuivirus/genética , Proteínas Virais/biossíntese , Proteínas Virais/metabolismo , Replicação Viral
6.
PLoS Pathog ; 10(2): e1003921, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24516387

RESUMO

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR ßC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the ßC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of ßC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the ßC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that ßC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.


Assuntos
Geminiviridae , Interações Hospedeiro-Parasita/fisiologia , Nicotiana/virologia , Proteínas de Plantas/metabolismo , Interferência de RNA/fisiologia , RNA Polimerase Dependente de RNA/biossíntese , Calmodulina/metabolismo , DNA de Plantas , Regulação da Expressão Gênica/fisiologia , Immunoblotting , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , RNA Polimerase Dependente de RNA/genética , Nicotiana/genética , Nicotiana/metabolismo
7.
J Med Chem ; 56(24): 10118-31, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24313730

RESUMO

The limited number of drug classes licensed for treatment of influenza virus (Flu), together with the continuous emergence of viral variants and drug resistant mutants, highlights the urgent need to find antivirals with novel mechanisms of action. In this context, the viral RNA-dependent RNA polymerase (RdRP) subunits assembly has emerged as an attractive target. Starting from a cycloheptathiophene-3-carboxamide derivative recently identified by us for its ability to disrupt the interaction between the PA and PB1 subunits of RdRP, we have designed and synthesized a series of analogues. Their biological evaluation led to the identification of more potent protein-protein interaction inhibitors, endowed with antiviral activity that also encompassed a number of clinical isolates of FluA, including an oseltamivir-resistant strain, and FluB, without showing appreciable toxicity. From this study, the cycloheptathiophene-3-carboxamide scaffold emerged as being particularly suitable to impart anti-Flu activity.


Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/enzimologia , RNA Polimerase Dependente de RNA/biossíntese , Tiofenos/farmacologia , Antivirais/síntese química , Antivirais/química , Benzamidas/síntese química , Benzamidas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Modelos Moleculares , Estrutura Molecular , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química
8.
PLoS One ; 8(9): e76090, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098767

RESUMO

Plant RNA-dependent RNA Polymerase 1 (RDR1) is an important element of the RNA silencing pathway in the plant defense against viruses. RDR1 expression can be elicited by viral infection and salicylic acid (SA), but the mechanisms of signaling during this process remains undefined. The involvement of hydrogen peroxide (H2O2) and nitric oxide (NO) in RDR1 induction in the compatible interactions between Tobacco mosaic tobamovirus (TMV) and Nicotiana tabacum, Nicotiana benthamiana, and Arabidopsis thaliana was examined. TMV inoculation onto the lower leaves of N. tabacum induced the rapid accumulation of H2O2 and NO followed by the increased accumulation of RDR1 transcripts in the non-inoculated upper leaves. Pretreatment with exogenous H2O2 and NO on upper leaf led to increased RDR1 expression and systemic TMV resistance. Conversely, dimethylthiourea (an H2O2 scavenger) and 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (an NO scavenger) partly blocked TMV- and SA-induced RDR1 expression and increased TMV susceptibility, whereas pretreatment with exogenous H2O2 and NO failed to diminish TMV infection in N. benthamiana plants with naturally occurring RDR1 loss-of-function. Furthermore, in N. tabacum and A. thaliana, TMV-induced H2O2 accumulation was NO-dependent, whereas NO generation was not affected by H2O2. These results suggest that, in response to TMV infection, H2O2 acts downstream of NO to mediate induction of RDR1, which plays a critical role in strengthening RNA silencing to restrict systemic viral infection.


Assuntos
Arabidopsis/imunologia , Peróxido de Hidrogênio/metabolismo , Nicotiana/imunologia , Óxido Nítrico/metabolismo , Doenças das Plantas/imunologia , RNA Polimerase Dependente de RNA/biossíntese , Vírus do Mosaico do Tabaco , Arabidopsis/virologia , Indução Enzimática/fisiologia , Fluorescência , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Nicotiana/virologia
9.
Anal Biochem ; 418(1): 50-7, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21782784

RESUMO

Hepatitis C virus (HCV) NS5B, an RNA-dependent RNA polymerase (RdRp), is an attractive target for antiviral agents. The in vitro RNA synthesis system based on radioisotopic readout is commonly used for polymerase inhibitor screening; however, this system generates large amounts of radioactive waste and is not amenable to high-throughput applications. To overcome this limitation, we generated pFLuc-(-)UTRΔC-RLuc, a bicistronic reporter vector, which allows effective and sensitive distinction of RdRp activity by using a cell-free coupled transcription/translation system. This reporter construct comprises the firefly luciferase (FLuc) and the Renilla luciferase (RLuc) genes in reverse orientation flanked by the two negative strands of the HCV 5'- and 3'-untranslated regions in which FLuc and RLuc reporter proteins are regulated by bacteriophage T7 polymerase and NS5B polymerase, respectively. The increase in RLuc activity was proportional to the amount of active RdRp. This cell-free dual reporter system was further validated using specific RdRp inhibitors. Hence, linear dose-response curves between RLuc activity and specific inhibitors were obtained, as was faster drug screening through real-time measurement of chemiluminescence. Moreover, this reporter system is suitable for robust in vitro screening because of a statistically acceptable Z' factor value of 0.79 under the antiviral screening condition in the 96-well format.


Assuntos
Genes Reporter , Hepacivirus/enzimologia , Luciferases de Renilla/genética , RNA Polimerase Dependente de RNA/genética , Transcrição Gênica , Antivirais/farmacologia , Cumarínicos/farmacologia , Hepacivirus/genética , Luciferases de Renilla/metabolismo , RNA Viral/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
10.
Biochem Biophys Res Commun ; 391(1): 570-4, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19932088

RESUMO

The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8mM, 8.0 and 25 degrees C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. K(m) (microM) were 10.74+/-0.26 (GTP), 33.22+/-3.37 (ATP), 28.93+/-0.48 (CTP) and 22.01+/-1.48 (UTP), and V(max) (fmol nucleotide/pmol RdRp/min) were 2.40+/-0.032 (GTP), 1.95+/-0.17 (ATP), 2.07+/-0.17 (CTP), and 1.52+/-0.38 (UTP), which agreed with high mutation of influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Virus da Influenza A Subtipo H5N1/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Animais , Células Cultivadas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Insetos/citologia , Cinética , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/isolamento & purificação , Proteínas Virais/biossíntese , Proteínas Virais/isolamento & purificação
11.
J Virol ; 83(23): 12279-89, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19759133

RESUMO

The RNA-dependent RNA polymerase 3D(pol) is required for the elongation of positive- and negative-stranded picornavirus RNA. During the course of investigating the effect of the transgenic expression of viral genes on the host immune response, we evaluated the viral load present in the host after infection. To our surprise, we found that 3D transgenic expression in genetically susceptible FVB mice led to substantially lower viral loads after infection with Theiler's murine encephalomyelitis virus (TMEV). As a result, spinal cord damage caused by chronic viral infection in the central nervous system was reduced in FVB mice that expressed 3D. This led to the preservation of large-diameter axons and motor function in these mice. The 3D transgene also lowered early viral loads when expressed in FVB-D(b) mice resistant to persistent TMEV infection. The protective effect of 3D transgenic expression was not altered in FVB-Rag(-/-).3D mice that are deficient in T and B cells, thus ruling out a mechanism by which the overexpression of 3D enhanced the adaptive immune clearance of the virus. Understanding how endogenously overexpressed 3D polymerase inhibits viral replication may lead to new strategies for targeting therapies to all picornaviruses.


Assuntos
Doenças Desmielinizantes/imunologia , RNA Polimerase Dependente de RNA/biossíntese , Theilovirus/imunologia , Animais , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora , RNA Polimerase Dependente de RNA/genética , Medula Espinal/patologia , Carga Viral
12.
Arch Virol ; 153(2): 315-21, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18193157

RESUMO

Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.


Assuntos
Citrus/virologia , Closterovirus/enzimologia , RNA Polimerase Dependente de RNA/biossíntese , Proteínas Virais/biossíntese , Western Blotting , Fracionamento Celular , Membrana Celular/química , Citrus/química , Closterovirus/genética , Citoplasma/química , Imunoprecipitação , RNA Polimerase Dependente de RNA/genética , Fatores de Tempo , Proteínas Virais/genética
13.
APMIS ; 116(12): 1089-96, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19133012

RESUMO

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.


Assuntos
Vírus Hantaan/fisiologia , Infecções por Hantavirus/virologia , RNA Polimerase Dependente de RNA/análise , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Virais/análise , Animais , Cloranfenicol O-Acetiltransferase/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Expressão Gênica , Genes Reporter , Genoma Viral , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Vírus Hantaan/genética , Humanos , RNA Polimerase I/metabolismo , RNA Viral/genética , RNA Polimerase Dependente de RNA/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Reversa , Células Vero/metabolismo , Proteínas Virais/biossíntese
14.
Nucleic Acids Res ; 36(4): 1220-6, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18160413

RESUMO

RNA-dependent RNA polymerase 6 (RDR6) catalyses dsRNA synthesis for post-transcriptional gene silencing (PTGS)-associated amplification and the generation of endogeneous siRNAs involved in developmental determinations or stress responses. The functional importance of RDR6 in PTGS led us to examine its connection to the cellular regulatory network by analyzing the hormonal responses of RDR6 gene expression in a cultured cell system. Delivery of dsRNA, prepared in vitro, into cultured rice (Oryza sativa cv. Japonica Dongjin) cells successfully silenced the target isocitrate lyase (ICL) transcripts. Silencing was transient in the absence of abscisic acid (ABA), while it became persistent in the presence of ABA in growth medium. A transcription assay of the OsRDR6 promoter showed that it was positively regulated by ABA. OsRDR6-dependent siRNA(ICL) generation was also significantly up-regulated by ABA. The results showed that, among the five rice OsRDR isogenes, only OsRDR6 was responsible for the observed ABA-mediated amplification and silencing of ICL transcripts. We propose that ABA modulates PTGS through the transcriptional control of the OsRDR6 gene.


Assuntos
Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas , Oryza/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Interferência de RNA , RNA Polimerase Dependente de RNA/genética , Isocitrato Liase/biossíntese , Isocitrato Liase/genética , Oryza/efeitos dos fármacos , Oryza/enzimologia , Proteínas de Plantas/biossíntese , RNA Polimerase Dependente de RNA/biossíntese
15.
Acta Virol ; 51(3): 195-201, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18076310

RESUMO

The RNA-dependent RNA polymerase (RdRp) in viruses of the family Flaviviridae plays an important role in the viral replication process and in the forming of a replicase complex. We used small interfering RNAs (siRNAs) corresponding to the highly conservative Motif V of RdRp gene of different viruses to examine their role in modulating the expression of RdRp. Evaluation of the expression of RdRps was performed by the fluorescence, flow cytometry, Western blotting, and real-time PCR. We found that Classical swine fever virus (CSFV) siRNA could completely block the transcription and expression of RdRp. Additionally, Hepatitis C virus (HCV) siRNA could cause effective inhibition of RdRp, whereas Japanese encephalitis virus siRNA did not show significant repression of corresponding RdRp. These results demonstrated that siRNAs inhibited the expression of tested RdRps at the transcription level or at the posttranscriptional processing to a different extent.


Assuntos
Vírus da Febre Suína Clássica/genética , Vírus da Encefalite Japonesa (Espécie)/genética , Inativação Gênica , Hepacivirus/genética , RNA Polimerase Dependente de RNA/biossíntese , Proteínas Virais/biossíntese , Animais , Fusão Gênica Artificial , Western Blotting , Linhagem Celular , Sequência Conservada/genética , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Proteínas Virais/genética
16.
Antiviral Res ; 76(1): 93-7, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17561278

RESUMO

Compound A-837093, a non-nucleoside HCV RNA-dependent RNA polymerase inhibitor, displayed nanomolar potencies against HCV genotypes 1a and 1b replicons. It also exhibited an excellent metabolic profile and achieved high plasma and liver concentrations in animals. In order to characterize the development of resistance to this anti-HCV agent, HCV subgenomic 1b strain N replicon cells were cultured in the presence of A-837093 with G418. Mutations S368A, Y448H, G554D, Y555C, and D559G in the NS5B polymerase gene were identified that led to substantial decreases in the susceptibilities of 1b genotype replicons to the inhibitor A-837093. However, the resistant mutants remained susceptible to HCV protease inhibitor BILN-2061 and alpha interferon as well as to a different class of non-nucleoside HCV polymerase inhibitor. In addition, each single resistant mutation identified significantly reduced the replication capacity of mutant compared to wild-type replicon. These findings provide a strategic guide for the future development of non-nucleoside inhibitors of HCV NS5B polymerase.


Assuntos
Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Linhagem Celular , Farmacorresistência Viral/genética , Genes Virais/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/química , Replicon/genética , Proteínas Virais/biossíntese , Proteínas Virais/química , Replicação Viral
17.
J Immunol ; 179(1): 64-70, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17579022

RESUMO

Ag-specific, CD8+ CTLs clear influenza A viruses from the lung via granzyme (Gzm) and perforin-dependent mechanisms. Ex vivo analysis of perforin-Gzm mRNA profiles demonstrated substantial heterogeneity in patterns of effector mRNA transcription of CD8+ D(b)NP(366)- or D(b)PA(224)-specific CTL. The only difference between the two epitope-specific sets was apparent very early after infection with similar molecular profiles seen in peak primary and secondary responses and in long-term memory. Surprisingly, memory T cells also expressed a diverse pattern of effector mRNA profile with an emphasis on GzmB and, surprisingly, GzmK. This analysis thus defines how naive, effector, and memory T cells differ in cytotoxic potential and provides novel insight into the molecular signatures of effector molecules observed at various stages after infection.


Assuntos
Epitopos de Linfócito T/imunologia , Memória Imunológica , Imunofenotipagem , Vírus da Influenza A Subtipo H3N2/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Doença Aguda , Animais , Citotoxicidade Imunológica , Feminino , Antígenos H-2/biossíntese , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Nucleocapsídeo , Nucleoproteínas/biossíntese , Nucleoproteínas/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/imunologia , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/imunologia , Linfócitos T Citotóxicos/enzimologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/imunologia , Proteínas Virais/biossíntese , Proteínas Virais/imunologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-17554171

RESUMO

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.


Assuntos
Enterovirus Humano B/enzimologia , RNA Polimerase Dependente de RNA/química , Cristalização , Enterovirus Humano B/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação
19.
Virus Genes ; 35(2): 395-8, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17356908

RESUMO

In the course of sobemovirus gene cloning the complete genome of Ryegrass mottle virus (RGMoV) was sequenced. Sequence analysis revealed differences including missing and extraneous nucleotides in comparison to the previously published sequence (Zhang, Toriyama, Takanashi, J. Gen. Plant Pathol. 67, 63 (2001)). A gene coding for a typical sobemovirus 3C-like serine protease was identified in ORF2a after multiple sequence alignment analysis. The newly identified 57-amino-acid stretch in ORF2a showed similarities ranging from 38.5 to 50.9% among sequenced genes of sobemovirus proteases. ORF analysis of the RGMoV polyprotein coding sequence demonstrated the arrangement of ORF2b coding for RNA-dependent RNA polymerase (RdRP) in the -1 frame in regard to ORF2a. The localization of conserved among sobemoviruses slippery sequence (UUUAAAC) at the 3'-end of ORF2a suggests the translation of RdRP via a -1 ribosomal frameshifting mechanism, allowing to include the RGMoV in the sobemovirus group with a Cocksfoot mottle virus-like (CfMV-like) genome organization.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Viral , Lolium/virologia , Vírus de Plantas/enzimologia , Vírus de Plantas/genética , Biossíntese de Proteínas , RNA Polimerase Dependente de RNA/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , RNA Polimerase Dependente de RNA/biossíntese , Serina Endopeptidases/biossíntese , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética
20.
J Virol ; 81(12): 6356-68, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17392363

RESUMO

Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually approximately 15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite Murina/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/fisiologia , Alanina/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cricetinae , Humanos , Imunoprecipitação , Cinética , Dados de Sequência Molecular , Mutação , Poliproteínas/química , RNA Polimerase Dependente de RNA/biossíntese , Homologia de Sequência de Aminoácidos
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