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1.
Sci Rep ; 6: 25806, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27194388

RESUMO

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.


Assuntos
Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Alcaloides/farmacologia , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Animais de Doenças , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Receptor Smoothened/metabolismo , Transcrição Genética/efeitos dos fármacos , Veratrum/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia , Alcaloides de Veratrum/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
2.
J Comp Pathol ; 124(4): 300-7, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11437506

RESUMO

The causative viruses of two diseases of rainbow trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN), exert much of their cytopathogenic effect in cell culture through the induction of apoptosis. In the present study, the TUNEL procedure was used to investigate the presence of apoptotic cells in different organs of rainbow trout infected with the viruses of VHS and IPN. VHS viral infection resulted in massive apoptosis in renal lymphoid tissue, where viral antigens were also detected. Large numbers of viral particles were observed in close proximity to apoptotic cells. Apoptosis was not detected in excretory cells of the renal tubules or in infected muscle cells. IPN virus did not induce apoptosis in the pancreas. However, the DNA degradation associated with apoptotic nuclei was observed in muscle lesions. Taken together, these results indicated that induction of apoptosis in vivo was critically influenced by the species of virus and the cell type. Moreover, it would seem likely that apoptosis contributed to the nature of the two diseases and to mortality.


Assuntos
Apoptose , Infecções por Birnaviridae/veterinária , Doenças dos Peixes/patologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Oncorhynchus mykiss/virologia , Pancreatopatias/veterinária , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Animais , Antígenos Virais/análise , Infecções por Birnaviridae/patologia , Fragmentação do DNA , Doenças dos Peixes/virologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Vírus da Necrose Pancreática Infecciosa/ultraestrutura , Túbulos Renais/patologia , Túbulos Renais/virologia , Microscopia de Fluorescência/veterinária , Músculo Esquelético/microbiologia , Músculo Esquelético/patologia , Miocárdio/ultraestrutura , Pancreatopatias/patologia , Pancreatopatias/virologia , Rhabdoviridae/isolamento & purificação , Infecções por Rhabdoviridae/patologia , Especificidade da Espécie
3.
J Virol ; 74(9): 3975-83, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10756009

RESUMO

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas do Nucleocapsídeo , Nucleocapsídeo/metabolismo , Vírus da Gastroenterite Transmissível/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Caspase 3 , Caspase 6 , Caspase 7 , Caspase 8 , Caspase 9 , Extratos Celulares , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/metabolismo , Nucleocapsídeo/genética , Oligopeptídeos/farmacologia , Receptores Virais/genética , Receptores Virais/metabolismo , Vírus da Gastroenterite Transmissível/fisiologia , Células Tumorais Cultivadas
4.
J Virol ; 72(6): 4918-24, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9573259

RESUMO

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Apoptose , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/patologia , Vírus da Gastroenterite Transmissível , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Transdução de Sinais , Suínos
5.
Virology ; 206(2): 817-22, 1995 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-7856095

RESUMO

The entire nucleotide sequence of cloned cDNAs containing the 5'-untranslated region and gene 1 of Purdue-115 strain of transmissible gastroenteritis virus (TGEV) was determined. This completes the sequence of the TGEV genome, which is 28,579 nucleotides long. The gene 1 is composed of two large open reading frames, ORF1a and ORF1b, which contain 4017 and 2698 codons, respectively (stop excluded). A brief, three-codon-long ORF is present upstream of ORF1a. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. In vitro experiments indicated that translation of the ORF1a/b polyprotein involves an efficient ribosomal frameshifting activity, as previously shown for other coronaviruses. Analysis of the predicted ORF1a and ORF1b translation products revealed that the putative functional domains identified in infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and human coronavirus 229E (HCV 229E) are all present in TGEV. The amino-terminal half of the ORF1a product exhibits greater divergence than the carboxyl-terminal half, including within the TGEV/HCV229E pair. The ORF1b protein is overall highly conserved among the above four coronaviruses, except a divergent region situated near the carboxy terminus.


Assuntos
Genes Virais , Vírus da Gastroenterite Transmissível/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Códon , Sequência Conservada , Coronaviridae/genética , Primers do DNA , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/genética , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/biossíntese
7.
J Biol Chem ; 268(8): 5431-7, 1993 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-8449904

RESUMO

Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.


Assuntos
Eritrócitos/metabolismo , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Transcrição Genética , Adulto , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Fator de Transcrição GATA1 , Células HeLa , Humanos , Leucemia Eritroblástica Aguda , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Deleção de Sequência , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Dedos de Zinco
8.
Eur J Biochem ; 212(3): 763-70, 1993 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-8385011

RESUMO

Previous studies have shown that a -112 to +78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid-specific activity. This PBGD-(-112 to +78) promoter contains a CCACC binding site (position -100), a GATA binding site (position -70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA-1 trans-activates the PBGD-(-112 to +78) promoter in non-erythroid cells. We show that, if trans-activation is abolished by mutations that destroy either the -100 CCACC binding or the -70 GATA binding sites, replacement of the -100 CCACC binding site by a simian-virus-40-protein-1 (Sp1) binding site maintains both the erythroid-specific activity of this promoter and the human GATA-1 trans-activation. Thus, human GATA-1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD-(-112 to +78) promoter is activated 20-fold by a cis-linked 5' hypersensitive site 2 (5'HS-2) of the human beta-globin locus control region. This activation depends on the -70 GATA and -100 CCACC or Sp1 binding sites. When a longer -714 to +78 fragment of the PBGD promoter is used, the -70 GATA mutant still displays erythroid-specific activity and is cis-activated by the 5'HS-2 enhancer, while the -100 CCACC mutant is completely inactive in the absence or in the presence of the 5'HS-2 enhancer. Thus, the -100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5'HS-2 enhancer, whereas the -70 GATA binding site can functionally be replaced by upstream cis-acting elements.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Vírus 40 dos Símios/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Hidroximetilbilano Sintase/metabolismo , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plasmídeos , Mutação Puntual , Proteínas Recombinantes de Fusão/metabolismo , TATA Box , Transfecção , Células Tumorais Cultivadas
9.
EMBO J ; 10(7): 1809-16, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2050118

RESUMO

The human GATA1, hGATA1 (previously called NF-E1, GF-1 or Eryf-1), a major sequence-specific DNA-binding protein of the erythrocytic lineage, is a member of a zinc-finger family of DNA-binding proteins. We report here the cloning of a human cDNA for a new member of this family. This member, called hGATA3, has 85% amino acid homology with hGATA1 in the DNA-binding domain and no homology elsewhere in the protein. Unlike hGATA1, hGATA3 is not localized on the X chromosome and we map it to the 10p15 band of the human genome. Northern blot analysis indicates that this factor is a T-cell specific transcription factor, present before activation and up-regulated during T-cell activation. The encoded hGATA3 protein, made in an in vitro transcription-translation assay, binds the WGATAR motif present in the human T-cell receptor (TCR) delta gene enhancer and, by transfection in HeLa cells, we show that hGATA3 can transactivate this TCR delta gene enhancer. Interestingly this enhancer binds and is also transactivated by hGATA1. Conversely, the promoter of the human glycophorin B (GPB), which is erythroid-specific and contains two WGATAR motifs, binds and is transactivated by hGATA1 and, to a lesser extent, by hGATA3. These results indicate that the activation of specific genes by hGATA1 or hGATA3 is partly governed by the lineage expression of these two factors during haematopoiesis and that, in the T-cell lineage, hGATA3 binds the human TCR delta gene enhancer and is involved in its expression.


Assuntos
Proteínas de Ligação a DNA/genética , Receptores de Antígenos de Linfócitos T/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 10 , Clonagem Molecular , Proteínas de Ligação a DNA/biossíntese , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Fator de Transcrição GATA1 , Genes Sintéticos , Células HeLa/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Megacariócitos/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/biossíntese , Ativação Transcricional
10.
J Biol Chem ; 265(36): 22090-2, 1990 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-2266112

RESUMO

We have studied the elements involved in the tetradecanoylphorbol acetate (TPA)-mediated extinction of erythroid-specific genes. We show that transcription driven by a -714/+78-base pair DNA fragment of the erythroid promoter of the human porphobilinogen deaminase gene is down-regulated upon TPA treatment of erythroleukemic cells. Examination of the DNA binding activity of trans-acting factors involved in the expression of the porphobilinogen deaminase erythroid promoter showed (i) a constitutive expression of the CACC binding proteins and (ii) a decrease in DNA binding activity of two tissue-specific factors, NF-E1 and NF-E2. Kinetics experiments indicated that NF-E2 was down-regulated after 1 h of TPA treatment whereas NF-E1 was down-regulated at the protein and mRNA levels only after 5 h of TPA treatment. These results suggest that different pathways, acting via different transcription factors, are involved in the TPA-mediated extinction of erythroid-specific genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Hidroximetilbilano Sintase/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sequência de Bases , Linhagem Celular , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Cinética , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Sondas de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética
11.
Nucleic Acids Res ; 18(22): 6509-15, 1990 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-2251113

RESUMO

Although the erythroid-specific promoter of human porphobilinogen deaminase [PBGD] gene has no TATA box, transcription is initiated at a single nucleotide. Using 5' and 3' deletions and point mutations, we have identified an element, located around the initiation site, which is necessary and sufficient for 'in vitro' accurate initiation of transcription. This 15 bp element extends 1 bp 5' and 14 bp 3' from the initiation site. It is composed of two regions, a proximal region centred on the cap site and a distal region which bears homology with the TdT initiator element. We show that a nuclear factor, present both in erythroid and non erythroid cells, binds the distal PBGD initiator element. Lack of heat inactivation suggests that initiation of transcription mediated by this element is not TFIID dependent. By transfection into erythroid cells, we also show that the proximal PBGD initiator element is essential for the selection of the initiation site but not for the regulation of transcription of the PBGD erythroid promoter during erythroid differentiation.


Assuntos
Células Precursoras Eritroides/enzimologia , Regulação da Expressão Gênica , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , DNA/química , Células HeLa , Humanos , Hidroximetilbilano Sintase/biossíntese , Camundongos , Dados de Sequência Molecular , Mutação , Homologia de Sequência do Ácido Nucleico , Transcrição Genética
12.
Proc Natl Acad Sci U S A ; 86(17): 6548-52, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2771941

RESUMO

Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.


Assuntos
Amônia-Liases/genética , Genes Reguladores , Genes , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/genética , Camundongos , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Transcrição Genética
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