RESUMO
Human immunodeficiency virus (HIV) regulatory protein Tat has pro-oxidant property, which might contribute to Tat-induced long terminal repeat region (LTR) transactivation. However, the intracellular mechanisms whereby Tat triggers ROS production, and the relationship between Tat-induced ROS production and LTR transactivation, are still subject to debate. The present study was undertaken to evaluate the specific effects of Tat on nicotinamide adenine denucleotide phosphate (NADPH) oxidase in MAGI cells, and to determine the specific role of NADPH oxidase in Tat-induced LTR transactivation. Application of Tat to MAGI cells caused increases in ROS formation that were prevented by both pharmacologic NADPH oxidase inhibitors and by siRNA Nox2, but not by other inhibitors of pro-oxidant enzymes or siRNA Nox4. Furthermore, inhibition of NADPH oxidase by both pharmacologic NADPH oxidase inhibitors and by siRNA Nox2 attenuated Tat-induced p65 phosphorylation and IKK phosphorylation. Phosphatidylinositol 3-kinase/Akt signaling pathway was involved in Tat-induced NADPH oxidase stimulation. Finally, NADPH oxidase inhibitors or Nox2 siRNA, but not control siRNA, inhibited Tat-induced LTR transactivation. Tat-induced HIV-1 LTR transactivation was inhibited in wortmannin or LY294002 treated cells compared to control cells. Together, these data describe a specific and biologically significant signaling component of the MAGI cells response to Tat, and suggest the PI3K/Akt signaling pathway might originate in part with Tat-induced activation of NADPH oxidase and LTR transactivation.
Assuntos
HIV-1/genética , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sequências Repetidas Terminais/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , NADPH Oxidase 2 , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo , Ativação TranscricionalRESUMO
Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV-1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide(+) (NAD(+))-dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the conversion of nicotinamide into NAD(+), which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat-regulated SIRT activity was studied in Hela-CD4-beta-gal (MAGI) cells. We demonstrated that Tat caused NAD(+) depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat-induced NAD(+) depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat-induced inhibition of SIRT1 activity was potentiated in Nampt-knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat-induced HIV-1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat-induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV-1 infection.
Assuntos
Repetição Terminal Longa de HIV/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Sirtuína 1/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Antígenos CD4/genética , Antígenos CD4/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Immunoblotting , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Interferência de RNA , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Estilbenos/farmacologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Influenza A virus is a successful parasite and requires host factors to complete its life cycle. Prop5 is an antisense oligonucleotide, targeting programmed cell death protein 5 (PDCD5). In this study, we tested the antiviral activity of prop5 against mouse-adapted A/FM/1/47 strain of influenza A virus in a mouse model. Prop5 intranasally administered the mice at dosages of 10 and 20 mg/kg/d at 24 h and 30 min before infection, provided 80% and 100% survival rates and prolonged mean survival days in comparison with influenza virus-infected mice (both p < 0.01). Moreover, viral titres in mice pretreated with prop5, at dose of 10 and 20 mg/kg/d, had declined significantly on day two, four, and six post-infection compared with the yields in infected mice (p < 0.05 or p < 0.01); lung index in mice pretreated with prop5 (20 mg/kg/d) had been inhibited on day six post-infection (p < 0.05). Western blotting and immunohistochemistry showed that prop5 could down-regulate the PDCD5 protein expression levels in lung tissues of infected mice. These data indicate that antisense oligonucleotide prop5 is a promising drug for prophylaxis and control influenza virus infections and provides an insight into the host-pathogen interaction.