RESUMO
Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-ß induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-ß mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-ß mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection.
Assuntos
Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatite C/virologia , Fator Regulador 3 de Interferon/antagonistas & inibidores , Proteínas do Core Viral/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos Básicos , Núcleo Celular/metabolismo , Genoma Viral , Células Hep G2 , Hepacivirus/genética , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Multimerização Proteica , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Small interfering RNA (siRNA) is a noncoding RNA with considerable potential as a new therapeutic drug for intractable diseases. siRNAs can be rationally designed and synthesized if the sequences of the disease-causing genes are known. In this paper, we describe the synthesis and properties of siRNAs modified with biaryl units. We found that incorporation of biaryl units into the 5' and 3' ends of sense and antisense strands of siRNA duplexes improved strand selectivity and nuclease resistance.
Assuntos
RNA Antissenso/química , RNA Antissenso/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ribonucleases/metabolismo , Animais , Sequência de Bases , Inativação Gênica , Hepacivirus/fisiologia , Luciferases/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismo , Replicação Viral/genéticaRESUMO
The mechanism of the innate immune response to hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We used HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins regulated by the Cre/loxP switching expression system, to examine the innate immune response to HCV structural proteins. Twelve hours after HCV transgene expression, HCV core protein levels in Tg mouse livers were 15-47 pg/mg. In contrast, in Tg mice with a depletion of natural killer (NK) cells, we observed much higher levels of HCV core proteins (1,597 pg/ml). Cre-mediated genomic DNA recombination efficiency in the HCV-Tg mice was strongly observed in NK cell-depleted mice between 0.5 and 1 day as compared to non-treated mice. These data indicated that NK cells participate in the elimination of core-expressing hepatocytes in the innate immune responses during the acute phase of HCV infection.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Hepatócitos/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Proteínas do Core Viral/imunologia , Animais , Citotoxicidade Imunológica , Hepatite C/patologia , Hepatite C/virologia , Hepatócitos/patologia , Hepatócitos/virologia , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
Persistent infection with hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Here we report that inhibition of heat shock protein 90 (Hsp90) is highly effective in suppressing HCV genome replication. In HCV replicon cells, HCV replication was reduced by Hsp90 inhibitors and by knockdown of endogenous Hsp90 expression mediated by small-interfering RNA (siRNA). The suppression of HCV replication by an Hsp90 inhibitor was prevented by transfection with Hsp90 expression vector. We also tested the anti-HCV effect of Hsp90 inhibition in HCV-infected chimeric mice with humanized liver. Combined administration of an Hsp90 inhibitor and polyethylene glycol-conjugated interferon (PEG-IFN) was more effective in reducing HCV genome RNA levels in serum than was PEG-IFN monotherapy. These results suggest that inhibition of Hsp90 could provide a new therapeutic approach to HCV infection.
Assuntos
Proteínas de Choque Térmico/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepatite C/prevenção & controle , Hepatócitos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Benzoquinonas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hepacivirus/crescimento & desenvolvimento , Hepatite C/sangue , Hepatite C/metabolismo , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Lactamas Macrocíclicas/farmacologia , Macrolídeos/farmacologia , Camundongos , Camundongos SCID , Polietilenoglicóis/farmacologia , RNA Interferente Pequeno/genética , RNA Viral/sangue , RNA Viral/genética , Proteínas Recombinantes , Replicon/efeitos dos fármacos , Rifabutina/análogos & derivados , Fatores de Tempo , Transfecção , Quimeras de Transplante/virologia , Transplante HeterólogoRESUMO
Serine palmitoyltransferase (SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin is an inhibitor of SPT and suppresses replication of the hepatitis C virus (HCV) replicon. However, it is still unknown whether this SPT inhibitor suppresses HCV replication in vivo. We investigated the anti-HCV effect of myriocin against intact HCV using chimeric mice with humanized liver infected with HCV genotype 1a or 1b. We administered myriocin into HCV infected chimeric mice and succeeded in reducing the HCV RNA levels in serum and liver to 1/10-1/100 of the levels prior to the 8 day treatment. Furthermore, combined treatment with pegylated interferon reduced the HCV RNA levels to less than 1/1000 of the control levels. We strongly suggest that suppression of SPT reduces HCV replication, and therefore that the SPT inhibitor is potentially a novel drug in the treatment of HCV infection.