Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

Intact JAK-STAT signaling pathway is a prerequisite for STAT1 to reinforce the expression of RIG-G gene

We previously reported that IRF-9/STAT2 functional interaction could drive the expression of retinoic acid-induced gene G (RIG-G), independently of STAT1 and the classical JAK–STAT pathway, providing a novel alternative pathway for interferons (IFN) to mediate their multiple biological properties. In addition, we also found that IRF-1 could regulate RIG-G induction as well as the expression of IRF-9 and STAT2 in some cases. But the mechanisms by which IRF-1 exerted its action remained to be elucidated. Here, we showed that STAT1 could significantly enhance the effects of the IRF-9/STAT2 complex or IRF-1 on RIG-G induction through an activated JAK–STAT pathway, though it was not essential for RIG-G expression. In STAT1-deficient U3A cells, IRF-1 could induce RIG-G expression via the IFN-stimulated response elements in the RIG-G gene promoter, but it failed to upregulate IRF-9 and STAT2 unless the U3A cells were reconstituted by exogenous STAT1. In STAT1-expressing cells, IRF-1 indirectly activated RIG-G expression through an IRF-9/STAT2-dependent manner. Taken together, we concluded that the expression of RIG-G was independent on the classical JAK–STAT pathway, but could be greatly increased by it. This work will be of great benefit to us for a better understanding of the mechanisms on RIG-G gene expression regulation.     

The PPAR-γ agonist troglitazone antagonizes survival pathways induced by STAT-3 in recombinant interferon-β treated pancreatic cancer cells

We have previously shown that cancer cells can protect themselves from apoptosis induced by type I interferons (IFNs) through a ras→MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-β and the PPAR-γ agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-β-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-γ with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-β and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. IFN-β alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT→mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-γ activation can counteract STAT-3-dependent escape pathways to IFN-β-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells.     

PIASy represses TRIF-induced ISRE and NF-kappaB activation but not apoptosis

The TIR domain-containing adapter protein TRIP is critically involved in TLR3-induced IFN-β production through activation of NF-κB and ISRE. In addition, TRIF also induces apoptosis when overexpressed in 293 cells. In this report, we demonstrate that PIASy, a member of the PIAS SUMO-ligase family, interacts with TRIP, IRF-3 and IRF-7. In reporter gene assays, PIASy dramatically inhibits TRIF-induced NF-κB, ISRE and IFN-β activation but not TRIF-induced apoptosis. Furthermore, PIASy also inhibits IRF-3, IRF-7 and Sendai virus-induced ISRE activation. Our results suggest that PIASy is an inhibitor of TRIF-induced ISRE and NF-κB activation but not apoptosis.     

Genomic structure, promoter analysis, and expression of the porcine (Sus scrofa) Mx1 gene

Allelic polymorphisms at the mouse Mx1 locus affect the probability of survival after experimental influenzal disease, raising the possibility that marker-assisted selection using the homologous locus could improve the innate resistance of pigs to natural influenza infections. Several issues need to be resolved before efficient large scale screening of the allelic polymorphism at the porcine (Sus scrofa) Mx1 locus can be implemented. First, the Mx1 genomic structure has to be established and sufficient flanking intronic sequences have to be gathered to enable simple PCR amplification of the coding portions of the gene. Then, a basic knowledge of the promoter region needs to be obtained as an allelic variation there can significantly alter absolute levels and/or tissue-specificity of MX protein expression. The results gathered here show that the porcine Mx1 gene and promoter share the major structural and functional characteristics displayed by their homologs described in cattle, mouse, chicken, and man. The crucial function of the proximal interferon-sensitive response elements motif for gene expression is also demonstrated. The sequence data compiled here will allow an extensive analysis of the polymorphisms present among the widest spectrum possible of porcine breeds with the aim to identify an Mx1 allele providing antiviral resistance.     

干扰素刺激基因（ISGs）的研究进展

干扰素刺激基因(ISGs)是干扰素作用机制研究的核心内容.干扰素与受体结合后,通过细胞内信号转换,激活胞浆转录调控因子与ISGs调控序列上的cis元件结合而诱导基因表达.     

Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains

PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor 3 (TLR3), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR3 expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR3 was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (Interferon-alpha/beta receptor) suppressed MV-induced TLR3 induction, indicating that type I IFN, IFN-alpha/beta, is critical for MV-mediated TLR3 induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR3 expression. A virus-responsive element that up-regulates TLR3 was identified in the TLR3-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -30bp of TLR3 promoter responded to MV to induce TLR3 expression. This further indicates the importance of type I IFN for TLR3 up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR3 had been induced beforehand. Thus, the MV-induced expression of TLR3 may reflect amplified IFN production that plays a part in host defense to viral infection.