DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication.

AIM: To investigate whether DNA-dependent activator of interferon-regulatory factors (DAI) inhibits hepatitis B virus (HBV) replication and what the mechanism is. METHODS: After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plasmid, viral protein (HBV surface antigen and HBV e antigen) secretion was detected by enzyme-linked immunosorbent assay, and HBV RNA was analyzed by real-time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 (IRF3) phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-κB (NF-κB) activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Transwell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines. RESULTS: Viral protein secretion was significantly reduced by 57% (P < 0.05), and the level of total HBV RNA was reduced by 67% (P < 0.05). The viral core particle-associated DNA was also dramatically down-regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-κB signaling was essential for DAI to elicit antiviral response in Huh7 cells. When the NF-κB signaling pathway was blocked by a NF-κB signaling suppressor (IκBα-SR), the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was independent of IRF3 signaling and secreted cytokines. CONCLUSION: This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is associated with activation of NF-κB but independent of IRF3 and secreted cytokines.

Hyper-activated IRF-1 and STAT1 contribute to enhanced interferon stimulated gene (ISG) expression by interferon alpha and gamma co-treatment in human hepatoma cells.

Previous reports suggest that type I and type II Interferon can co-operatively inhibit some virus replication, e.g. HCV, SARS-CoV, HSV-1. To find out the molecular mechanism underlying this phenomenon, we analyzed the transcription profile stimulated by IFN-alpha and IFN-gamma in Huh-7 cells and found that the transcription of a subset of IFN stimulated genes (ISGs) including BclG, XAF1, TRAIL and TAP1 was enhanced when IFN-alpha and gamma were both present. Promoter analysis of BclG revealed that IRF-1 and STAT1 were both required in this process. Enhanced IRF-1/DNA complex formation was observed in interferon co-treatment group by gel shift analysis. Furthermore, IRF-1 activation was found to be generally required in this cluster of ISGs. STAT1 tyrosine phosphorylation was elevated by IFN combination treatment, however, only the hyper-transactivation of GAS but not ISRE was observed. In conclusion, hyper-activation of IRF-1 and elevated STAT1 dimer formation may be two general switches which contribute to a much more robust antiviral symphony against virus replication when type I and type II IFNs are co-administered.

Gene expression profiles in an hepatitis B virus transfected hepatoblastoma cell line and differentially regulated gene expression by interferon-alpha.

AIM: To study interactions between hepatitis B virus (HBV) and interferon-alpha in liver- derived cells. METHODS: mRNAs were separately isolated from an HBV-transfected cell line (HepG(2)2.2.15) and its parental cell line (HepG(2)) pre- and post-interferon-alpha (IFN-alpha) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis. RESULTS: Compared to HepG(2) cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG(2)2.2.15 cells prior to IFN-alpha treatment. After interferon-alpha treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG(2)2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-alpha treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions. CONCLUSION: The up-regulated genes in HepG(2)2.2.15 cell line suggested that under IFN-alpha treatment, these repressed cellular genes in HBV infected hepatocytes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.

Analysis of gene expression in hepatitis B virus transfected cell line induced by interferon.

Infection of hepatitis B virus (HBV) continues to be a significant health problem. alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma) have been proven to be effective in inhibiting HBV replication. To study the global effect of HBV persistent existence on IFN induced cellular gene expression, cDNA microarrays dotted with 14 112 human genes were used to examine the transcriptional changes between an HBV DNA transfected cell line (HepG2.2.15) and its parental cell line (HepG2) after the treatment of IFN-alpha or IFN-gamma for 6 h. The results showed that many genes related to cell cycle, proliferation, apoptosis and new ESTs were regulated by IFN-alpha and/or IFN-gamma. Many genes involved in kinase and signal transduction, transcription regulation, antigen presentation and processing were differentially regulated between these two cell lines post IFN-alpha or IFN-gamma treatment. Interestingly, several IFN-differentially regulated genes, such as MyD88 and Diubiquitin, were found to inhibit HBV gene expression, and MyD88 was proved to inhibit HBV replication. Taken together, our results revealed the global effects of HBV persistent existence on IFN-induced cellular gene expression. The novel antiviral genes identified by microarray could be potentially developed as new anti-HBV drugs or for novel therapies.

[Establishing DNA chip technique for detecting hepatitis C virus genotypes and primary application].

OBJECTIVE: To establish a new method based DNA chip technique for detecting HCV genotypes. METHODS: Genotyping probes were designed according to the sequence of HCV 5' NCR to generate DNA chip. The probes on DNA chip contains 5 major genotypes and 8 subtypes. The DNA fragment amplified by labeling Cy5 fluorescence was hybridized with DNA chip. RESULTS: Fifty-five out of 65 isolates detected by DNA chip belonged to 1b- DNA sequencing of form a part of the isolates was used as the control. The results of both were completely consistent. CONCLUSION: The method is simple and rapid with high specificity and sensitivity. It can be applied in detection of HCV RNA and genotypes.