Tumor Necrosis Factor-Inducible CIAP₂ Protein Inhibits Hepatitis B Virus Replication

Infection of hepatitis B virus (HBV) continues to be a significant health problem. It is estimated that there are approximately 350 million chronic hepatitis B patients worldwide. These patients have a high risk of developing liver cirrhosis and hepatocellular carcinoma. But until now, there is no efficient therapeutic measure. Recently, studies showed that HBV-specific cytotoxic T lymphocytes can abolish HBV gene expression and replication through a noncytopathic mechanism mediated by tumor necrosis factor-α (TNF-α). Some studies have been focused on investigating the complex mechanisms responsible for the antiviral activity of TNF-α, but only parts of them have been defined.The antiviral activation of TNF-α is the outcome of the interaction between HBV and host cells. Upon binding to its specific cell surface receptor, TNF-α activates the expression of a number of cellular genes, through the distinct, yet not completely understood pathways of signaling cascade in a rapid and direct manner. HBV may also decrease the expression of some TNF-α induced gene to escape the antiviral effect of the TNF-α system.To examine TNF-α induced cellular responses and identify genes involved in anti-HBV activity, cDNA microarrays dotted with 14 112 human genes were used to examine the transcriptional changes in HepG2 after treatment with TNF-α for 6 h. Compared to no TNF-α treatment, among 14,112 genes, 31 cellular genes were regulated including 8 up-regulated genes and 23 down-regulated genes. The functional categories of these genes include ligands and receptors, signal transduction, mitochondrial and ribosomal proteins, factors involve in transcription regulation, and TNF-α inducible proteins. Further Real-Time PCR analysis confirmed the results of microarray analysis and showed that the transcription of cIAP₂, B4-2, diubiquitin and MAD3 could be dose-dependently increased by TNF-α among which cIAP₂ was the most strongly induced.CIAP2, known as the inhibitor of apoptosis (IAP) family of proteins, has three BIRs in the N-terminal portion of the molecule and a RING finger at the C-terminus. CIAP2 can directly bind to activated caspase-3 and -7 and inhibit their activities and also inhibit activated caspase-9 by binding pro-caspase 9 and preventing its activation. They were recruited to the TNFR by interactions between their BIRs and TRAF (TNFR-associated factor)-1 and -2, which directly bind to the TNFR. Furthermore, CIAP2 has E3 activity and are not only able to catalyze their own ubiquitination in vitro and in transfected cells, but may also modulate the amount of their (putative) substrates. Utill now it is not clear whether CIAP2 possess anti-virus activity. As CIAP2 belong to NF-kB signaling pathway and can be induced by TNF-a in different cell lines, this gene may be the potential NF-KB-stimulated cellular genes that act to inhibit HBV gene replication and expression.To examine the role of CIAP2 in the antiviral activity of TNF-a against HBV, CIAP2 expression plasmids were transiently co-transfected with HBV replication competent plasmid. HBV gene replication and expression were analyzed. Compared to comtrol empty plasmid transfected cells, when different doses of CIAP2 plasmids (0.5 , 1 > 2ug) were co-transfected with 0.5 ug HBV replicative plasmid, a dose-dependent reduction of HBeAg and HBsAg expression were observed(19%, 38% and 47% for HBeAg, and 26%, 44% and 41% for HBsAg in culture supernatants). Besides, a dose-dependent reduction of about 26% and 59% for total viral transcripts was observed in Northern blot analysis when 2, 4ug of CIAP2 expression plasmids were co-transfected with of HBV replicative plasmids(2 ug). Similar to above results, a dose-dependent reduction of HBV replicative intermediate DNA in HBV core particles was also observed by Real-time PCR and Southern blot analysis(52% and 72% with 0.5, lug cIAP2 and 0.5 ug HBV in Real-time PCR analysis and 49%, 81% with 2, 4ug CIAP2 and 2 ug HBV in Southern blot analysis). The above results indicated that the over-expression of CIAP2 could effectively reduce the synthesis of HBV proteins, inhibit RNA transcription and DNA replication. We next examined whether siRNA directed against CIAP2 could reduce the inhibitory effect of TNF-a on HBV. Results showed that the introduction of CIAP2 targeted siRNA reduced the inhibitory effect of TNF-a on HBV gene replication. These results indicated that CIAP2 protein play an important anti-HBV role on TNF-a inhibiting HBV replication. Further studies with different domains of CIAP2 showed that RING domain of cIAP2 alone could decrease HBV gene expression (74% for HBeAg, 82% for HBsAg in...