Molecular Mechanism(s) Of The Inhibition Of HBV Gene Replication By MyD88 Protein.

Infection of hepatitis B vims (HBV) continues to be a significant health problem. The chronic hepatitis B patients have a high risk of developing liver cirrhosis and hepatocellular carcinoma with high mortality rate. Interferon alpha (IFN-α) has been proven to be effective in the treatment of chronic hepatitis B patients, which can decrease viral titer and improve liver function. However, the antiviral mechanisms of IFN are not clear. Our previous studies reported that IFN-αinducible myeloid differential primary response protein (MyD88) could inhibit Hepatitis B virus (HBV) replication with a significant reduction of total and cytoplasmic HBV RNAs at posttranscriptional level. MyD88 protein is a key cytoplasmic adaptor of Toll-like receptors, interleukin 1 (IL-1) and interleukin 18 (IL-18) induced signaling cascades, which results in the activation of NF-κB-dependent signal transcription. Until now, a growing body of evidence has shown that some interferon-inducible protein or cytokines affect HBV replication at a post-transcriptional level. Whether IFN-αinducible MyD88 share the same strategies, including the blockade of viral RNA transport or post-transcriptional degradation of RNA to inhibit HBV replication, remain to be further investigated.To study the association between the inhibition of HBV replication by MyD88 and NF-κB activation, two MyD88 truncated versions, M (1-151) and M (152-296) were first constructed. Huh7 cells were transiently transfected with HBV, MyD88 or truncated versions. The results showed that the expression of the full length MyD88, M (1-151) and M (152-296) could induce NF-κB activity to a different extent, which were consistent with their inhibitory effect on viral protein synthesis and core particle-associated HBV DNA replication. To further determine the role of NF-κB activation in the inhibition of HBV by MyD88, the Huh7 cells were transfected with HBV DNA, plasmid MyD88, or with NF-κB super-repressor IκBα-SR. Results showed that, compared to that the expression of MyD88 resulted in the significant reduction of core protein, the co-expression of IκBα-SR dramatically restored the core protein level. Consistent results were observed on the restoring effects of IκBα-SR on inhibition of HBeAg, HBsAg and HBV replicative intermediate DNA levels by MyD88. Furthermore, plasmid IKKα/IKKβwhich can strongly activate NF-κB signaling was co-transfected into Huh7 cells with the HBV replicating plasmid. Results showed that over-expression of IKKα/IKKβled to a dose-dependent reduction of HBeAg, HBsAg and HBV core particle DNA. Finally, SB203580, a specific inhibitor of p38 MAPK and PD98059, a specific inhibitor of ERK1/2, were used to study if MAPKs and ERK pathways played role in the inhibition of HBV gene replication by MyD88. Results showed that, after addition of SB203580 or PD98059, over-expression of MyD88 still significantly suppressed the HBV protein synthesis and DNA replication. These results indicated that the suppression of the HBV DNA replication was independent of the p38 MAPK and ERK1/2 signaling pathway. All the above results supported the role of NF-κB signaling activation in inhibition of HBV replication by MyD88.To further investigate the mechanism(s) of the antiviral action of MyD88 against HBV, HBV promoter controlled luciferase reporter assay was first employed. Results showed that over expression of MyD88 did not affect HBV promoter activity, which excludes the possibilities that MyD88 reduced viral mRNA levels by suppressing the activity of HBV promoters. To analyze the possible influence of MyD88 on the distribution of HBV RNA in nuclear and cytoplasm, HBV replication competent DNA was cotransfected with empty vector or pcDNA-MyD88 into Huh7 cells. RNA slot and quantitative real-time PCR was used to analyze the levels of nuclear and cytoplasmic HBV RNAs. Results showed that, compared with empty vector transfected group, the levels of nuclear and cytoplasmic RNAs was both lower in the pcDNA-MyD88 transfected cells. However, there was an almost 2 to 3 fold increase for the pcDNA-MyD88 transfected group in ratio of nuclear RNA to cytoplasmic RNA, indicating the possibility of nucleus retention of HBV RNA by MyD88. Furthermore, the well-characterized posttranscriptional regulation element (PRE) reporter system was used to investigate the possibility of interference of MyD88 protein with HBV RNA nuclear retention. Results showed that, CAT expression was reduced to 2.5 to 3.0 fold in MyD88-expressing clones. Similar result was obtained in pcDNA-MyD88 transfected Huh7 cells. These results strongly suggested that MyD88 protein could interfere with nuclear export of HBV RNAs mediated by PRE sequence. Finally, a tetracycline-controlled HBV system was used to examine the half-life of HBV RNA. It was observed that MyD88 protein could lead to a shorter half-life of HBV RNA and promoted RNA degradation. These results support that MyD88 inhibits HBV replication at a posttranscriptional level, by inhibiting the nuclear export of viral RNAs and contributing to degradation of HBV RNA stability.In conclusion, this study confirmed that MyD88 has inhibitory activity on HBV gene replication and further demonstrated that the anti-HBV effect exerted by MyD88 is mediated by the activation of the NF-κB signaling pathway. The above results also suggested a novel mechanism for the inhibition of HBV replication by IFN-αinducible MyD88 protein, one on blockade of the HBV RNA nuclear export and the other related to posttranscriptional degradation of HBV RNA. Studies are ongoing to investigate the precise mechanism involved in MyD88's antiviral effect against HBV, such as the identification of inhibitory factor(s), which may lead to new approaches for controlling chronic HBV infection.