Mechanism(s) Of The Inhibition Of HBV Replication By Myeloid Differentiation Primary Response Protein 88

The hepatitis B virus (HBV) belongs to the family of Hepadnaviradae with a unique genome structure and life cycle. Hepatitis B is the most common serious liver infection in the world. Current antiviral therapies involve the use of nucleoside analogs and interferon-alpha (IFN-a). Importantly, the precise antiviral mechanism of IFN-a and the biological functions of many IFN-stimulated genes (ISGs) have not been fully elucidated. We and others have shown that the expression of Myeloid differentiation primary response protein 88 (MyD88) can be induced by IFN-a, and that MyD88 has an antiviral activity against HBV in hepatoma cells that is mediated by nuclear factor-kappaB (NF-κB) activation. To counteract its inhibition, the HBV polymerase dampens activation of the MyD88 promoter by blocking nuclear translocation of Statl, thereby reducing IFN-a-inducible MyD88 expression, further suggesting a critical role for MyD88 in antiviral activity against HBV. The aim of the present study was to further investigate the antiviral activity of MyD88 and the mechanism of action.To determine the effect of MyD88 on established HBV replication, a cell line stably transformed with replicating HBV genomic DNA, HepG2.2.15, was infected with adenovirus expressing MyD88 (Ad-MyD88). The amount of viral RNA and core particle-associated DNA were determined by northern and southern blot analyses, respectively. The results showed that viral DNA levels were decreased to the same extent as viral RNA levels by Ad-MyD88 infection, suggesting that the main primary antiviral target of MyD88 was most likely the viral RNA, which was verified using using the pCIdA-HBV construct, which is capable of viral gene expression and incapable of viral geneome replication. In addition, consistent with in vitro results, MyD88 significantly reduced the levels of viral core particle-associated DNA and RNA in a mouse model of acute HBV infection established using hydrodynamic-based transfection.To investigate the antiviral activity of MyD88 in physiological condition, Huh7 cells were transfected with siRNA targeting MyD88, and the antiviral activity of IFN-a was tested. The results showed that HBV showed resistant to IFN-a treatment in the MyD88 knock-down cells to some extent, suggesting that MyD88 plays an active antiviral role in the IFN-a-mediated inhibition of HBV replication.To determine whether this inhibition occurs transcriptionally or posttranscriptionally, we first employed reporter plasmids in which the luciferase reporter gene was under control of HBV promoters/enhancers. The results showed that MyD88 had little inhibitory effect on the activity of the viral promoters/enhancers. But MyD88 inhibited the expression of pregenomic RNA from pCMV-HBV in which the transcription of viral pregenomic RNA is under control of CMV promoter. All these results suggest that MyD88 posttranscriptionally reduces the levels of HBV RNA.Because the inhibition of pregenomic RNA expression is a posttranscriptional event, we investigated whether the decrease in RNA was due to an accelerated turnover rate of the pregenomic RNA using the Tet-off system. The results showed that MyD88 accelerated the decay of the pregenomic RNA. Furthermore, cytoplasmic and nuclear fractionation analysis showed that MyD88-induced decay of the pregnomic RNA occurred in the cytoplasm, and not in the nucleus. In addition, we blocked the 5'-3'and 3'-5'mRNA decay pathways by siRNAs and found that the two mRNA decay pathways were required for the MyD88-induced decay of viral pregenomic RNA.It has been reported that the La protein contributes to HBV pregenomic RNA stability through specific binding to the viral RNA. In this study, we showed that the MyD88-induced decay of viral pregenomic RNA was independent of the interaction of La with viral pregenomic RNA. The mapping analysis showed that HBV(1804-2454) and HBV(1151-1684) are MyD88-responsive regions of the pregenomc RNA. To investigate the relative contribution of the two MyD88-responsive sequences to MyD88-induced decay of viral pregenomic RNA, we constructed deletion mutants of these sequences and tested their response to MyD88. Our results showed that the HBV(1151-1684) deletion mutant retained sensitivity to MyD88, while the HBV(1804-2454) deletion mutant was resistant to MyD88. These results define the HBV(1804-2454) region as a crucial cis-regulatory sequence for MyD88-induced decay of viral pregenomic RNA.The HBV(1151-1684) region, which is located in the 3'overlapping region of the pregenomic RNA and preS/S RNAs, was not required for MyD88-induced decay of the pregenomic RNA, we determined whether it selectively conferred sensitivity of preS/S RNAs to MyD88. The results showed that the HBV(1151-1684) region selectively mediated the decay of viral preS/S RNAs. Furthermore, cytoplasmic and nuclear fractionation analysis showed that MyD88-induced decay of the preS/S RNAs occurred in the nucleus, and not in the cytoplasm.Interestingly, we found that the HBV(1151-1684) region overlaps with an RNA cis element termed the posttranscriptional regulatory element (PRE). The PRE selectively mediates the nuclear export of viral preS/S RNAs. In addition, viral preS/S RNAs fail to translocate to the cytoplasm and degrade in the nucleus with an elusive mechanism. We evaluated whether the decay of preS/S RNAs in the nucleus was associated with a deficiency in nuclear transport mediated by the PRE using the pRSV138PRE-CAT construct. The results showed that CAT activity derived from the PRE-containing transcript was significantly decreased by MyD88. In addition, MyD88 did not further diminish CAT expression, when co-expressed with NES(-)RanBP1, an inhibitor of the PRE-mediated nuclear export. Expression of polypyrimidne tract-binding protein (PTB), an export factor for PRE-containing RNA, almost fully restored the function of the PRE. Given that the CAT assays only represent an indirect measure of RNA levels, we also performed northern blot analysis for CAT RNA in the nucleus and cytoplasm. The results showed that the changes in RNA levels are in good agreement with the observed changes in CAT activity. Therefore, we conclude that MyD88 inhibits the nuclear export of HBV preS/S RNAs mediated by the PRE.To uncover the mechanism underlying the impaired PRE function in nuclear export, we evaluated the expression of PTB in MyD88-overexpressing cells. The results showed that MyD88 has an equally inhibitory effect on both PTB protein and mRNA levels, suggesting that PTB mRNA may be the target of MyD88. We investigated the effect of MyD88 on the stability of PTB mRNA. The results showed that the expression of MyD88 could not accelerate the degradation of PTB mRNA. Therefore, MyD88 transcriptionally inhibits the expression of PTB.In conclusion, our results provide further insights into the mechanism of MyD88 antiviral activity. Elucidation of this antiviral pathway may ultimately lead to the development of new therapeutics for HBV infection.