Mechanism (s) Of The Inhibition Of HBV Replication By Cellular Inhibitor Of Apoptosis Protein 2

Mechanism(s) of the inhibition of HBV replication by cellular inhibitor of apoptosis protein 2Worldwide more than 350 million people are chronically infected with Hepatitis B virus (HBV) and chronic hepatitis B causes significant morbidity and mortality. It has been manifested that virus and host interaction determines viral clearance or persistent infection. And during acute HBV infection, HBV clearance is mainly achieved in a noncytolytic manner. It is reported that some inflammatory cytokines such as TNF-α, IFN-α, IFN-βor IFN-γmediated this effect. However, how these cytokines playing their anti-HBV role is still not fully understood. Previously, we screened TNF-αinducible genes and found that cIAP2 was involved in TNF-αmediated anti-HBV response, but the actual mechanism remains to be elucidated. This study thus focused on how cIAP2 exerted its anti-HBV activity.We confirmed the inhibitory effects of cIAP2 on HBV replication in pCMV-HBV replicon system in which the transcription of HBV pregenomic RNA is driven by the CMV-IE promoter. Results showed that cIAP2 signifcantly reduced HBV replication intermediates DNA level, but had no effects on HBV pregenomic RNA and core protein levels. Moreover, we found endogenous cIAP2 in Huh7 cells could inhibit HBV replication, since knock-down of endogenous cIAP2 expression increased HBV replication. Above results suggested that cIAP2 inhibited HBV replication through interfering with the converstion of pregenomic RNA to DNA.To determine how cIAP2 performs its function, we mapped cIAP2 functional domains. cIAP2 and its trancated mutants were cotransfected with HBV replicon plasmid, and results showed that cIAP2 lost its antiviral activity as the C-terminal CARD and RING domains were deleted, while cIAP2 N-terminal domains deletion just attenuated its antiviral activity. Besides, the E3 ligase activity mutants cIAP2* completely abolished its anti-HBV ability, suggesting that its E3 ligase activity is required for cIAP2 inhibiting HBV replication.Since E3 ligase usually mediates protein ubiquitylation related degradation, it is possible that cIAP2 decreased the expression of some HBV replication related proteins. So, HBV protein expressing plasmids were cotransfected with cIAP2 or cIAP2* and the expression of HBV proteins were analyzed. Results showed that cIAP2 but not cIAP2* selectively decreased polymerase expression. Whether endogenous cIAP2 reduced polymerase expression, was also examined, and results showed that endogenous cIAP2 expression knock-down increased polymerase expression.The steady state level of protein is balanced by the synthesis and degradation rate. To discriminate whether polymerase reduction occurs at synthesis or degradation level, the synthesis of polymerase was first examined. Northern blot analysis demonstrated that cIAP2 had no inhibitory effects on polymerase RNA level. To rule out the possibility that cIAP2 mediated polymerase reduction was a result of polymerase overexpression induced protein unfolding, we transfected low amount of polymerase expressing plasmid into Huh7 cells and found that cIAP2 could still decrease lowly expressed polymerase. And cIAP2 also had no effects on the expression of polymerase associated molecular chaperones. Therefore, we determined the turnover rate of polymerase using CHX chase assay to see if polymerase degradation was affected. Data showed that cIAP2 significantly accelerated the degradation of polymerase, and shorten its half-life from 75 min to 25 min.There are two pathways for protein degradation, lysosome pathway and the proteasome pathway. To discriminate which pathway is involved, their specific inhibitors were applied and results showed that the degradation of polymerase could be restored by proteasome inhibitor but not by lysosome inhibitor. We therefore proposed that polymerase was degradated via the ubiquitin-proteasome pathway. In vivo ubiquitination assay was performed, and showed that cIAP2 promoted the ubiquitylation of polymerase, confirming that cIAP2 promoted polymerase degradation through the ubiquitin-proteasome pathway.Substrate ubiquitination requires the interaction between enzyme and substrate, so we examined if cIAP2 binds to polymerase. Co-immunoprecipitation and GST pull-down assay domenstrated that cIAP2 could bind to polymerase both in vivo and in vitro, and the N-terminal BIR domains of cIAP2 and the TP, RT and RH domains of polymerase participated in the interaction.Polymerase plays an important role in HBV life cycle, as it recognizes the pregenomic RNA, cooperates with core protein to initiate the encapsidation of pregenomic RNA, and then catalyzes the conversation of pregenomic RNA into DNA in the nucleocapsid. To verify whether polymerase degradation by cIAP2 accounted for the HBV replication inhibiton, we examined the effects of cIAP2 on HBV pregenomic RNA encapsidation. Results showed that cIAP2 overexpression decreased HBV capsids associated RNA levels, while endogenous cIAP2 knock-down increased HBV capsids associated RNA levels. Besides, cIAP2 had no inhibitory effects on HBV capsids levels. The above results suggested that cIAP2 interfered with HBV encapsidation process.In conclusion, we provide a novel explanation of inhibiting HBV replication by cIAP2, though promoting the degradation of polymerase via the ubiquitin-proteasome pathway, and further interrupting the encapsidation of pregenomic RNA. Elucidation of this antiviral pathway may ultimately lead to the development of new therapeutics for HBV infection.