Ciap2 Cut The Hepatitis B Virus Polymerase Protein Level Mechanism, A Preliminary Study

Hepatitis B virus (HBV) is a serious pathogen that is harmful to human’health.Persistent infection with HBV is easy to develop to liver cirrhosis or liver cancer.Currently drugs available for treatment of HBV infection mainly include interferon (interferon, IFN), nucleotide analogs (lamivudine lamivudine) and immunomodulatory agents, but there are no effective ways to clear the virus completely.Many data have showed that TNFa can play role in anti-HBV through a noncytopathic mechanism. Previous study undertaken by Dr. Liu Xiaoying in our lab found that clAP2 (cellular inhibitor of apoptosis protein) can be significantly up-regulated in the TNFα- treated HepG2 cells. Moreover clAP2 protein could inhibit HBV replication. As clAP2 protein is an E3 ligase, plasmids expressing clAP2 and HBV proteins were co-transfected to cells and results demonstrated that clAP2 could downregulate the expression of HBV polymerase specifically.To further study its related mechanism, the inhibitory efffec of clAP2 on HBV polymerase was confirmed firstly. When the clAP2 and HBV polymerase expressed plasmids were co-transfected into Huh7 cells, the level of HBV polymerase can be significantly reduced compared to negative control. After endogenous clAP2 protein was knocked down by siRNA in Huh7 and Hela cells, the polymerase levels could be partially restored which further supported that the clAP2 can downregulate the expression of HBV polymerase.It is known that clAP2 protein contains three BIR domains, a CARD domain and a RING domain. To further determine which domain of the clAP2 was responsible for the down-regulation of polymerase level, the truncated mutants were cloned into pCMV-myc vector respectively and then cotransfected with the HBV polymerase.Results showed that BIR2 and the RING domain were related to the role of downregulation. It has been reported that C-terminal RING domain of clAP2 protein is an E3 ligase and it could promote ubiquitination and degradation of specific substrates. To investigate the involvement of E3 ligase activity in the inhibitory effect, plasmids encoding, the RING domain deleted mutation of clAP2 and polymerase were co-transfected into Huh7 cells. Results showed that the level of polymerase could not be lowered in the absense of C terminal RING domain, suggesting that the down-regulation was related to its C terminal RING domain.There should be an interaction between the two proteins if clAP2 is the E3 ligase of HBV polymerase. To study this possibility, clAP2 and a series of truncated mutant were cloned into pGEX4T and purificated by Gluetathitone beads after induced by IPTG. GST-pull down experiment showed that clAP2 and HBV polymerase had a direct interaction in vitro, and the BIR2 and BIR3 domain of clAP2 was responsible for this interaction. Furthermore, immunofluorescence results showed that the two proteins were co-localized in the cytoplasm partially.To study whether clAP2 is the E3 ligase of HBV polymerase, we utilized in vivo ubiquitination assay and found clAP2 could promote the ubiquitination of HBV polymerase. It has been known that there are two modes of ubiquitin attatchment to their substrates:through the lysine sites on the substrate or N-terminal base. As the instability of HBV polymerase was mainly due to its TP and RH domains, the lysine sites on the TP and RH domains were mutated into arginine. Results showed that the KO mutants could still be degradated by clAP2, suggesting the N-side might be duo to the instability of polymerase. Results supported the above notion as linking the large tag molecule to polymerase’s N-terminal rather than the C-terminal the polymerase could make the polymerase more stable.Taken together, clAP2 protein could reduce the levels of HBV polymerase and the BIR2 and RING domain play an important role in this degradation. There is an interaction between clAP2 and HBV polymerase, and mainly due to the BIR2 and BIR3 domains. As an E3 ligase, clAP2 protein can lead HBV polymerase to ubiquitination and degradation by linking ubiquitin to the HBV-polymerase N terminal. The above results partially explain the molecular mechanism of inhibitng of HBV viral polymerase by a new ubiquitin-associated protein and will provide theoretical and experimental basis for the future development of more potential antiviral drug for HBV infection.