Deneddylation by SENP8 Restricts Hepatitis B Virus Propagation

概要

Hepatitis B Virus (HBV) infection is strongly associated with acute and chronic liver disease and a major global health problem. The World Health Organization (WHO) estimates that around 2 billion people are infected with HBV, and more than 240 million people are chronically infected patients. Chronic infection of HBV is a risk factor for developing liver cirrhosis and hepatocellular carcinoma (HCC). Although reverse transcriptase inhibitors and interferon alpha are approved for HBV treatment and can suppress viral replication, these two strategies have limitation and viral cure is rarely achieved. Therefore, discovery of novel candidates for the treatment of viral infections is required.

A number of cellular factors had been reported to be involved in HBV propagation including CDKN2C, SRSF10, and JMJD5. Post-translational modifications (PTMs) such as phosphorylation, acetylation, methylation, and ubiquitination affects newly synthesized protein function through a various mechanisms, such as regulating cellular localization, protein stability, protein conformation and protein interaction with other proteins. PTMs are known to have crucial role for regulating various biological processes at each stage of HBV life cycle. However, it remains unclear which ubiquitin-like proteins (UBLs) are involved in HBV life cycles.

In this study, I set up the screening system to identify the UBLs to regulate HBV propagation. Huh7 cells was lentivirally transduced with shRNA targeting UBLs, which is known to be expressed in the liver and established stable knock-downed (KD) Huh7 cells. Next, I transfected HBV expression plasmid (pUC19 HBV_JPNAT) to each of the KD Huh7 cells and the levels of intracellular core incorporated HBV DNA were determined by qPCR. I found that transduction of shNEDD8 significantly suppressed HBV DNA compared to control cells. In addition, I found that overexpression of NEDD8 enhanced HBV propagation. Conjugation of NEDD8 to the substrates is called as neddylation. Next, to examine the role of neddylation on HBV life cycle, I focus on the SENP8, which is a protease that specifically remove NEDD8 from its substrates. Overexpression of SENP8 suppressed intracellular HBV DNA in HBV plasmid transfection model, HepG2.2.15-7 cells, which persistently produce HBV particles and HBV infection model by using HepG2 NTCP-C4 cells, susceptible to HBV infection. In order to confirm the effects of SENP8 expression on HBV propagation in vivo, I utilized a hydrodynamic injection model of HBV replication in mice. The pAAV-HBV vector was injected into mice together with a SENP8 or GFP vector and serum HBV DNA was determined by qRT-PCR after 3 days. I found that the amount of HBV DNA in mouse serum was lower in the mice injected with the SENP8 vector when compared to control mice. To study more details of involvement of SENP8 on HBV life cycle, I established SENP8KO Huh7 cell clones using the CRISPR/Cas9 system. The levels of HBV DNA were significantly higher in all of SENP8KO Huh7 cell clones when compared to parent Huh7 cells. In order to confirm the effects of inhibition of neddylation on HBV propagation, I utilized a MLN-4924, a potent and selective inhibitor of NEDD8-activating enzyme. Treatment of MLN-4924 reduced HBV propagation in transfection and infection model suggest that neddylation is required for sufficient HBVV propagation. In addition, we demonstrated that suppression of HBV propagation via SENP8 overexpression was independent of HBx and HBV promoter activity. Therefore, my data suggested that neddylation plays an important role in the late stages of HBV life cycles.