Proteomic Analysis of Nuclear Hepatitis B Virus Relaxed Circular DNA-Associated Proteins Identifies UV-Damaged DNA Binding Protein as a Host Factor Involved in Covalently Closed Circular DNA Formation

Abstract

Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify said host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the co-precipitated proteins by mass spectrometry. The DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported shRNA screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Trans-complementation of wild type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA-binding, DDB1-binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified a handful of host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNAi screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay we discovered a novel relationship between the UV-DDB complex and cccDNA formation, hence, providing a proof-of-concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.

Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication

“…HBV infectious particles were harvested from the supernatants of gRNA/Cas9‐treated HepDE19 cells and used for the infection of HepG2‐NTCP cells following a previously published method 24,25 . Briefly, the supernatants of gRNA RNP‐treated HepDE19 cells were harvested and concentrated using a PEG concentration kit (Cat# ab102538, Abcam).…”

“…HBV infectious particles were harvested from the supernatants of gRNA/Cas9-treated HepDE19 cells and used for the infection of HepG2-NTCP cells following a previously published method. 24,25 Briefly, the supernatants of gRNA RNP-treated HepDE19 cells were harvested and concentrated using a PEG concentration kit (Cat# ab102538, Abcam). For HBV infection, 2.5 × 10 5 HepG2-NTCP cells were seeded in a 12-well plate and mixed with 10 µL concentrated HBV supernatants in 1 mL hepatocyte maintenance medium (HMM) (with 8% PEG8000% and 5% DMSO) at 37°C for 16 h. The culture media were changed daily with fresh HMM.…”

A protein with broad functions: damage-specific DNA-binding protein 2

“…The hepatitis B virus (HBV) relies on the host DNA repair mechanism to convert virus-relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA) in the nucleus [ 99 ]. Marchetti et al found that UV-DDB, especially the binding activity of DDB2 to DNA and DDB1, and its ubiquitination activity, play an indispensable role in HBV infection [ 100 ]. DDB2 must combine with DDB1 to scan the nuclear HBV rcDNA, sense DNA damage, and be ubiquitinated to transfer the rcDNA to the next factor in the cccDNA formation pathway.…”

CRISPR/Cas9 for hepatitis B virus infection treatment

“…7 , 8 Another compact arrangement is to embed regulatory elements such as promoters and enhancers in the virus ORF. 9 , 10…”

“…Naturally, extra chromosomal DNA (such as cccDNA) is a target for transcriptional silencing through the maintenance of the cell structure of chromosome complex 7,8 . Another compact arrangement is to embed regulatory elements such as promoters and enhancers in the virus ORF 9,10 …”