Nucleotide excision repair and poly(ADP-ribose) polymerase 1 cooperate in HBV cccDNA synthesis: A potential viral therapeutic target.

Blocking the synthesis of HBV covalently closed circular (ccc)DNA is crucial for achieving a viral cure. Previous work showed that inhibiting nucleotide excision repair (NER) reduced cccDNA levels in HBV-producing liver cells. Here, we aimed to clarify the role of NER endonucleases in addressing the flap structure on relaxed circular DNA (rcDNA) and to explore an antiviral strategy that disrupts viral replication reservoirs. We investigated the need for NER endonucleases for cccDNA synthesis in liver cells by examining the interactions between the complex of excision repair cross complementation group 1 (ERCC1)-xeroderma pigmentosum group F (XPF) and that of poly(ADP-ribose) polymerase 1 (PARP1)-tyrosyl-DNA phosphodiesterase 1 (TDP1). We also assessed the impact of a PARP1 inhibitor on cccDNA synthesis and developed a digital PCR method for quantifying cccDNA in human liver tissues with high sensitivity. Finally, we evaluated the correlation between the levels of ERCC1, XPF, TDP1, and PARP1 and cccDNA. ERCC1-XPF NER endonuclease and TDP1 increased cccDNA in liver cells and were shown to directly bind each other to cleave the rcDNA flap. This was demonstrated through various protein interaction and in vitro rcDNA cleavage analyses. PARP1 inhibition effectively blocked cccDNA formation and was dependent on ERCC1, XPF, and TDP1. The interactions among these complexes suggest that inhibiting PARP1 could reduce cccDNA production. Furthermore, these complexes were found to associate with the HBV genome in human liver tissues, showing correlations between their levels and cccDNA concentration (p 0.003-0.034; r 0.31-0.43). ERCC1-XPF and TDP1-PARP1 complexes work in tandem to achieve cccDNA synthesis, indicating PARP1 inhibition as a promising therapeutic strategy for clearing cccDNA. Blocking HBV cccDNA synthesis is imperative for reaching the goal of a cure for HBV. This study delineated the involvement of host NER endonucleases in resolving the flap structure on rcDNA and seeking an antiviral strategy by blocking the production of viral replication reservoirs. The NER ERCC1-XPF endonuclease and the topoisomerase 1-associated TDP1-PARP1 complex directly bind to the HBV genome in the human liver, where these factors correlate with the cccDNA levels. The ERCC1-XPF and TDP1-PARP1 complexes function in tandem to cleave the HBV rcDNA flap so that cccDNA synthesis can proceed. Thus, PARP1 inhibition could be a promising therapeutic strategy for cccDNA clearance.