Involvement of Host ATR-CHK1 Pathway in Hepatitis B Virus Covalently Closed Circular DNA Formation

Abstract

The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5′ truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production. IMPORTANCE Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth.

Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation

“…Because of the damaged-DNA-like structure of rcDNA, Luo and colleagues assessed the role of two DNA damage repair pathways (DDR), ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR), in cccDNA formation using inhibitors and silencing approaches. Their results supported the role of ATR in HBV cccDNA formation [ 80 ]. Because all of the results were generated mostly in hepatoma cells, one may argue that the expression of repair proteins and pathway activity in quiescent cells such as hepatocytes may differ from those in replicative cells, as was shown for neurons or myoblasts [ 81 , 82 ].…”

Topoisomerase II-binding protein 1 promotes the progression of prostate cancer via ATR-CHK1 signaling pathway

“…At the beginning of DDR, cell-cycle arrest will be activated, and then cells will attempt DNA [13]. ATM/Chk2 pathway and ATR/CHk1 pathway is known to be DDR regulator in many cancers including prostate cancer [14][15][16]. NKX3.1, one of the prostate cancer suppressor, can interact with ATM leading to activate ATM, enhance the DDR and thus contribute to DNA integrity in prostate epithelial cells [17].…”

Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

“…Tetracyclin was withdrawn from the culture medium for two days to initiate HBV expression. Phosphonoformic acid (PFA, 2 mM) was then added for four days to arrest HBV DNA synthesis at the single-stranded DNA stage [28,63,64]. Subsequently, PFA was removed and the PP2A inhibitor fostriecin (20 μM, 40 μM, 60 μM) was added.…”

“…Core linker and PP2A in HBV replication uncoating to release RC DNA for CCC DNA formation [28]. Using the inducible HepAD38 cell HBV replication system that allows rapid and synchronous NC maturation and CCC DNA formation, based on reversible PFA arrest of HBV reverse transcription [28,59,63,64], we tested if inhibition of PP2A could affect NC maturation or CCC DNA formation (Fig 11A ). Following the brief one-day treatment with the PP2A inhibitor fostriecin, HBc extracted from the treated cells was analyzed by SDS-PAGE and western blotting using the mAb 25-7 to detect the dephosphorylated (S178) HBc and mAb 1D8 targeting the HBc NTD (Fig 2A ) to detect total HBc.…”