Abstract
Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell’s DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.
Highlights
Hepadnavirus specifies a group of hepatotropic viruses that carry a single copy of the partially double stranded relaxed circular viral DNA genome in the enveloped virion particle [1]
Hepadnavirus cccDNA is the persistent form of viral genome, and in terms of human hepatitis B virus (HBV), cccDNA is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure with current medications
We screened a total of 107 cellular DNA repair genes and identified DNA ligase 1 and 3 as key factors for cccDNA formation from viral relaxed circular DNA
Summary
Hepadnavirus specifies a group of hepatotropic viruses that carry a single copy of the partially double stranded relaxed circular (rc) viral DNA genome in the enveloped virion particle [1]. Upon infection of an hepatocyte, the hepadnaviral rcDNA genome is delivered into the nucleus and converted into an episomal covalently closed circular (ccc) DNA, which exists as a minichromosome and serves as viral mRNA transcription template [5, 6]. The mature nucleocapsid either acquires viral envelope proteins for virion egress, or recycles the viral DNA to the nucleus to replenish the cccDNA reservoir [8]. The available drugs for treatment of chronic hepatitis B are rarely curative due to their failure to eliminate cccDNA [10]. There is an urgent unmet need to fully understand HBV cccDNA biology and develop novel effective treatments to directly target cccDNA formation and maintenance [11, 12]
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