Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.

TitleCore components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.
Publication TypeJournal Article
Year of Publication2020
AuthorsWei, L, Ploss, A
JournalNat Microbiol
Volume5
Issue5
Pagination715-726
Date Published2020 05
ISSN2058-5276
KeywordsCell Line, DNA Ligase ATP, DNA Replication, DNA, Circular, DNA, Viral, Flap Endonucleases, Genome, Viral, Hepatitis B virus, Hepatocytes, Humans, Proliferating Cell Nuclear Antigen, Replication Protein C, Virion, Virus Replication
Abstract

<p>Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.</p>

DOI10.1038/s41564-020-0678-0
Alternate JournalNat Microbiol
PubMed ID32152586
PubMed Central IDPMC7190442
Grant ListR01 AI138797 / AI / NIAID NIH HHS / United States