TY - JOUR
T1 - Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation
AU - Wei, Lei
AU - Ploss, Alexander
N1 - Funding Information:
We thank X. Zhao (MSKCC) for sharing wild-type yeast stains and yeast-tagging plasmids; P. Modrich (Duke University) for sharing plasmids for expression of the RFC and POLδ complexes; B. Wan (MSKCC) for discussions on yeast-tagging strategies; the Zakian and Shenk lab (Princeton University) for sharing equipment and reagents for the yeast work; S. Port and S. Travis (Hughson lab, Princeton University), X. Gong (Yan lab, Princeton University), M. Estrella (Korennykh lab, Princeton University), R. Alfaro-Aco (Petry lab, Princeton University) and the Remus lab (MSKCC) for the their advice on protein purification and extract preparation; and B. Bratton and J. Sheehan (Gitai lab, Princeton University) for their advice on P1 phage transduction. We thank J. Gaska and other members of the Ploss laboratory for providing critical feedback on this manuscript. This work was supported in part by grants from the National Institutes of Health (grant no. R01 AI138797 to A.P.), a Research Scholar Award from the American Cancer Society (grant no. RSG-15-048-01-MPC to A.P.), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to A.P.) and funding from Princeton University. L.W. is a recipient of a postdoctoral fellowship award from the New Jersey Commission on Cancer Research (NJCCR, award no. AWD1005321, sponsor award no. DFHS17PPC011).
Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41564-020-0678-0
DO - 10.1038/s41564-020-0678-0
M3 - Article
C2 - 32152586
AN - SCOPUS:85081276837
SN - 2058-5276
VL - 5
SP - 715
EP - 726
JO - Nature Microbiology
JF - Nature Microbiology
IS - 5
ER -