# Mechanisms of hepatitis B virus cccDNA formation

> **NIH NIH R01** · PRINCETON UNIVERSITY · 2020 · $554,139

## Abstract

Project summary
Chronic hepatitis B virus (HBV) infection results 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
depends on elusive host factors for its generation. Using a yeast extract screen, we identified five core
components of lagging strand synthesis –PCNA, the replication factor C (RFC) complex, DNA polymerase δ
(POLδ), FEN-1, and DNA ligase 1 (LIG1) – as essential for cccDNA formation. We reconstituted cccDNA
formation with purified human homologs, establishing these as a minimal set of factors necessary and
sufficient for cccDNA formation. We further demonstrated that inhibiting POLδ significantly diminishes
cccDNA formation. In this proposal, we will build on these findings to determine the precise kinetics of
cccDNA formation, delineating the role of each factor at every step of the repair process. In understanding the
dynamics of rcDNA to cccDNA repair, we can identify potential rate-limiting steps that could be novel
therapeutic targets for disrupting cccDNA formation and maintenance. Using a series of innovative techniques
in both cell culture and mouse model systems, we will be able to test our findings in physiologically relevant
platforms that will strengthen the impact of our data. Factors found to be critical for rc- to cccDNA
conversion will be disrupted in these systems by a degron-mediated approach that will allow for fine-tuned
control of expression to alleviate any potential cytotoxicity. We can then monitor the effect of each factor in
turn on cccDNA formation or the maintenance of established cccDNA pools in chronically infected cells. To
increase the resolution of such studies, we will also examine at the single-cell level how the expression levels
of a given factor correlate with that of cccDNA. Altogether, these data will give us a far more comprehensive
view of this process critical to the persistence of HBV in chronically infected individuals.

## Key facts

- **NIH application ID:** 10032771
- **Project number:** 1R01AI153236-01
- **Recipient organization:** PRINCETON UNIVERSITY
- **Principal Investigator:** Alexander Ploss
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $554,139
- **Award type:** 1
- **Project period:** 2020-05-15 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10032771

## Citation

> US National Institutes of Health, RePORTER application 10032771, Mechanisms of hepatitis B virus cccDNA formation (1R01AI153236-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10032771. Licensed CC0.

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