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Principal Investigator: Alexander Ploss
Organization: PRINCETON UNIVERSITY
Fiscal Year: 2024
Award: $30,051
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <A1 protein><Affect><Animal Model><Animal Models and Related Studies><Anti-viral Therapy><Antibodies><Assay><Bioassay><Biochemical><Biologic Models><Biological Assay><Biological Models><CRISPR><CRISPR/Cas system><Cell Body><Cell Culture Techniques><Cell Extracts><Cell Line><Cell Nucleus><CellLine><Cells><Cessation of life><Chimp><Chimpanzee><Chronic><Chronic Hepatitis B><Circular DNA><Clustered Regularly Interspaced Short Palindromic Repeats><Co-culture><Cocultivation><Coculture><Coculture Techniques><Complex><DNA Damage Repair><DNA Ligases><DNA Polymerase III><DNA Polymerase delta><DNA Repair><DNA lesion><DNA-Dependent DNA Polymerase III><Data><Death><Exposure to><FEN-1><Fen1 Protein><Flap Endonuclease 1><GeneHomolog><Generations><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genome><Goals><HBV><HLA-DR Associated Protein II><Health><Hep G2><HepG2><HepG2 cell line><Hepatic Cancer><Hepatic Cells><Hepatic Disorder><Hepatic Parenchymal Cell><Hepatitis B Virus><Hepatocyte><Homolog><Homologous Gene><Homologous Serum Hepatitis Virus><Homologue><Host Factor><Host Factor Protein><Human><IGAAD><In Vitro><Individual><Infection><Inhibitor of GZMA-Activated DNase><Integration Host Factors><Kinetics><Knock-out><Knockout><Learning><Lesion><Life Cycle><Life Cycle Stages><Liver><Liver Cells><Liver diseases><Lytotoxicity><Maintenance><Malignant neoplasm of liver><Mediating><Methods><Mice><Mice Mammals><Model System><Modern Man><Molecular><Monitor><Murine><Mus><Mutation><Nature><Nucleus><Pan Genus><Pan Species><Patients><Persons><Phosphatase 2A Inhibitor I2PP2A><Physiologic><Physiological><Pol III><Polydeoxyribonucleotide Ligases><Polydeoxyribonucleotide Synthetases><Polymerase><Process><Public Health><RAD2 Homolog-1 Nuclease><RF-C protein><RTH-1 Nuclease><Recombinants><Refractory><Regimen><Relaxation><Resolution><Risk><Role><SET Translocation Inhibitor-2 of Protein Phosphatase-2A><Series><Set protein><Strains Cell Lines><System><Techniques><Template Activating Factor I Beta><Testing><Therapeutic Intervention><Transcript><Unscheduled DNA Synthesis><Vaccination><Viral><Viral Diseases><Viral Genome><Viral Receptor><Virion><Virus><Virus Diseases><Virus Particle><Virus Receptors><Work><Yeasts><cell culture><cell cultures><chronic HBV infection><chronic hepatitis B virus infection><cultured cell line><cytotoxicity><design><designing><experience><genome mutation><hepatic body system><hepatic disease><hepatic organ system><hepatoma cell><hepatopathy><high risk><innovate><innovation><innovative><intervention therapy><knock-down><knockdown><life course><liver cancer><liver disorder><liver malignancy><malignant liver tumor><model of animal><mouse model><murine model><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><overexpress><overexpression><pathogenic virus><prevent><preventing><purge><purges><purging><reconstitute><reconstitution><repair><repair endonuclease><repair enzyme><repaired><replication factor C><resolutions><self assembly><shRNA><short hairpin RNA><small hairpin RNA><social role><viral infection><viral infectious disease treatment><viral pathogen><virus genome><virus infection><virus pathogen><virus-induced disease>