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Published on: July 16, 2012
Replenishment of Hepatitis B Virus cccDNA Pool Is Restricted by Baseline Expression of Host Restriction Factors In
Sergey Brezgin1,2, Anastasiia Kostyusheva1, Ekaterina Bayurova3,4
1National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia.
Insights
Hepatitis B virus (HBV) replication increases DNA methyltransferase DNMT3A, which elevates HBV cccDNA levels and DNA damage. Host factors APOBEC3A and APOBEC3B may suppress HBV cccDNA formation.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) drives viral persistence in chronic infections.
- Understanding HBV cccDNA stability mechanisms is crucial for developing new hepatitis B therapies.
Purpose of the Study:
- Investigate the role of DNA methyltransferase DNMT3A in HBV cccDNA maintenance.
- Identify additional mechanisms contributing to HBV cccDNA persistence.
Main Methods:
- Utilized HBV-expressing cell lines and DNMT3A-expressing plasmids.
- Assessed HBV replication via real-time PCR and HBsAg assays.
- Employed CRISPR/Cas9 to silence APOBEC3A and APOBEC3B expression.
Main Results:
- DNMT3A suppressed HBV transcription but increased HBV cccDNA levels (4-6 fold).
- DNMT3A accelerated cell proliferation, ruling out cell cycle arrest in cccDNA expansion.
- Silencing APOBEC3A/APOBEC3B mimicked DNMT3A's effect, increasing cccDNA levels.
Conclusions:
- Host factors APOBEC3A and APOBEC3B may partially suppress HBV cccDNA formation.
- HBV and DNMT3A induce DNA damage in infected hepatocytes.
Background:
Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance.
Methods:
HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR.
Results:
Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression.
Conclusion:
Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.
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