TY - JOUR
T1 - Distribution of hepatitis B virus nuclear DNA
AU - Li, Mingming
AU - Sohn, Ji A.
AU - Seeger, Christoph
N1 - Publisher Copyright:
© 2017 American Society for Microbiology.
PY - 2018/1/1
Y1 - 2018/1/1
N2 - Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.
AB - Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.
KW - Animals
KW - Antiviral Agents/pharmacology
KW - Cell Division/genetics
KW - DNA Replication/drug effects
KW - DNA, Circular/isolation & purification
KW - DNA, Viral/drug effects
KW - Hepatitis B Virus, Duck/genetics
KW - Hepatitis B virus/genetics
KW - Hepatitis B, Chronic/drug therapy
KW - Hepatocytes/virology
KW - In Situ Hybridization, Fluorescence/methods
KW - Virus Replication
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U2 - 10.1128/JVI.01391-17
DO - 10.1128/JVI.01391-17
M3 - Article
C2 - 29046450
SN - 0022-538X
VL - 92
JO - Journal of Virology
JF - Journal of Virology
IS - 1
M1 - e01391-17
ER -