A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures
Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research.
Key wordsHBV cccDNA Hirt extraction Southern blot
We thank Dr. Andrea Cuconati (Institute for Hepatitis and Virus Research) for critical reading of the manuscript. This work was supported by the NIH grants (R01AI094474, R21AI088424) and Hepatitis B Foundation through an appropriation of the Commonwealth of Pennsylvania. Haitao Guo is the Bruce Witte fellow of the Hepatitis B Foundation.
- 16.Cai D, Mills C, Yu W, Yan R, Aldrich CE, Saputelli JR, Mason WS, Xu X, Guo JT, Block TM, Cuconati A, Guo H (2012) Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation. Antimicrob Agents Chemother 56:4277–4288PubMedCrossRefGoogle Scholar
- 21.Margeridon S, Carrouee-Durantel S, Chemin I et al (2008) Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA. Antimicrob Agents Chemother 52:3068–3073PubMedCrossRefGoogle Scholar