Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR

Abstract

Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate "cccDNA copies per (infected) cell".

Keywords: Copy number per cell; Covalently closed circular DNA; Hepatitis B Virus; Quantitative PCR; T5 exonuclease; cccDNA.

Figure 1.. Working flowchart of this protocol.

Susceptible cells (PHH, HepaRG^NTCP, HepG2^NTCP) infected with high mges (> 300) of HBV are lysed. A. Lysates are incubated at 70 °C in the presence of proteinase K and later loaded on silica column and eluted. B. T5 exonuclease (5 units/1 h) removes host genomic DNA (black) and HBV replicative intermediates (rcDNA, dslDNA, etc.) (green) and preserves cccDNA (red) intact. C. cccDNA is amplified by p1040/p1996 and detected by probe p1085.

Figure 2.. Schematic diagram of the calculation of absolute cccDNA copy numbers.

A. On the day of experiment, cell number in the well is determined. B. Total DNA samples are extracted as suggested in Procedure. C. Here shows a proper T5 exonuclease digestion as shown in Procedure. D. Absolute cccDNA copies and E level of human β-globin are quantified, respectively as shown in Procedure. “cccDNA copies/cell” is “cccDNA copies/well” divided by “cell numbers”. Optional: F. If calculation of copies per infected cell is required, additional wells have to be arranged in parallel on the same plate during the infection. On the day of DNA extraction, cells in the wells are fixed and subjected to an immunofluorescence assay (HBcAg visualization using DAKO B0586 antibody, etc.) to determine infectivity (%: number of HBcAg positive cells divided by number of total cells) ( Qu et al., 2018 ). “cccDNA copies/infected cell” is the “cccDNA copies/cell” divided by “infectivity (%)”.

Figure 3.. cccDNA levels in three in vitro models and upon IFN-α-2a and Myrcludex B treatment.

A. PHH, differentiated HepaRG^NTCP and HepG2^NTCP cells were infected with HBV at mges/cell of 500. Myrcludex B (1 μM), an entry inhibitor blocking cccDNA formation, was co-administered with HBV inoculum during infection. On Day 7 post infection, cccDNA copies per infected cell were determined. Data shown in triangles were collected from three independent experiments. B. HepG2^NTCP cells were infected with HBV at mges/cell of 500 with mock treatment (untreated), co-treated with Myrcludex B (1 μM) during infection, or co- and post-treated with IFN-α-2A at 100 ng/ml purchased from PeproTech and PBL Assay Science. On Day 7 post infection, cccDNA copies per infected cell were analyzed. Statistics: P < 0.01 untreated versus IFN-treated; P = 0.828 PeproTech versus PBL.