T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

Abstract

Chronic infection with the human hepatitis B virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular DNA (cccDNA), the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular DNA [rcDNA]), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and Southern blotting or selective quantitative PCR (qPCR) methods allowing discrimination of cccDNA and rcDNA. In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases for targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximately 1-kb fragment permits reliable quantification of cccDNA without the requirement of prior nucleus enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicities of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance. IMPORTANCE cccDNA elimination is a major goal in future curative regimens for chronic HBV patients. However, PCR-based assays for cccDNA quantification show a principally constrained specificity when high levels of input virus or replicative intermediates are present. Here, we characterized T5 exonuclease as a suitable enzyme for mediumthroughput in vitro assays that preserves cccDNA but efficiently removes rcDNA prior to PCR-based quantification. We compared T5 exonuclease with the previously described exonuclease III and showed that both nucleases are suitable for reliable quantification of cccDNA by PCR. We substantiated the applicability of our method through examination of early cccDNA formation and stable accumulation in several in vitro infection models and analyzed cccDNA stability after administration of anti-HBV drugs. Our results support the use of T5 exonuclease for fast and convenient rcDNA removal, especially for early cccDNA quantification and rapid drug testing in in vitro studies. Citation Qu B, Ni Y, Lempp FA, Vondran FWR, Urban S. 2018. T5 exonuclease hydrolysis of hepatitis B virus replicative intermediates allows reliable quantification and fast drug efficacy testing of covalently closed circular DNA by PCR. J Virol 92:e01117-18. https://doi .

De novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA

“…Total cellular DNA was extracted from Huh7-NTCP cells after infection with 200 mge of WT or ΔHBc HBV and analysed by inverse nested PCR at 7dpi ( Figure S4). We found that WT and ΔHBc HBV integrated at a geometric mean frequency (± geometric SD factor) of 4.82 x 10 -5 (±2.36) and 5.97 x [6,36,38]. Our finding also confirms previous work that demonstrated that a more than genome length construct carrying a HBc stop mutation can be rescued by trans-complementation with a HBc expressing plasmid [39] to produce replication-deficient virions.…”

“…Wild-type and ΔHBc HBV establish comparable levels of cccDNA in HBV-susceptible cells and then analysed intracellular cccDNA levels at 7 and 14dpi by Southern blot (Figure 3A). As shown previously [36], cccDNA was detectable at 7 and 14dpi with WT virus without a significant increase within the second week. Formation of cccDNA could be blocked by MyrB, confirming receptor mediated entry as a prerequisite for cccDNA formation.…”

Study on the structure optimization and anti‐hepatitis B virus activity of novel human La protein inhibitor HBSC11

“…Quantitative analysis of HBV DNA and HBV RNA was performed using Hepatitis B Viral DNA/RNA Quantitative Fluorescence Diagnostic Kit (ShenXiang, China) according to the manufacturer's protocol. Hirt extraction and T5 exonuclease digestion for cccDNA PCR quantification . Cytotoxicity was assessed by CCK‐8 (Bimake) and flow cytometer.…”

“…Parallel to this, compound 1 (567 mg, 3.5 mmol) was suspended in dry toluene (20 mL), and three drops DMF and oxalyl chloride (1.0 mL, 11.8 mmol) were added at RT, then warmed to 50°C and stirred for to afford the desired product as shown in supplement (S1). 14,15 Cytotoxicity was assessed by CCK-8…”

MafF is an antiviral host factor that suppresses transcription from Hepatitis B Virus core promoter

“…International HBV meeting (62). Levels of cccDNA were measured by quantitative real-time PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems), specific primers, and probe as described previously (63). Data were processed as 2 (−ΔΔCt) for quantification of cccDNA using GAPDH (via primer-probe set Hs04420697_g1; Applied Biosystems) as an internal normalization control.…”