Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for viral persistence in the natural course of chronic HBV infection and during prolonged antiviral therapy and serves as the template for the production of HBV pregenomic RNA (pgRNA), the primary step in HBV replication. In this study, we have developed and applied sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays for the measurement of intrahepatic concentration, pgRNA production, and replicative activity of cccDNA in liver biopsy samples from 34 non-treated patients with chronic hepatitis B (CHB); 12 hepatitis B e antigen (HBeAg)(+) and 22 HBeAg(−). Median copy number for cccDNA was 1.5 per cell and for pgRNA significantly higher, 6.5 copies per cell, with a good correlation between cccDNA and pgRNA levels in all samples. In HBeAg(−) patients, median values of cccDNA and pgRNA levels were 10-fold and 200-fold lower than in HBeAg(+), respectively, reflecting the differences in viral activity and clinical characteristics of the two groups. Furthermore, the replicative activity of intrahepatic cccDNA was significantly lower in HBeAg(−) patients harboring mutant HBV strains than in HBeAg(+) patients: median 3.5 versus 101 pgRNA copies per cccDNA molecule. In conclusion, the levels of both HBV cccDNA, a marker of HBV persistence, and pgRNA, an indicator of viral replication, in the liver of chronically infected patients correlate with viral activity and the phase of HBV infection. The combined measurement of cccDNA and pgRNA levels provides valuable information on the presence and replicative activity of intrahepatic HBV cccDNA. (HEPATOLOGY 2006;44:694–702.)

Infection with the hepatitis B virus (HBV) leads to either acute, self-limiting, or fulminant hepatic disease, or to chronic hepatitis B (CHB) infection with a wide spectrum of viral activity, clinical manifestations, and disease progression.1–3 The factors and molecular events that influence the course and the outcome of chronic infection remain poorly understood. Persistent HBV infection of the hepatocyte is characterized by the presence of a covalently closed circular (ccc) DNA episome, whereas the degree of viral replicative activity is primarily determined by the transcriptional state of cccDNA molecules and the production of pregenomic (pg) RNA.4–6

On entry of the HBV virion into the hepatocyte, the viral genome is translocated to the nucleus and converted into a supercoiled cccDNA viral minichromosome.7–9 The cccDNA episome is the template for the HBV mRNA transcripts; and the 3.5-kb pg RNA, template for reverse transcription and synthesis of the viral genome. Transcription of pgRNA from cccDNA is a key event in HBV replication, and the process is under the control of the HBV core promoter (CP),10 which directs the synthesis of two species of overlapping 3.5-kb viral transcripts that only differ at their 5′ initiation sites.11, 12 Translation of the longer precore (preC) mRNA leads to the synthesis of the precore protein, precursor of the HBV e antigen (HBeAg).13 The shorter pg RNA serves as mRNA for the core and the polymerase genes and, after encapsidation, as the template for reverse transcription to generate viral DNA. Intracellular cycling of DNA-containing nucleocapsids to the nucleus is necessary for cccDNA pool maintenance.9, 14

During the course of chronic HBV infection and during prolonged antiviral therapy, development and selection of mutations in the HBV genome may become clinically relevant and influence the outcome of infection.15–17 CP mutations clustering in the basic CP (BCP) region are commonly found among chronically infected patients.18, 19 CP mutations may affect HBV gene expression, replication, and pathogenicity by altering the levels and balance between pgRNA and precore mRNA production20–23 and affecting the transcriptional activity of HBV cccDNA molecules. HBV cccDNA is believed to be responsible for viral persistence and reactivation.24–26 However, the factors that determine the transcriptional state of cccDNA in hepatocytes are not well understood, and widespread HBV mutant strains have different transcriptional and replicative activities.20, 23, 27, 28 Therefore, measuring in vivo pgRNA levels in conjunction with cccDNA levels could provide a more precise method for evaluating HBV persistence and activity in infected patients than measuring cccDNA alone.

Despite the generation and publication of methodology on HBV cccDNA detection,29–34 few in vivo data are currently available from human liver samples,32–35 and most of the existing knowledge is derived from animal models.36–39 Even less in vivo information is available on cccDNA replicative activity and pgRNA production,22, 37 because most HBV expression studies have been performed in vitro or on heterologous systems.

We have designed and used quantitative assays for the measurement of both concentration and, for the first time, the replicative activity of intrahepatic cccDNA in liver biopsy samples of patients with CHB. Our data show that the combined measurement of cccDNA and pgRNA levels provides valuable information by monitoring the presence, as well as the activity of intrahepatic HBV cccDNA in chronically infected patients.

Abbreviations

HBV, hepatitis B virus; CHB, chronic hepatitis B; cccDNA, covalently closed circular DNA; pgRNA, pregenomic RNA; CP, core promoter; HBeAg, hepatitis B e antigen; BCP, basic core promoter; rcDNA, relaxed circular DNA; RT-PCR, reverse transcription polymerase chain reaction.

Patients and Methods

Patients and Biological Samples.

We studied 34 patients with CHB; 12 HBeAg(+), 7 men and 5 women, median age 36 (range, 18-58) years and 22 HBeAg(−), 12 men and 10 women, median age 42 (27-65) years. All patients had active liver disease with persistent biochemical activity [serum alanine aminotransferase (ALT) levels >2× the upper limit of normal] and had been on long-term follow-up on an outpatient basis. Degree of hepatic inflammation and fibrosis was assessed according to Ishak et al.40: 7 (21%) had established cirrhosis [1 HBeAg(+), 6 HBeAg(−)], 17 (50%) had fibrosis score ≥3 [4 HBeAg(+), 13 HBeAg (−)], and the mean necro-inflammatory score was 8 (range, 4-14). All patients were negative for serological markers for hepatitis D virus, hepatitis C virus, and HIV infection. None was on treatment or had received nucleoside analog treatment in the past. None was consuming alcohol or taking immunosuppressive drugs. Serological markers of HBV infection, hepatitis B surface antigen, HBeAg, and anti-HBe were tested by radioimmunoassay (Sorin Biomedica S.p.A, Saluccia, Italy). Liver biopsies and synchronous sera samples were stored at −70°C until analysis.

This study was approved by the Ethics Committees of the Evgenidion Hospital of Athens University and Henry Dunant Hospital.

HBV cccDNA Detection and Quantitation.

Liver biopsy material from each patient was homogenized and divided into two parts, one for DNA and one for RNA/DNA extraction. Total liver DNA was extracted from biopsy samples using the Qiamp DNA Mini Kit (QIAGEN GmbH, Germany). Real-time polymerase chain reaction (PCR) was performed with the LightCycler FastStart DNA Master Hybridization Probes kit (Roche Diagnostics GmbH, Mannheim, Germany), using 3′ antisense primer BC1 (5′-GGAAAGAAGTCAGAAGGCAA, nt1974-1955) and 5′ primers CCC (5′-GTGCCTTCTCATCTGCCGG nt1555-1573) or PGP (5′-CACCTCTGCCTAATCATC nt1826-1843) that specifically detect cccDNA and total HBV DNA, respectively (Fig. 1). FRET hybridization probes 0.4 mmol/L hbvLC (5′-TGGAGGCTTGAACAGTAGGACATGAAC nt1874-1848) labeled with LC Red 640 at the 5′ terminus and 0.2 mmol/L hbvFL (5′-CYAAAGCCACCCAAGGCACAGC nt1897-1876) labeled with fluoresceine at the 3′ terminus (Fig. 1). After 10 minutes' incubation at 95°C amplification was performed for 45 cycles (95°C 10 seconds, 52°C 16 seconds, and 72°C 10 seconds). Plasmid-Safe DNase (Epicentre, Madison, WI) was used according to the manufactures' instructions, in testing designed to validate the specificity of the assay. Cell number in liver biopsies was estimated by measuring β-globin gene levels using the LightCycler Control Kit DNA (Roche Diagnostics GmbH, Mannheim, Germany); results were normalized to 1 × 106 cells.

Figure 1.

Schematic representation of the hepatitis B virus (HBV) double-stranded relaxed circular (rc) DNA and covalently closed circular (ccc) DNA and organization of the core promoter and precore/core regions. Direct repeats (DR) 1 and 2 used in HBV replication are shown as black boxes. Regulatory elements that constitute the core promoter and their corresponding nucleotide positions are shown, negative regulatory element (NRE) nt 1613-1636, core upstream regulatory sequences (CURS) nts 1636-1742 and basic core promoter (BCP) nts 1742-1849. Precore mRNA and pregenomic RNA transcripts are shown with horizontal arrows indicating the origin and direction of transcription. Real-time polymerase chain reaction (PCR) primers used for cccDNA detection (CCC and BC1) and for the transcript-specific reverse transcription (RT)-PCR assay (M3, PCP, PGP, and BC1) are shown as horizontal arrows. The position of the LightCycler hybridization probes hbvLC and hbvFL is also shown. Shaded triangles indicate the translation initiation codons for precore and core and the open triangle the polyadenylation (PolyA) signal for HBV RNA transcripts.

Pregenomic RNA Quantitative Detection/Transcript-Specific RT-PCR.

Total liver RNA (and subsequently total liver DNA) was extracted from liver biopsy samples using a modified guanidinium thiocyanate-phenol procedure (TriPure, Roche Diagnostics, GmbH). RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) at 1 U/μg nucleic acid for 1 hour at 37°C, to remove contaminating DNA. One microgram liver RNA was used for cDNA synthesis with antisense primer BC1 and M-MLV reverse transcriptase (RT). Extracted liver RNA (1 μg) was incubated for 40 minutes at 42°C in 60 μL reaction mixture containing 50 pmol cDNA primer, 50 mmol/L Tris (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, 1 mmol/L dNTPs, 20 U Rnasin (Promega), and 300 U M-MLV RT (Promega). The cDNA product was used in each of three separate amplification reactions with BC1 as the common 3′ primer and 5′ primers: (1) PCP (5′-GGTCTGCGCACCAGCACC nt1796-1813) for the specific detection of precore mRNA transcripts, (2) PGP (5′-CACCTCTGCCTAATCATC nt1826-1843) for monitoring total CP-directed transcription, and (3) M3 (5′-CTGGGAGGAGTTGGGGGAGGAGATT nt1730-1754) for detecting contaminating HBV DNA molecules (Fig. 1). FRET hybridization probes and real-time PCR conditions were as described. HBV cccDNA replicative activity was expressed as molecules of pgRNA synthesized per HBV cccDNA molecule. DNA extracted subsequently was used to estimate cell number as described.

Serum HBV DNA Extraction, PCR Amplification, and Sequencing.

HBV DNA was extracted from 200 μL serum amplified and sequenced as described previously.22 Serum cccDNA and total HBV DNA were quantified by real-time PCR as described.

Statistical Analysis.

Statistical analyses were performed using SPSS10.0 (Chicago, IL). Correlation between two variables was tested by using Pearson's analysis after logarithmic transformation. Statistical significance was denoted as P < .05.

Results

Specificity and Sensitivity of cccDNA and pgRNA Quantitative Assays.

A major concern in designing and evaluating cccDNA-specific detection assays is the complex structure of the HBV genome, which may result in nonspecific background signal compromising the integrity of data. Because the complexity and heterogeneity of HBV genomic DNA is not reflected in cloned HBV sequences, we have tested the performance of the assay on HBV DNA extracted from serum and liver biopsy samples.

Liver biopsy specimens and synchronous serum samples randomly selected from three HBeAg(+) (patients 2, 5, and 6) and six HBeAg(−) (patients 13, 15, 20, 25, 27, and 34) patients were used initially to determine the specificity of the cccDNA detection assay. Total HBV DNA and cccDNA levels were measured in liver and serum samples, and the results are shown in Table 1. Serum HBV DNA ranged from 3.7 × 104 to 1.9 × 109 copies per milliliter; cccDNA was detected in seven of nine serum samples tested and ranged from 9.4 × 102 to 4.1 × 105 copies per milliliter. Corresponding intrahepatic total HBV DNA levels ranged from 2.2 × 105 to 2.1 × 109 per 1 × 106 cells and cccDNA, detected in all liver samples ranged from 1.8 × 104 to 1.4 × 107 per 1 × 106 cells. Total serum HBV DNA levels were 1,000-fold (800- to 6,500-fold) higher than the corresponding serum cccDNA levels, which represents 0.1% (range, 0.02%-0.12%) of serum HBV DNA. In contrast, high levels of cccDNA were detected in all liver samples, representing a significant proportion of total intrahepatic HBV DNA, 6% on the average (range, 0.65% to 22%), with total HBV DNA levels being 17-fold (5-fold to 154-fold) higher.

Table 1. Specific Detection of HBV Total DNA and cccDNA in Synchronous Serum and Biopsy Samples From Nine Randomly Selected Patients
Patient NumberHBeAg StatusSerum HBV DNA (copies/mL)Liver HBV DNA (copies/106 cells)
Total DNAcccDNA% cccDNATotal DNAcccDNA% cccDNA
  1. Abbreviation: UN, undetectable.

2+1.9 × 1093.0 × 1050.022.1 × 1091.4 × 1070.7
5+1.5 × 1071.5 × 1040.107.1 × 1086.5 × 1060.9
6+8.9 × 1072.4 × 1040.032.5 × 1083.6 × 1061.5
134.1 × 1084.1 × 1050.101.1 × 1084.0 × 1063.6
156.0 × 1075.5 × 1040.098.0 × 1075.6 × 1067.0
203.2 × 1063.8 × 1030.122.2 × 1051.8 × 1048.0
254.5 × 105UN3.0 × 1062.9 × 1059.6
271.6 × 1069.4 × 1020.066.3 × 1053.7 × 1045.9
343.7 × 104UN1.5 × 1063.4 × 10522.0

Although other investigators have reported the presence of HBV cccDNA33 and naked HBV nucleic acids41, 42 in the bloodstream, it could still be argued that measured serum cccDNA is a result of nonspecific detection of the assay. Even in this case, a background of nonspecific cccDNA detection of 1:1,000 molecules would not affect the integrity of the liver data.

To dismiss this possibility, the specificity of the cccDNA detection assay was further validated by treatment with Plasmid-Safe DNase, which selectively degrades linear DNA or circular single-stranded DNA without affecting closed or nicked circular double-stranded DNA. Total HBV DNA samples extracted from four liver biopsies and synchronous serum samples were subjected to DNase treatment, and detected cccDNA levels were compared with those measured in mock-treated samples (Table 2). HBV cccDNA levels were resistant to DNase treatment in all samples tested, demonstrating the high specificity of our cccDNA detection assay.

Table 2. Effect of Plasmid-Safe DNase Treatment on cccDNA-Specific Detection in Synchronous Biopsy and Serum Samples
Patient NumberHBeAg StatusLiver HBV cccDNA (copies/106 cells)Serum HBV cccDNA (copies/mL)
DNase TreatedMock TreatedDNase TreatedMock Treated
10+3.8 × 1053.0 × 1056.8 × 1044.5 × 104
12+1.5 × 1041.6 × 1042.8 × 1041.9 × 104
142.1 × 1041.8 × 1041.1 × 1057.8 × 104
212.7 × 1034.5 × 1032.1 × 1042.5 × 104

The sensitivity of the real-time assay was determined using serially diluted samples of serum and liver HBV DNA. The lower limit of detection was 10 copies/assay for total HBV DNA detection and 100 copies/assay for cccDNA detection, with a dynamic range of 6 orders of magnitude. This is in part attributable to a less efficient amplification by the cccDNA-specific primers, which amplify a longer DNA fragment than the primers used in the detection of total HBV DNA (420 bp vs. 149 bp). Taking into account the cell number of the liver biopsy specimens and necessary sample dilutions, intrahepatic detection limits were 2 × 10−4 and 2 × 10−3 copies/cell, respectively, for total HBV DNA and cccDNA assays.

The specificity of the methodology used in the transcript-specific RT-PCR assay for the differential detection of precore mRNA and pgRNA transcripts has been previously described.22 Application of RT-PCR technology has improved the sensitivity of the method to 10 copies/assay for either precore mRNA or total BCP-directed transcription.

Quantitative Detection of cccDNA in the Liver.

HBV cccDNA levels were measured in all liver biopsy specimens from 22 HBeAg(−) and 12 HBeAg(+) patients. Total liver DNA was analyzed by template-specific real-time PCR for the presence of cccDNA and total intracellular HBV DNA, which also includes immature and mature viral genomic DNA. Cell number in the liver biopsy samples was calculated by measuring β-globin gene levels, and results were normalized to 1 × 106 cells (number of copies/cell is also given to facilitate comparisons with other studies).

The median copy number of cccDNA per 1 × 106 cells was 1.5 × 106 ranging from 3.2 × 103 to 4.0 × 107 (median, 1.46; range, 0.003-40 copies/cell). High levels of cccDNA were detected in HBeAg(+) patients with a median of 3.0 × 106, ranging from 1.2 × 106 to 4.0 × 107 (median, 3.0; range, 1.24-40 copies/cell). Significantly lower levels of cccDNA were detected in HBeAg(−) patients with a median of 3.1 × 105, ranging from 3.2 × 103 to 6.8 × 106 (median, 0.31; range, 0.003-6.8 copies/cell) (Fig. 2).

Figure 2.

Intrahepatic cccDNA, pgRNA and total intracellular HBV DNA levels in HBeAg(+) (open squares) and HBeAg(−) (closed circles) patients.

The levels of total intracellular HBV DNA in these patients had a median copy number of 3.1 × 107, ranging from 6.5 × 103 to 9.9 × 109 per 1 × 106 cells (median, 31; range, 0.007-9,890 copies/cell). Total intracellular HBV DNA levels were higher in HBeAg(+) patients, with a median of 3.4 × 108 (range, 9.5 × 107-9.9 × 109) than in HBeAg(−) patients, with a median of 2.1 × 106 (range, 6.5 × 103-1.1 × 108) per 1 × 106 cells (Fig. 2).

A strong correlation was seen between intrahepatic total HBV DNA and cccDNA levels (r = 0.945) (Fig. 3A); however, the percentage of intrahepatic HBV DNA in the form of cccDNA is inversely related to the total intrahepatic and serum HBV DNA levels (partially shown in Table 1).

Figure 3.

Correlation between various parameters measured in liver biopsy samples of hepatitis B e antigen (HBeAg)(+) (open squares) and HBeAg(−) patients (closed circles). (A) Intrahepatic total hepatitis B virus (HBV) DNA versus cccDNA, correlation coefficients: r = 0.910 for HBeAg(+) and r = 0.964 for HBeAg(−) patients. (B) Pregenomic RNA vs cccDNA, correlation coefficients: r = 0.726 for HBeAg(+) and r = 0.867 for HBeAg(−) patients. (C) Pregenomic RNA versus intrahepatic total HBV DNA, correlation coefficients: r = 0.855 for HBeAg(+) and r = 0.928 for HBeAg(−) patients.

Quantitative Analysis of pgRNA Transcripts in the Liver.

Total liver RNA was analyzed by transcript-specific real-time RT-PCR, which allows specific detection and quantification of precore mRNA and simultaneously monitors total RNA transcription (precore mRNA plus pgRNA) directed by the core promoter in the liver of infected patients. The levels of pgRNA transcripts were calculated by subtracting precore mRNA levels from total CP-directed transcription.

HBV pgRNA transcript levels per 1 × 106 cells had a median copy number of 6.5 × 106 ranging from 9.6 × 103 to 8.7 × 109 (median, 6.5; range, 0.01-8,730 copies per/cell). High levels of pgRNA transcripts were measured in HBeAg(+) patients having a median of 2.7 × 108 and ranging from 9.6 × 107 to 8.7 × 109 (median, 266; range, 96-8,730 copies/cell). In contrast, low levels of pgRNA were detected in the liver of HBeAg(−) patients, with a median of 1.4 × 106, ranging from 9.6 × 103 to 4.5 × 107 (median, 1.4; range, 0.01-45 copies/cell) (Fig. 2).

Precore mRNA transcripts were detected in only 22 patients, and their levels were significantly lower than the corresponding pgRNA levels. Precore mRNA was measured in all HBeAg(+) patients with a median value of 2.4 × 106 and ranging from 8.5 × 105 to 3.2 × 107 (median, 2.4; range, 0.85-32 copies/cell), representing approximately 1% of total CP-directed transcription. In contrast, precore mRNA was detectable in only 10 of 22 HBeAg(−) patients, with a median of 1.8 × 105 and ranging from 2.6 × 103 to 1.4 × 107 (median, 0.18; range, 0.003-14 copies/cell). Eleven of 12 HBeAg(−) patients harboring the A1762T/G1764A mutation had undetectable precore mRNA levels. Four patients with either one of A1762T, G1764A, or other mutations in the BCP promoter had intermediate precore mRNA levels, whereas five HBeAg(−) patients without any BCP mutations expressed high precore mRNA levels.

The levels of pgRNA in the liver were found to correlate very well with cccDNA (r = 0.863) (Fig. 3B) and the levels of total intrahepatic HBV DNA (r = 0.964) (Fig. 3C). Generally good correlation was seen between all HBV nucleic acid replication parameters. Serum HBV DNA levels show significant correlation with intrahepatic cccDNA levels (r = 0.744) and highly correlate with intrahepatic pgRNA and total HBV DNA levels (r = 0.874 and r = 0.840, respectively).

Replicative Activity of Intrahepatic cccDNA.

Production of pgRNA from transcriptionally active cccDNA molecules is a key step in HBV replication and therefore a measure of the replicative activity of intrahepatic cccDNA. The simultaneous measurement of cccDNA and pgRNA allows us to measure for the first time the activity of cccDNA molecules in the liver of patients with CHB.

Good correlation between cccDNA and pgRNA levels was found in all samples tested (Fig. 3B); however, considerable differences were found in the median values for cccDNA and pgRNA levels between the two groups (Fig. 2). In HBeAg(−) patients median cccDNA levels were 10-fold (3.1 × 105 vs 3.0 × 106) and median pgRNA levels 200-fold (1.4 × 106 vs 2.7 × 108) lower than in HBeAg(+) patients. Thus the replicative activity of the cccDNA intermediate, calculated as the number of pgRNA copies produced per cccDNA molecule, was significantly different between the HBeAg(+) and the HBeAg(−) patients. Median replicative activity of cccDNA in HBeAg(+) patients was 101 (range, 31-404) versus 3.5 (range, 0.5-32) in HBeAg(−) patients. Most (12 of 22) HBeAg(−) patients had very low replicative activity, less than 5 pgRNA transcripts per cccDNA molecule, whereas only 6 of 22 patients had replicative activity greater than 15 pgRNA/cccDNA (Fig. 4).

Figure 4.

HBV cccDNA Replicative Activity (pgRNA transcripts produced per cccDNA molecule) in HBeAg(+) (open squares) and HBeAg(−) (closed circles) patients.

The reduced CP transcriptional activity of cccDNA in HBeAg(−) patients can be at least in part attributed to the accumulation of mutations in the CP and precore regions of the viral genome. Although all HBeAg(+) patients had wild-type CP and precore sequences, HBeAg(−) patients were found to harbor the precore stop codon mutation G1896A and additional mutations in the CP region, most prevalent being the double A1762T/G1764A BCP mutation.

Discussion

Persistent hepatocyte infection by HBV is achieved by the establishment and maintenance of a stable cccDNA pool in the nuclei of infected cells.7–9 Replenishment of the cccDNA nuclear reservoir, as well as initiation of viral replication, require the synthesis of pgRNA transcripts from episomal viral genomes.4–6, 14 In this study, we have developed and applied sensitive assays for the quantitative analysis of HBV cccDNA and pregenomic RNA in liver biopsy samples of chronically HBV infected patients, which enable us to measure both the concentration and the replicative activity of intrahepatic cccDNA.

In designing the cccDNA-specific real-time PCR detection assay, we have positioned the amplification primers at conserved regions flanking the direct repeats DR1 and DR2 and the nicks in both (−) and (+) strands (Fig. 1). However, that both 3′ primer and FRET detection probes are placed downstream from DR1 and that probes have (−) polarity are critical, to avoid a nonspecific signal resulting from asymmetric amplification of either the (−) or (+) strands, which would obscure the measurement of the low-concentration intrahepatic cccDNA component. Perhaps for this reason other studies report significant background from relaxed circular (rc) DNA in their experiments, necessitating enzymatic DNase digestion before cccDNA detection in their methodology.32, 34 Our methodology is specific by design, and, as we have shown, it does not depend on DNase removal of other HBV DNA molecules (Table 2).

Transcript-specific detection of pgRNA and precore mRNA is achieved, first, by positioning the 3′ primer downstream from the common polyadenylation site, unique sequences present at the 5′ end of the CP produced 3.5-kb RNAs allow distinction between them and the rest of the HBV transcripts (Fig. 1). Second, specific detection of the longer precore transcript is accomplished by positioning the 5′ primer in the small region between the precore mRNA and the pgRNA start sites. A second 5′ primer positioned downstream from both start sites monitors total CP-directed transcription (Fig. 1).

Our in vivo study demonstrated the presence of HBV cccDNA in all included patients with CHB at low median levels of approximately 1 copy/cell, but exhibiting a wide dynamic range of four orders of magnitude. In general, cccDNA levels correlate with HBeAg status and viral activity. HBeAg(+) patients with CHB had high cccDNA copy numbers, approximately 1 to 40 copies/cell, and high levels of total intracellular HBV DNA (95-9,890 copies/cell) and serum HBV DNA (107-109 copies/mL).

A far more complex situation exists within the HBeAg(−) group, where median cccDNA levels are 10-fold lower compared with HBeAg(+) patients. Intrahepatic cccDNA levels show significant variability (0.003–6.8 copies/cell), consistent nonetheless with the heterogeneity of this group in terms of viral genomic sequences, replication, and disease activity. In HBeAg(−) patients with low viremia, chronic infection appears to be maintained by a very low cccDNA copy number (<0.1 copies per cell), in agreement with the study of Zhang et al.38 The accumulation of mutations in the precore region and the BCP appears to adversely affect intrahepatic cccDNA levels in HBeAg(−) patients, suggesting that the cccDNA pool might be affected by pgRNA production. This is further supported by the good correlation of intracellular cccDNA and pgRNA levels (r = 0.863). This could be a result of specific processes regulating HBV replication and intracellular trafficking of nucleocapsids43, 44 or it simply could be attributable to lower pgRNA levels, which would result in a reduced pool of precursor genomic molecules available for the replenishment of nuclear cccDNA.

The levels and range of intrahepatic cccDNA, as well as total liver HBV DNA, in HBeAg(+) and HBeAg(−) patients in this study are consistent with and support the available data in the literature.33, 34 However, serum cccDNA levels were found to be lower than those reported by Wong et al.33 (0.1% vs. 3.5%), perhaps reflecting differences in methodology or the characteristics of patients and viral strains in the two studies.

Although pregenomic RNA is important both for viral production and replenishment of the cccDNA reservoir,9, 14 little in vivo information is currently available.22 This is the first report that accurately measures in vivo pgRNA levels in the liver of patients with CHB. Our study showed an extremely wide range of pgRNA levels (0.01-8,700 copies/cell), which nonetheless correlate with viral activity and phase of HBV infection, as is the case with cccDNA levels. Pregenomic RNA levels were consistently high at the early HBeAg(+) phase and more variable but significantly lower (200-fold) at the later HBeAg(−) phase. PgRNA levels were significantly higher than the corresponding levels of cccDNA, 90-fold higher in HBeAg(+) patients and fivefold higher in HBeAg(−) patients. Therefore, measurement of pg RNA levels in biopsy samples may provide more accurate information on viral replication as well as higher sensitivity, particularly in HBeAg(−) patients with low viremia and cccDNA levels. Furthermore, because precore mRNA contribution to CP-directed transcription is low, it may be possible to perform a single assay for monitoring pgRNA levels; however, a larger number of samples should be analyzed before making this recommendation. The observed differences and variability of pgRNA levels can be attributed only in part to differences in the cccDNA template content in the liver of chronic HBV patients, and they must also be accounted for by changes in the transcriptional activity of intrahepatic cccDNA molecules.

A unique result of this study is the assessment of the replicative activity of intrahepatic cccDNA by simultaneously measuring cccDNA and its pgRNA transcript levels in the same tissue sample of patients with CHB. This allows us to evaluate the transcriptional and replicative activity of cccDNA molecules in vivo.

HBV cccDNA replicative activity was high in all HBeAg(+) patients harboring wild-type HBV strains. The intensive pgRNA production coupled with high cccDNA levels in this group results in a highly productive infections with median viremia 2.3 × 108 copies/mL serum. In contrast, in HBeAg(−) patients, cccDNA replicative activity was dramatically lower. Furthermore, although the correlation between cccDNA, pgRNA, and viremia is significant, the correlation coefficients are relatively low, suggesting that other factors, such as the accumulation of mutations and changes in the host immune response, may significantly affect HBV production and disease activity in the HBeAg(−) phase.

Mutations in the BCP have been shown to suppress precore mRNA production and alter the ratio between pgRNA and precore mRNA synthesis.20–23 In this study, BCP mutants had low or undetectable precore mRNA levels and lower replicative activity than wild-type strains, as did HBV G1896A precore mutants. Given the variability in viral activity characterizing HBeAg(−) CHB, significant fluctuations in the cccDNA pool or its replicative activity may occur during the course of infection. However, to what extent activation or sequestering of cccDNA activity is determined by mutations accumulating in the CP promoter or other regions of the HBV genome remains undetermined. High levels of viral production may not necessitate a relative increase in the cccDNA pool but rather enhanced levels of transcriptional activity mediated by other modulatory factors. In agreement with this hypothesis is the observation that three HBeAg(−) patients had relatively high replicative activity (>20) in the absence of obvious differences in cccDNA levels and mutation profile. Reduced replicative activity and inefficient replenishment of the nuclear cccDNA reservoir could explain differences in the intrahepatic cccDNA levels between HBeAg(+) and HBeAg(−) patients reported in this and other studies.33, 34

Collectively our data demonstrate that differences in the natural course of HBV infection between HBeAg(+) and HBeAg(−) patients are not limited to decreased intrahepatic cccDNA levels. On the contrary, although a significant reduction occurs in cccDNA concentration, a much stronger decrease is seen in corresponding intrahepatic pgRNA levels, reflecting a markedly reduced replicative activity of cccDNA in HBeAg(−) patients.

In conclusion, in the liver of chronically infected patients, the levels of both HBV cccDNA, a marker of HBV persistence and stability, and pgRNA, an indicator of viral replication, correlate with viral activity and the phase of HBV infection. The combined measurement of cccDNA and pgRNA levels may provide valuable information on HBV natural history and the efficacy of long-term antiviral therapy by monitoring the persistence and replicative activity of both wild type and mutant HBV strains.

Acknowledgements

The authors thank Dr. Peter Karayiannis for helpful discussions and critical reading of the manuscript.

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