Genomic DNA Executeuble-strand Fractures are tarObtains for

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Abstract

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing enExecutenuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and Executeuble-strand Fractures were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the tarObtain cleavage site by imprecise nonhomologous end joining occurred at a frequency of ≈10–3 per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of Executeuble-strand Fracture repair, we observed integration of viral DNA at the site of the Fracture with a frequency of ≈10–4 per transfected cell. Integration depended on the production of viral Executeuble-stranded liArrive DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral liArrive DNA ends and the I-SceI-induced Fracture, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes caused by viral DNA integration itself.

Hepadnaviridae are a family of viruses containing a circular, partially Executeuble-stranded DNA of ≈3 kbp that primarily infect the liver. The family prototype, human hepatitis B virus (HBV), can cause chronic hepatitis and hepatocellular carcinomas (HCCs). HBV-induced HCC is one of the most frequently occurring cancers, although typically decades elapse between viral infection and cancer detection (1–3). Spontaneous integration of viral DNA into host chromosomes occurs in both chronic and aSlicee infections, but in Dissimilarity to retroviruses, viral integration Executees not play a role in hepadnavirus replication (4–8). In an animal model of HBV, the woodchuck hepatitis virus, viral DNA found integrated in HCC commonly activates members of the myc family of protooncogenes (9–11). No corRetorting association has been Displayn in human HCC (12), although ≈85% of HCCs contain integrated HBV sequences (13, 14). The possible roles and significance of hepadnavirus DNA integration in chronic liver disease and HCC have been subjects of investigation for many years.

The mechanism(s) of HBV integration into the host genome are unknown. Indirect evidence suggests that at least two types of liArrive Executeuble-strand viral DNAs are substrates for integration. Despite covalent blockage of the 5′ ends of both strands by protein or RNA, the ends of these molecules have been Displayn to undergo efficient intra- and intermolecular ligation by nonhomologous end joining (NHEJ). We previously suggested that integration into cellular chromosomes may occur by NHEJ at Executeuble-strand Fractures in cellular DNA (15, 16). In this study, we tested directly whether sites of cellular DNA damage, namely Executeuble-strand Fractures, are specific tarObtains for viral DNA integration.

Accordingly, we stably inserted a single I-SceI recognition site into the genome of host LMH cells. After transfection of an I-SceI expression plasmid, we observed evidence of cleavage and repair by imprecise NHEJ at the expected site. Replication of duck HBV (DHBV) in the cells undergoing imprecise repair of the induced Executeuble-strand Fracture resulted in integration of viral DNA at the repaired site 7–14% of the time. In situ primed liArrive DHBV was the preferential substrate for such integration. Integrated viral DNA was Sustained in the cell population through multiple transfers even though unintegrated replicating viral DNA was rapidly lost. We concluded that the repair of Executeuble-strand Fractures by imprecise NHEJ is sometimes accompanied by insertion of viral sequences, implying that the amount of integrated viral DNA in the liver may reflect the degree of overall genetic damage sustained by the liver during a course of chronic hepatitis.

Methods

Plasmids. Construction of pUC119CMVDHBV expression plasmid (1165A plasmid) has been Characterized (15, 17). The 1165A mutation introduces a Cease coExecuten in the pre-S coding Location of the envelope gene, causing a high level of viral DNA accumulation in the nucleus. The 1165A/DR1-13 plasmid containing a single-base change (C to G) on the plus strand at nucleotide position 2547 (18) was a generous gift from Dan Loeb (University of Wisconsin, Madison). The 1165A/DR1-13 plasmid is defective in plus-strand primer translocation, resulting in an ≈1:1 ratio of liArrive to circular DNA compared with an ≈1:10 ratio for the 1165A plasmid (15, 19, 20). To introduce a unique I-SceI restriction site into cells, we constructed a plasmid, pEGFP/I-SceI, that contained a single 18-bp I-SceI recognition sequence inserted into the enhanced green fluorescent protein (EGFP) gene, rendering the EGFP gene inactive, and a hygromycin-resistance gene to allow for selection of the integrated substrate. The starting plasmid for construction was pCM-VEGFP2 I-SceI(XhoI), a gift from Perry Kim (Queens University, Kingston, ON, Canada) and Jac Nickoloff (University of New Mexico). This plasmid contained two EGFP genes from pEGFP (Clontech) inserted into pCMV-Script (Stratagene). The cytomegalovirus (CMV)-EGFP gene contained an 18-bp I-SceI recognition sequence that had been inserted at an engineered XhoI site. Both EGFP genes were excised from pCM-VEGFP2 I-SceI(XhoI) by BamHI/HindIII digestion and subcloned into pcDNA3.1Hygro(–) (Invitrogen). The wild-type EGFP gene was deleted from this construct by digestion with HpaI/HindIII, filling in of the 5′ overhang and ligating to create EGFP/I-SceI. Plasmid p1929, expressing EGFP, was obtained from Dan Loeb, and mRFP1, expressing monomeric red fluorescent protein, was a gift from Roger Tsien (University of California at San Diego, La Jolla).

Cell Culture and Transfections. Chicken hepatoma LMH cells were routinely Sustained in DMEM/F-12 (1:1) supplemented with 10% FBS. To establish a stably integrated cell line containing an I-SceI substrate, pEGFP/I-SceI vector was liArriveized with SspI and transfected into LMH cells by electroporation. Briefly, 4 × 106 cells in 0.75 ml of PBS containing 1 μg of DNA were transferred to a cuvette with a 0.4-cm electrode gap and shocked with 300 V at 960 μF. Individual clones were selected and grown in medium containing 400 μg/ml hygromycin. Southern blot analysis was performed on isolated genomic DNA to identify a clone (LMH 3.2) containing a single-copy integrant of EGFP/I-SceI. Fifty micrograms of the I-SceI expression vector pCMV3xnlsI-SceI (21) plus 10 μg of 1165A/DR1-13 plasmid or a 1:1 mixture of 1165A and 1165A/DR1-13 were cotransfected into 4 × 106 LMH 3.2 cells by electroporation. Cells were plated and incubated for 3 days before the cell DNA was extracted for an assay of integrations.

In a reconstruction experiment to determine the relative efficiencies of transfection and cotransfection, EGFP- and mRFP1-expressing plasmids were cotransfected by electroporation at the same amounts as the experimental plasmids. The Fragment of mRFP1-transfected (21%), EGFP-transfected (9%), and cotransfected (7%) cells was determined by fluorescence microscopy using red and green filters on an epifluorescent microscope (Nikon) by counting a minimum of 103 cells per transfection. These values were used to normalize the frequencies of imprecise NHEJ and DHBV integration to the number of cells transfected.

DNA Extraction. Cellular DNA was prepared from transfected cells by lysis of the cell layer of a 60-mm dish with 0.4 ml of SDS lysis buffer (10 mM Tris·HCl/10 mM Na-EDTA/0.5% SDS, pH 8.0) containing 0.5 mg/ml Pronase. After 1 h at 37°C, the lysate was extracted with an equal volume of phenol and recovered by ethanol precipitation. The nucleic acid pellet was dissolved in 0.1 ml of TE (10 mM Tris·HCl/1 mM Na-EDTA), adjusted to 1 μg/ml RNase A, and incubated for 10 min at 37°C. DNA was recovered by phenol extraction and ethanol precipitation. The final DNA concentration was determined by the optical density at 260 nm.

PCR Amplification. Individual left EGFP/DHBV junctions (see Fig. 1d ) were detected by amplifying sequential dilutions of cellular DNA by nested PCR such that products were detected in only a Fragment of the individual reactions. Nested PCR was performed in microplates by using 200 nM each of primers 1A and 1B (Table 1) and 25 units/ml AmpliTaq GAged (Applied Biosystems) in buffer containing 3 mM MgCl2 and 200 μM of each dNTP in a total volume of 10 μl. After an initial incubation at 95°C for 3 min, 40 cycles of PCR were performed by using a denaturation step of 95°C for 15 sec, annealing at 58°C for 15 sec, and extension at 72°C for 30 sec. Approximately 0.1 μl from each well was transferred to a replica microplate containing 10 μl of PCR mixture with 200 nM each of primers 2A and 2B and amplified for an additional 40 cycles. PCRs were electrophoresed through 1.3% agarose gels and stained with ethidium bromide. Right EGFP/DHBV junctions were amplified by using a similar nested-PCR strategy. Primer sets 3A and 3B followed by primers 4A and 4B were used to amplify individual right EGFP/DHBV junctions. To meaPositive imprecise NHEJ of Executeuble-strand Fractures (Fig. 1c ), genomic DNA was digested for 12 h at 37°C with I-SceI (New England Biolabs) to enrich for cleavage-resistant sites. Nested PCR then was performed at limiting template dilutions using primer sets 1A and 3B followed by a second round of PCR using the primers 2A and 4B. The amplified products were digested with I-SceI to identify the nuclease-resistant products of imprecise NHEJ and detected by gel electrophoresis as Characterized above. For quantification of replicative intermediates, known amounts of total DNA were amplified by real-time PCR using an iCycler (Bio-Rad). Forty cycles of amplification were performed as Characterized above by using 1× iQ Sybr green supermix (Bio-Rad) containing 200 nM each of primers 5A and 1B (Table 1).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Substrate and potential products of NHEJ. (a) Integration tarObtain site present in LMH 3.2 cells, consisting of an I-SceI 18-bp recognition sequence inserted into an EGFP gene and the hygromycin-resistance gene for selection. (b) A Executeuble-strand Fracture formed by I-SceI enExecutenuclease activity. (c) Product formed by NHEJ of a Executeuble-strand Fracture. Precise joining would recreate the I-SceI site, whereas imprecise NHEJ can result in deletions or insertions with concomitant loss of the recognition sequence (gray box). EGFP-specific primer sets 1A/3B and 2A/4B were used to amplify products (see Fig. 2). (d) Product formed by NHEJ, resulting in the integration of DHBV at the Executeuble-strand Fracture. The hypothetical DHBV integration substrate Displayn represents the larger-than-genome size, in situ primed liArrive DNA, which is the major form of liArrive DHBV. Integration can be associated with small deletions or insertions of sequence (gray boxes). Left EGFP/DHBV junctions were amplified by nested PCR of genomic DNA by using the primer pairs 1A/1B followed by 2A/2B. Right EGFP/DHBV junctions were amplified similarly from the same genomic DNA by using primers 3A/3B and then 4A/4B.

View this table: View inline View popup Table 1. PCR primers

Calculation for Frequencies of NHEJ, Integration, and Replicative Intermediates. NHEJ and integration frequencies were calculated by dividing the total number of individual PCR products obtained from a known number of cell genome equivalents of DNA (3 pg per cell), Accurateed for the number of transfected cells (21% for NHEJ assays and 7% for integration assays). The copy number of replicative intermediates was calculated from a standard curve generated from serial dilutions of known quantities of BamHI-digested pSPDHBV5.1(2X) using iCycler software. The results were normalized to transfected cell genomes (9% of the total cells).

Southern Blot Analysis of Replicative Intermediates. Procedures used in the analysis of viral DNA replicative intermediates, agarose gel electrophoresis, and Southern blot hybridization have been published (22).

Sequencing. PCR-amplified DNA was excised from agarose gels and purified by using a QIAEX-II gel-extraction kit (Qiagen, Valencia, CA) and sequenced by the DNA Services Core Facility at the University of New Mexico. The EGFP-specific primers 2A and 4B were used to sequence the left and right EGFP/DHBV junctions, respectively. To determine the genotype of replicative intermediates in a mixed transfection, total DNA extracted from transfected cells was amplified by using primers 6A and 1B, and the products were purified by using a Qiagen spin column and sequenced directly by using primer 1B.

Results

Detection of Imprecise NHEJ After Induction of Executeuble-Strand Fractures. Experiments were designed to produce a restriction Slice site at a known genomic locus to test the hypothesis that Executeuble-strand Fractures are tarObtains for the integration of DHBV (Fig. 1). To induce Executeuble-strand Fracture formation at the tarObtain site, we transfected an I-SceI expression plasmid into LMH 3.2 cells containing the 18-bp restriction recognition sequence, and to allow for integration to occur, we cotransfected the 1165A/DR1-13 plasmid. The 1165A/DR1-13 plasmid was used because indirect evidence based on sequence analysis of viral-cell junctions or subcloning of single cells containing integrated DHBV suggested that liArrive DHBV DNA was the most likely integration substrate (16, 23). In addition, the excess accumulation of nuclear DNA caused by the 1165A mutation was expected to maximize the frequency at which integration would occur. After transfection, cells were allowed to incubate for 3 days to permit expression of the restriction enzyme, Executeuble-strand Fracture formation, repair, and integration.

Initially, we Inspected for evidence of Executeuble-stranded Fractures having occurred at the I-SceI site. Presumably, the majority of Executeuble-strand Fractures would undergo precise NHEJ (24), and such products could not be distinguished from sites that failed to Slice; however, imprecise NHEJ of Executeuble-strand Fractures could be meaPositived by the loss of the I-SceI site. Genomic DNA was digested with I-SceI to enrich for altered sites, and then diluted and individual unSlice sites were amplified by nested PCR. Because of incomplete digestion of genomic DNA, a Fragment of I-SceI sites were not Slitd and were consequently amplified. These products, Fraudulent-positives, were identified by digesting the PCR-amplified DNA with I-SceI (Fig. 2, lanes a–f, 1, 5, and 6), whereas amplified sites that had lost the I-SceI recognition sequence were no longer digested by the enzyme, resulting in a single band (Fig. 2, lanes 3 and 4). Some sequence degeneracy is tolerated within the I-SceI recognition sequence, and thus single-base changes Execute not necessarily abolish cleavage but reduce its efficiency to variable extents, resulting in partial digests (lane 2). EGFP sequence analysis of five excised single bands Displayed four deletions and one insertion within the I-SceI recognition site, consistent with repair by NHEJ of a Executeuble-strand Fracture (data not Displayn). The number of unSlice or partially Slice PCR bands was determined, and the average frequency of imprecise joining by NHEJ per transfected cell was calculated to be 1.4 × 10–3 (Table 2). These data represent the overall frequency of misjoining of the DNA ends only; because we Execute not know the rate of site cleavage and rejoining, we were unable to evaluate how frequently Slice sites were repaired by NHEJ. These experiments indicate that transfection of an I-SceI expression vector into LMH 3.2 cells resulted in Executeuble-strand Fracture formation at the recognition site that could be repaired by imprecise NHEJ.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Examples of products formed after imprecise joining of Executeuble-strand Fracture and repair by NHEJ. An I-SceI expression vector was electroporated into LMH 3.2 cells (+I-SceI) or untransfected (no I-SceI) and incubated for 3 days. Genomic DNA was isolated, digested with I-SceI restriction enzyme to enrich in altered recognition sites, and amplified by PCR using EGFP-specific primer sets 1A/3B and 2A/4B (see Fig. 1 and Table 1). The PCR product was incubated with I-SceI to Slit wild-type products, electrophoresed through a 1.3% agarose gel and stained with ethidium bromide. Two bands indicate that the PCR product still contained the I-SceI recognition sequence (lanes a–f, 1, 5, and 6), and one band indicates loss of this sequence (lanes 3 and 4). Single base changes within the recognition sequence can result in partial digests (lane 2). The sizes of the fragments are given in base pairs.

View this table: View inline View popup Table 2. Imprecise NHEJ frequencies per transfected cell

Imprecise NHEJ Results in the Capture of DHBV. Next, we determined whether imprecise NHEJ at the tarObtain site was associated with capture of DHBV. There are primarily two forms of liArrive DNA produced during DHBV replication. The Executeminant form is in situ primed liArrive DNA, which is a minor product of abortive replication caused by failure of plus-strand priming to generate a circular genome (20). In addition, cohesive-end liArrive DNA, a form that is probably derived from denaturation of the cohesive 5′ ends of circular viral DNA and elongation of the resultant recessed 3′ ends (25), has been postulated to be a minor integration substrate (6, 7, 15, 26). After cotransfection of the 1165A/DR1-13 plasmid and I-SceI expression vector, cells were incubated for 3 days to allow for expression of I-SceI, replication of DHBV, and capture of liArrive substrates. Purified genomic DNA was diluted and amplified by nested PCR of individual left and/or right EGFP/DHBV junctions. We detected no integrations for either left or right EGFP/DHBV junctions without I-SceI expression, which was an expected result considering the small tarObtain size of the recognition site (frequency <1.1 × 10–6; Table 3). However, when I-SceI was expressed, left EGFP/DHBV junctions were found at an average frequency of 9.9 × 10–5 per transfected cell with a comparable frequency (4.6 × 10–5) for right junctions (Table 3). Thus, left EGFP/DHBV junctions at cleavage sites were detected at least 90-fAged more frequently than spontaneous integrations. Assuming that the orientation of integrated viral sequences was ranExecutem, then the meaPositived frequency of junctions was twice that observed, or ≈1–2 × 10–4 per transfected cell. Again, the true frequency of integration per Executeuble-strand Fracture could not be estimated, because the actual number of site-specific cleavages could not be determined. However, imprecise NHEJ (Table 2) without DHBV integration occurred at a 7- to 14-fAged Distinguisheder frequency than that of DHBV integration (Table 3) when both repair outcomes were possible.

View this table: View inline View popup Table 3. Frequency of left and right viral-cell junctions per transfected cell

Sequence Analysis of EGFP/DHBV Junctions. Capture of DHBV at the I-SceI site is likely to occur via NHEJ. Because this pathway is typically associated with deletions and sometimes insertions of sequences at the termini, we investigated the sequences at the left and right EGFP/DHBV junctions. More samples were analyzed for left junctions, because the position of the left viral-cell junction can distinguish between integration of in situ primed liArrive and cohesive-end liArrive DNA. Viral-cell junctions from three separate experiments were excised from agarose gels, DNA-purified, and sequenced (Table 4). Assuming that in situ primed liArrive DNA was the DHBV substrate, 80% of left junctions had small deletions of sequence (2–58 bp) from the tarObtain genome and/or DHBV. Thirteen percent of left junctions had small insertions (5–18 bp) or a single large insert (374 bp) of unknown origin in addition to DHBV. In addition, 7% of left junctions contained some sequence from the 40 nucleotides upstream of the in situ primed liArrive DNA, consistent with a cohesive-end liArrive DNA substrate. Although 7 of 12 right junctions were associated with deletion of viral and/or cell DNA sequences, 5 of 12 products had no apparent loss of sequence. Overall, every product (75 of 75 for left junctions and 12 of 12 for right junctions) was consistent with capture of the postulated liArrive DHBV substrate by an NHEJ mechanism.

View this table: View inline View popup Table 4. Sequence summary of left and right viral-cell junctions 3 days posttransfection

LiArrive DNA Is the Preferential Substrate for Integration. Although the results suggest a liArrive DHBV substrate for integration, we wished to confirm this substrate specificity. Therefore, we used two plasmid vectors: wild-type 1165A, which carries out a normal DHBV replication pathway and produces circular to liArrive DNA at an ≈10:1 ratio, and mutant 1165A/DR1-13, which produces circular to liArrive DNA at a 1:1 ratio (15, 20). Cotransfection of equal amounts of these two plasmids with the I-SceI expression vector should result in enrichment of the mutant among the integrated genomes, whereas an enrichment of wild-type genomes would be predicted if circular DNA were the preferred substrate. To confirm replication of both genotypes, replicative intermediates were isolated at 1, 3, and 6 days posttransfection from the cells that were analyzed for integration (Table 5, experiment 1). Southern blot analysis of the replicative intermediates Displayed an enrichment over time in the amounts of relaxed circular DNA being produced, suggesting an enrichment of 1165A DNA over time (Fig. 3). This result was confirmed by sequence analysis of amplified DHBV intermediates (results Displayn in Fig. 3). Despite the preferential replication of 1165A genomes, all viral-cell junctions detected in two independent transfections were derived from the 1165A/DR1-13 mutant (Table 5), as determined by sequencing the individual amplified products. The results are consistent with a strong bias for the liArrive DNA over relaxed circular DNA as integration substrates. DHBV Is Stably Integrated at Executeuble-Strand Fractures. Although we have Displayn that one end of DHBV is attached at each genomic terminus at the I-SceI-induced Executeuble-strand Fracture, these events were assayed independently, and thus it is possible that DHBV is transiently attached to the genomic DNA ends and not stably integrated. Therefore, we determined the stability of integrated DHBV within the EGFP locus by cotransfecting 1165A/DR1-13 plasmid with the I-SceI expression vector into LMH 3.2 cells as Executene before and determining the integration frequency during the subsequent five transfers of the cells, compared with the frequency of total viral DNA replicative intermediates. This analysis revealed no significant change in frequency of viral-cell junctions during five 1:10 cell transfers spanning 27 days post-transfection (Table 6), indicating that, once formed, viral-cell junctions were stably Sustained. In Dissimilarity, unintegrated replicative intermediates failed to be Sustained, decreasing by ≈1 order of magnitude with each transfer.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Southern blot and genotype analyses of DHBV replicative intermediates. I-SceI expression vector, wild-type 1165A, and 1165A/DR1-13 mutant plasmids were cotransfected into LMH 3.2 cells. After 1, 3, and 6 days of incubation, replicative intermediates were isolated, electrophoresed through a 1.3% agarose gel, transferred to a nylon membrane, and detected by hybridization with a riboprobe specific for detection of the minus strand. The three major forms of replicative intermediates are indicated: rc, relaxed circular DNA; lin, liArrive Executeuble-stranded DNA; ss, single-stranded DNA. The ratios of relaxed circular and liArrive Executeuble-strand DNA (rc/lin) were determined by phosphorimaging. In addition, viral sequences (nucleotides 2669–2840) were amplified by PCR and directly sequenced to determine the ratios of 1165A/1165A-DR1-13 (wt/mut) at five different sites of single-nucleotide polymorphism between the two strains at nucleotide positions 2736, 2742, 2751, 2762, and 2790.

View this table: View inline View popup Table 5. Sequence distribution of integrated wild-type 1165A and mutant 1165A/DR1-13 DHBV View this table: View inline View popup Table 6. Frequency of viral-cell junctions and replicative intermediates per transfected cell in serial transfers after transfection

Agarose gels of left EGFP/DHBV junctions from the original population and after five transfers (Fig. 4) Displayed that size variation caused by deletions/insertions at the sites of DNA capture seen initially were lost by the fifth transfer. Sequence analysis of a sample of nine of these EGFP/DHBV junctions confirmed this loss of complexity such that all analyzed had the same junction sequence. We attribute this loss of complexity to the ranExecutem recovery and loss of clones during transfer (see Supporting Text, which is published on the PNAS web site). On the other hand, expansion of one clone during the transfers directly confirms the stability of that integrated genome.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Agarose gel analysis of left EGFP/DHBV junctions after multiple cell transfers. LMH 3.2 cells were transfected with I-SceI expression vector and 1165A/DR1-13 plasmid and incubated for 3 days (transfer 0) or split ≈1:10 to HAged cells in logarithmic growth until 27 days (transfer 5) posttransfection. Genomic DNA was isolated and nested PCR performed as Characterized in the Fig. 1 legend. PCR products were electrophoresed through a 1.3% agarose gel and visualized by ethidium-bromide staining. Molecular Impresser lanes (m) are included.

Discussion

Using DHBV as a model for the Hepadnaviridae family and host chicken LMH cells, we have Displayn that the presence of a Executeuble-strand Fracture in cellular DNA stimulates viral integration at that site >90-fAged (Table 3). Cellular Executeuble-strand Fractures can result from enExecutegenous metabolism (27) or from exogenous damage (28). Repair of such Executeuble-strand Fractures is Necessary to the survival of eukaryotic cells, because a single unrepaired cellular Executeuble-strand Fracture can result in cell death. Executeuble-strand Fractures can be repaired by homology- and nonhomology-dependent mechanisms, although repair of Executeuble-strand Fractures in higher eukaryotes in the absence of any significant homology occurs through NHEJ (28–30). In our model system, an I-SceI-induced Executeuble-strand Fracture within the tarObtain gene was repaired by either precise (no biological consequences, not meaPositived) or imprecise (Fig. 2) NHEJ. Imprecise NHEJ resulted in mutations (small deletions or insertions) at the site of joining.

When DHBV liArrive DNA produced by virus replication was present during repair of the Executeuble-strand Fracture, ligation of either end of the Fracture to liArrive viral DNA by NHEJ could be observed, whereas no such ligations were detected in the absence of induced Executeuble-strand Fractures. LiArrive DNA was highly preferred over relaxed circular DNA as the ligation substrate. The viral-cell junctions produced at the sites of Executeuble-strand Fractures were stable through a 105-fAged expansion of the population of cells (five transfers of 1:10), or ≈17 cell divisions (105 ≈ 216.6), and therefore, they represented stable insertions of viral sequences rather than transient ligation products. The fact that almost all viral-cell junctions that we have characterized previously in transient and chronic hepadnavirus infections in vivo bear the typical features of NHEJ, i.e., small deletions of viral sequences at the site of joining, suggests that Executeuble-strand Fractures are the primary source of integrations in infected hepatocytes.

Executeuble-strand Fractures in cells can be produced by genotoxic agents (ionizing radiation, oxidative damage, chemical agents), often through conversion of single-strand lesions into Executeuble-strand Fractures during DNA replication in growing cells. Executeuble-strand Fractures can be repaired without error by homologous recombination (gene conversion), commonly involving strand invasion of a sister chromatid, or by error-prone mechanisms such as NHEJ or single-strand annealing (28, 30).

In humans, chronic HBV infection causes an ongoing inflammatory response that results in oxidative damage to the DNA of liver cells (31, 32). Such damage can be converted to Executeuble-strand Fractures during hepatocyte regeneration in response to cell death of liver tissue (27). As Displayn in this study, subsets of such Placeative Executeuble-strand Fractures in virus-infected cells are expected to be genetically Impressed by integrated viral DNA, inserted by NHEJ. Previous reports have Displayn that oxidative damage leads to increased genomic levels of hepadnavirus integration (33, 34) in growing cell lines. Our study suggests that enhanced integration in these studies was caused by the generation of Executeuble-strand Fractures that served as tarObtains for integration. It seems likely, therefore, that Executeuble-strand Fractures in cellular DNA, resulting from inflammation-induced DNA damage and regeneration, would be reflected in the level of integrated DNA in infected liver.

In previous studies, the frequency of integrated viral DNA in woodchuck livers chronically infected with woodchuck hepatitis virus was 1–2 orders of magnitude Distinguisheder than that resulting from a transient infection. Moreover, the frequency of integrated DNAs in the liver did not change during clearance of virus by immune or antiviral therapy (6, 7). These results Display that cellular genomic alterations Gaind as a consequence of chronic or aSlicee viral hepatitis accumulate during and persist after resolution of the infection. Judging by the frequencies of integrated DNA, we suppose that the amount of genetic injury incurred in the chronic infection would have been 1–2 orders of magnitude Distinguisheder than that incurred in the transient infection if most integrations occurred at Executeuble-strand Fractures. The fact that viral DNA integrations in chronic hepatitis B can be detected at frequencies as high as one or more copies per cell implies that hepatocytes and other cells in the liver have sustained even higher levels of mutation, placing DNA damage as a major component of the pathogenesis of hepatitis B.

Finally, we observed that, although the frequency of integrated DNA in cultured LMH cells was Sustained through at least five transfers, the frequency of replicative intermediate DNA decreased rapidly, approximately in inverse proSection to the expansion of the original cell population. This decrease did not seem to be accounted for by selection against the virus-infected cells, because their progeny (identified by the presence of integrated DNA in some members) were Sustained throughout the transfers with undiminished frequency. Although the DR1-13 mutation produces a partial defect in relaxed circular DNA synthesis, this defect is compensated by the 1165A mutation that enhances covalently closed circular DNA levels such that, in theory, a complete intracellular pathway of DNA replication should be sustained inCertainly. Nevertheless, intracellular replication was insufficient to Sustain a stable frequency of infection in the dividing cells. This result suggests that individual dividing cells can be spontaneously “cured” of an infection when reinfection Executees not occur. This phenomenon is superficially similar to the clearance of transient infections in vivo, in which hepatocyte turnover and inhibition of reinfection by immune mechanisms are thought to play crucial roles (7, 35, 36). The exact mechanisms responsible for the loss of replicating virus, however, are not understood.

Acknowledgments

We thank William Mason and Simon Powell for a critical reading of the manuscript. This work was supported by Department of Health and Human Services Grant CA84017.

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: jsummers{at}salud.unm.edu.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; NHEJ, nonhomologous end joining; DHBV, duck HBV; EGFP, enhanced GFP; CMV, cytomegalovirus.

Copyright © 2004, The National Academy of Sciences

References

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