肝病毒DNA整合的关键是染色体DNA双螺旋的断裂

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Table 3. Frequency of left and right viral-cell junctions per transfected cell

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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 linear and cohesive-end linear DNA. Viral-cell junctions from three separate experiments were excised from agarose gels, DNA-purified, and sequenced (Table 4). Assuming that in situ primed linear DNA was the DHBV substrate, 80% of left junctions had small deletions of sequence (2–58 bp) from the target 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 linear DNA, consistent with a cohesive-end linear 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 linear DHBV substrate by an NHEJ mechanism.

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Table 4. Sequence summary of left and right viral-cell junctions 3 days posttransfection

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Linear DNA Is the Preferential Substrate for Integration. Although the results suggest a linear 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 linear DNA at an 10:1 ratio, and mutant 1165A/DR1-13, which produces circular to linear 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 showed 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 shown 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 linear DNA over relaxed circular DNA as integration substrates. DHBV Is Stably Integrated at Double-Strand Breaks. Although we have shown that one end of DHBV is attached at each genomic terminus at the I-SceI-induced double-strand break, 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 done 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 maintained. In contrast, unintegrated replicative intermediates failed to be maintained, decreasing by 1 order of magnitude with each transfer.

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Table 5. Sequence distribution of integrated wild-type 1165A and mutant 1165A/DR1-13 DHBV

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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, linear double-stranded DNA; ss, single-stranded DNA. The ratios of relaxed circular and linear double-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.

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Table 6. Frequency of viral-cell junctions and replicative intermediates per transfected cell in serial transfers after transfection

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Agarose gels of left EGFP/DHBV junctions from the original population and after five transfers (Fig. 4) showed 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 random 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.

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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 keep cells in logarithmic growth until 27 days (transfer 5) posttransfection. Genomic DNA was isolated and nested PCR performed as described in the Fig. 1 legend. PCR products were electrophoresed through a 1.3% agarose gel and visualized by ethidium-bromide staining. Molecular marker lanes (m) are included.

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[B]Discussion  [/B]

Using DHBV as a model for the Hepadnaviridae family and host chicken LMH cells, we have shown that the presence of a double-strand break in cellular DNA stimulates viral integration at that site >90-fold (Table 3). Cellular double-strand breaks can result from endogenous metabolism (27) or from exogenous damage (28). Repair of such double-strand breaks is important to the survival of eukaryotic cells, because a single unrepaired cellular double-strand break can result in cell death. Double-strand breaks can be repaired by homology- and nonhomology-dependent mechanisms, although repair of double-strand breaks in higher eukaryotes in the absence of any significant homology occurs through NHEJ (28–30). In our model system, an I-SceI-induced double-strand break within the target gene was repaired by either precise (no biological consequences, not measured) or imprecise (Fig. 2) NHEJ. Imprecise NHEJ resulted in mutations (small deletions or insertions) at the site of joining.
When DHBV linear DNA produced by virus replication was present during repair of the double-strand break, ligation of either end of the break to linear viral DNA by NHEJ could be observed, whereas no such ligations were detected in the absence of induced double-strand breaks. Linear DNA was highly preferred over relaxed circular DNA as the ligation substrate. The viral-cell junctions produced at the sites of double-strand breaks were stable through a 105-fold 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 double-strand breaks are the primary source of integrations in infected hepatocytes.

Double-strand breaks in cells can be produced by genotoxic agents (ionizing radiation, oxidative damage, chemical agents), often through conversion of single-strand lesions into double-strand breaks during DNA replication in growing cells. Double-strand breaks 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 double-strand breaks during hepatocyte regeneration in response to cell death of liver tissue (27). As shown in this study, subsets of such putative double-strand breaks in virus-infected cells are expected to be genetically marked by integrated viral DNA, inserted by NHEJ. Previous reports have shown 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 double-strand breaks that served as targets for integration. It seems likely, therefore, that double-strand breaks 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 greater 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 show that cellular genomic alterations acquired as a consequence of chronic or acute 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 greater than that incurred in the transient infection if most integrations occurred at double-strand breaks. 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 maintained through at least five transfers, the frequency of replicative intermediate DNA decreased rapidly, approximately in inverse proportion 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 maintained 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 indefinitely. Nevertheless, intracellular replication was insufficient to maintain 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 does 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.