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20楼
发表于 2004-8-25 01:59
[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. |
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