肝细胞短暂病毒感染后的转变

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Table 3. Integrated WHV DNA in infected liver


Table 4. Enrichment of defective cccDNA after clearance

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Proliferation of Infected Cells Containing Integrated WHV DNA During Resolution of the Infection. As previously reported for duck hepatitis B virus (31), the viral sites of joining of viral DNA to nonviral (cell) DNA were clustered in a region corresponding to the left ends of linear DNA forms (see Fig. 4, which is published as supporting information on the PNAS web site). This distribution of recombination sites is consistent with a nonhomologous end joining reaction between the left end of linear DNA and a cellular DNA end. To determine whether viral cell junctions in infected hepatocytes could serve as unique markers of hepatocyte lineage, we examined the sequences of 127 viral cell junctions identified in 4- and 8-week biopsy samples of infected liver (Table 3 and data not shown) for the appearance of repeated copies of the same sequence. Of 127 viral cell junctions identified, all but 2 were unique (the repeated junctions were found in the same biopsy), indicating that integration of viral DNA did not occur at highly preferred sites. For this reason we assumed that the occurrence of multiple copies of the same virus cell junction would have been derived from the division of a cell that contained that particular junction. To estimate the amount of cell division that occurred in the population of infected cells during the resolution of infection we extracted DNA from small pieces of the liver containing 105 to 106 cells and amplified and sequenced all of the viral cell junctions contained therein. The population of sequences was then examined for the occurrence of unique vs. repeated copies. A summary of these data are shown in Table 5.

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Table 5. Complexity of viral cell junctions Evidence for cell proliferation, i.e., multicopy junctions, can be seen in all three postrecovery liver samples. The data were used to calculate the "complexity" of the population of viral cell junctions, i.e., the number of different sequences detected in the sample divided by the total number of junctions detected. The complexity of the population of cells with integrated DNA was 0.5, 0.5, and 0.6 in the three woodchucks, where a complexity of 1.0 would correspond to a sample with no multicopy junctions. A complexity of <1.0 is direct evidence of hepatocyte proliferation, induced presumably by an equivalent amount of cell death. The presumed turnover giving rise to the observed complexity was related quantitatively to that expected from a process of random cell turnover. Using a model (see Supporting Text) that assumes a starting cell population size corresponding to the total number of viral cell junctions, we calculated the effect of sequential random cell killing and division cycles on the distribution of multicopy junctions and the complexity of the population of junctions after different amounts of turnover. From this model, we estimated that the reduced complexity of the postrecovery samples (0.5, 0.5, and 0.6) corresponded to 1, 1, and 0.7, respectively, complete turnovers of the population of infected cells. Incorporating into this model the effect of the efficiency of detection of viral cell junctions (Table 6, which is published as supporting information on the PNAS web site), we estimated the turnover expected to give rise to the reduced complexity, assuming an efficiency of detection of 50%, as 2, 2, and 1.4 complete turnovers of the population of infected cells. Selection of Defective Viral Genomes During cccDNA Clearance. Viral cccDNA in the nucleus of infected cells consists of a mixture of two types of molecule. The predominant type is derived from a precursor circular double-stranded DNA molecule that is generally a complete copy of the genome and active for expression of all viral proteins. A minor type of cccDNA is derived by nonhomologous end joining reactions between the ends of linear double-stranded DNA, resulting in gross deletions of parts of the viral genome extending in both directions from near the start site of pregenome transcription (32–34). Most such genomes are predicted to be defective in replication and protein expression and may be recognized by their reduced size. We observed that, after resolution of the infection in the livers of transiently infected woodchucks, the residual amount of cccDNA was greatly enriched in defective molecules. Single copies of cccDNA molecules were amplified by nested PCR of samples diluted so that individual reactions contained an average of less than one molecule, and the PCR products were analyzed by agarose gel electrophoresis to identify products derived from grossly deleted cccDNA templates. An example of a collection of such products amplified from a 16-week sample is shown in Fig. 3B. Grossly deleted molecules whose mobility was shifted constituted up to one-half of all molecules in the 16-week sample but were infrequent in the 4-week biopsies. A summary of the data for the three woodchucks is seen in Table 4. It is clear that the reduction of full-length cccDNA molecules was substantially greater than the reduction in defective molecules. One explanation for this selective destruction of full-length cccDNA would be that cells containing full-length molecules were removed by an antigen-dependent process, according to their ability to produce the full array of viral proteins. An alternative explanation would be that cccDNA molecules were destroyed by a process that depended on their physical target size.

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[B]Discussion  [/B]


The transient WHV infection seen after inoculation of three adult woodchucks followed the typical course described by us and others (9–11). Infection occurring in >95% of hepatocytes was cleared over a period of 4–8 weeks without any apparent life-threatening crisis and with levels of inflammation, necrosis, and apoptosis in two animals that was similar to that previously reported, whereas in one animal (WC377) five times higher levels of apoptosis were observed. Histological changes in the liver (data not shown) ranged from relatively mild (WC378) to moderate or severe (WC380 and WC377, respectively). Evidence for hepatocyte destruction, seen in biopsies from the clearance phase, consisted of necrosis, apoptosis, and regeneration. Viral markers in the liver, i.e., replicative intermediates, cccDNA, and infected cells, decreased approximately in parallel, as would be expected if infected cells were being removed primarily by killing during the inflammatory process. However, it was not apparent from the histological observations how to estimate the total amount of killing that occurred during the resolution phase of the infection.

To address this question, a quantitative assay for integrated DNA was developed to follow the fate of the infected cell population, using integrated DNA as a genetic marker specific to that population of cells. Had the infected cells been killed and replaced by uninfected stem cells, the integrated DNA characteristic of the infected cell population would have disappeared along with the infected cells. Because we did not observe such a gross disappearance of integrated DNA, we concluded that a substantial fraction of the cells of the recovered liver were derived from the population of infected cells. This result allowed us to estimate the amount of proliferation that these cells in the recovered liver had undergone during the recovery phase. Our estimate was derived from measurements of the distribution of unique and multicopy viral cell junctions within small samples of liver. The estimate depends on the validity of two assumptions. The first is that all of the progeny of a single dividing hepatocyte were found in the same immediate vicinity, in a cluster, and therefore that entire clusters were recovered in the small piece of liver analyzed. BrdUrd labeling experiments in the three woodchucks revealed that 42–85% of cells labeled after a single injection could be found as pairs in thin sections after 4 weeks (9). This result implies that multiple cell divisions would result in clustering of progeny. If such clusters did exist their dimensions would be only a fraction of the total dimensions of the piece of liver analyzed. For example, even a large clonal cluster of 100 hepatocytes would make up only 1/1,000 of the volume of a sample containing 105 cells, making it improbable that dissection of the liver tissue would result in dissection of the cluster.

A second assumption of our estimate of proliferation is that infected cells with integrated DNA behaved in all ways similar to infected cells without integrated DNA. At least, in the initial stages of resolution, antigen-dependent cell killing would be directed primarily by presentation of antigens produced as a result of virus replication rather than from integrated DNA, a minor template in the infected cell that may or may not produce sufficient antigenic peptides to be presented by the cell in competition with endogenous peptides. In addition, the target sites for integration are numerous, and we expect that integration into only a small fraction of these sites would affect cell growth or viability. This assumption implies that the population of cells with integrated DNA is representative of the whole population of infected cells.

Proliferation was likely in response to hepatocyte death. During the course of the infection each of the woodchucks actually declined in body mass (data not shown), arguing against normal growth and in favor of hepatocyte turnover as a source of proliferation. The amount of turnover estimated by this method depends on the efficiency with which any particular viral cell junction was detected. The turnover values of 0.7 and 1 were calculated by assuming that every type of viral cell junction that was detected in the assay was detected with 100% efficiency. It would be difficult to argue that we detected each viral cell junction with 100% efficiency, however, because failure to prepare and amplify a viral cell junction could occur at four different steps: extraction of cell DNA, circularization, recovery of templates, and amplification (assuming that all three restriction enzyme digestions were 100% complete). For example, although each step may be as much as 85% efficient, the combined efficiency of detection would be 52%, and the efficiency may be different for each viral cell junction. With this in mind, the estimated turnover must be considered to be the minimum estimate, but the actual turnover was likely to be higher.

Past lack of evidence of a high degree of hepatocyte turnover during clearance of infection has been used to support a significant role for noncytolytic "purging" of virus infection, including cccDNA, from individual cells (13, 35). Elimination of cccDNA from infected cells is crucial to cell curing because it is cccDNA that is the source of sustained virus production. Our analysis showed that cumulative hepatocyte turnover was extensive and similar in all three animals, irrespective of the histological evaluations. Therefore, consideration of an alternative mechanism must be revived (21), namely that a large fraction of infected hepatocytes was killed, and uninfected cells arose from the proliferation of remaining infected hepatocytes. This scenario requires that de novo infection of uninfected hepatocytes be prevented, a role that might be ascribed to the antiviral effects of inflammatory cytokines.

Studies of antiviral therapy with inhibitors of viral DNA synthesis in natural infections of woodchucks and ducks (36–40) have shown that inhibition of viral DNA synthesis alone can result in decreases in the number of infected hepatocytes, in the apparent absence of a role for the immune system. These reductions are thought to occur by elimination of infected hepatocytes through cell turnover and compensatory cell proliferation, leading to reduction of cccDNA copy number and eventual segregation and accumulation of uninfected cells. Significantly, in one study in duck hepatitis B virus-infected ducks treated with an antiviral drug that caused liver cell toxicity (41), the loss of infected hepatocytes was markedly accelerated, suggesting that cell death and proliferation are key components in virus elimination. Although cytokine-induced purging of virus from infected cells would be a truly noncytolytic process, the segregation and accumulation of uninfected hepatocytes through repeated cell division could not be considered noncytolytic because it is cell death that drives the proliferation. Our data do not distinguish between these two alternatives because both scenarios predict that integrated DNA would survive clearance, and both scenarios provide for hepatocyte turnover.

Thus, although resolution of the WHV infections was accompanied by an amount of death and regeneration equivalent to the entire liver or more, whether this turnover was essential for resolution of the infection even in the presence of antiviral cytokines is unclear. Our data argue, however, that the amount of killing and regeneration involved must have played a significant role in the direct elimination of infected cells (21).

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  Acknowledgements  

We thank Darren Miller for technical support and Robert Lanford for a critical reading of the manuscript. A.R.J. was supported by a project grant from the National Health and Medical Research Council of Australia. J.S. and W.S.M. were supported by grants from the National Institutes of Health.



    Footnotes  

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 1, 2001.

Abbreviations: cccDNA, covalently closed circular DNA; HBV, hepatitis B virus; WHV, woodchuck hepatitis virus; p.i., postinoculation; SDH, sorbital dehydrogenase; PCNA, proliferating cell nuclear antigen; TE, Tris/EDTA.

To whom correspondence should be addressed. E-mail: [email protected].

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