Conditional Replication of Duck Hepatitis B Virus in Hepatoma Cells

206

209

We have previously shown that hepatitis B virus (HBV) replication is controlled by noncytolytic mechanisms that depend primarily on the effector functions of the CD8 ؉ T cell response, especially the production of IFN-␥ in the liver. The mechanisms that control the nuclear pool of viral covalently closed circular DNA (cccDNA) transcriptional template of HBV, which must be eliminated to eradicate infection, have been difficult to resolve. To examine those mechanisms, we quantitated intrahepatic HBV cccDNA levels in acutely infected chimpanzees whose virological, immunological, and pathological features were previously described. Our results demonstrate that the elimination kinetics of the cccDNA are more rapid than the elimination of HBV antigen-positive hepatocytes during the early phase of viral clearance, and they coincide with the influx of small numbers of IFN-␥ producing CD8 ؉ T cells into the liver. In contrast, terminal clearance of the cccDNA is associated with the peak of liver disease and hepatocellular turnover and with a surge of IFN-␥ producing CD8 ؉ T cells in the liver. Collectively, these results suggest that cccDNA clearance is a two-step process mediated by the cellular immune response. The first step reduces the pool of cccDNA molecules noncytolytically, probably by eliminating their relaxed circular DNA precursors and perhaps by destabilizing them. The second step enhances this process by destroying infected hepatocytes and triggering their turnover. Surprisingly, despite this multipronged response, traces of cccDNA persist indefinitely in the liver, likely providing a continuous antigenic stimulus that confers lifelong immunity.H epadnaviruses are small enveloped hepatotropic DNA viruses containing a relaxed circular DNA genome, which is converted into a covalently closed circular DNA (cccDNA) episome in the nucleus of infected cells (1). The cccDNA molecule serves as the transcriptional template for production of viral RNAs that are exported to the cytoplasm and encode the viral structural and nonstructural proteins, including the reverse transcriptase͞DNA polymerase. Reverse transcription of the viral pregenomic (pg)RNA and second-strand DNA synthesis occurs in the cytoplasm within viral capsids formed by the HBV core protein (HBcAg). Capsids containing the mature relaxed circular DNA genome are either enveloped and exported as virions, or they deliver their DNA content to the nucleus, thereby amplifying the pool of cccDNA molecules to 10-50 copies per cell (2-5). The factors that regulate the cccDNA content of infected cells are not very well understood. In the duck hepatitis B virus (DHBV) model, the cccDNA level is regulated by the viral envelope proteins (6). Evidence from the woodchuck hepatitis virus (WHV) system suggests that cccDNA clearance is tightly associated with the destruction and turnover of infected hepatocytes (7,8). The possibility that cccDNA may also be susceptible to control by immune-mediated noncytolytic mechanisms is an open question at this time, at least partiall...

Section: Discussionsupporting

confidence: 57%

“…Experiments in DHBV-infected primary duck hepatocytes and in a conditional DHBV replication system suggest that the half-life could be as short as 2-5 days (21,22). However, cccDNA has been reported to be very stable with a half-life of at least 33-50 days in WHV-infected woodchucks and hepatocyte cultures (3,23).…”

Section: Discussionmentioning

confidence: 99%

Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees

Wieland

Spangenberg²,

Thimme³

et al. 2004

206

209

Section: Hemotherapy Of Hepatitis B Virus (Hbv) Infections Hasmentioning

confidence: 99%

“…It has been proposed that the removal of cccDNA occurs by hepatocyte turnover, and that dilution of cccDNA copy numbers in dividing hepatocytes results in eventual segregation of uninfected progeny cells (12). Alternatively, uninfected hepatocytes may arise by differentiation of uninfected progenitors (13) or be generated through a slow loss of cccDNA from infected hepatocytes (14,15).During hepadnavirus infection, a small fraction of hepatocytes undergo recombination with viral DNA molecules in the nucleus and acquire an integrated viral DNA that may be stably retained in that hepatocyte lineage (16)(17)(18)(19)(20). The common precursor to integrated viral DNA is a full-length linear double-stranded DNA, with a left end just upstream of the viral core gene, that is formed as a by-product of viral genome synthesis (21,22).…”

mentioning

confidence: 99%

Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy

Summers

Mason

2003

We determined the frequency of integrated viral DNA in the livers of three woodchucks chronically infected with the woodchuck hepatitis virus before and during 30 weeks of therapy with the nucleoside analog L-FMAU [1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl)uracil, clevudine]. We found that although viral covalently closed circular DNA declined 20-to 100-fold, integrated viral DNA showed no discernable decrease over the course of treatment. Thus, chemotherapeutic clearance of covalently closed circular DNA did not involve the replacement of the infected hepatocyte population with uninfected progenitors, but rather, uninfected hepatocytes in the treated liver were derived from the infected hepatocyte population. The frequency of integrated DNA in chronically infected woodchucks was found to be 1 or 2 orders of magnitude higher than that in transiently infected woodchucks, implying that integration and other genomic damage accumulate over the duration of infection. Our results indicate that genetic changes from this damage remain in the liver even while virus infection is cleared and argue for early antiviral intervention in chronic hepatitis. C hemotherapy of hepatitis B virus (HBV) infections hasfocused primarily on a strategy of inhibiting viral DNA synthesis through the use of nucleoside analogs that are highly specific for the viral DNA polymerase (1-9). Treatment of patients with such inhibitors can be remarkably effective in reducing viremias to very low levels (2, 3). The reduction of viremia has been shown to occur with biphasic kinetics, with the first phase of rapid reduction attributed to the inhibition of virus production by infected cells, combined with viral clearance from the blood by mechanisms probably unrelated to the action of the drug. The slower second phase has been attributed to the elimination of infected cells from the liver, by either normal or immune-enhanced mechanisms, and the accumulation of uninfected cells (10, 11). The gradual replacement of infected by uninfected hepatocytes has been observed during antiviral therapy of woodchucks chronically infected with the woodchuck hepatitis virus (WHV), an animal model for HBV (7). In 1-(2-f luoro-5-methyl-beta, L-arabinofuranosyl)uracil (L-FMAU)-treated woodchucks, the onset of the reduction in infected cells lags behind the decline of the covalently closed circular DNA (cccDNA), the viral transcriptional template in the nucleus. It has been proposed that the removal of cccDNA occurs by hepatocyte turnover, and that dilution of cccDNA copy numbers in dividing hepatocytes results in eventual segregation of uninfected progeny cells (12). Alternatively, uninfected hepatocytes may arise by differentiation of uninfected progenitors (13) or be generated through a slow loss of cccDNA from infected hepatocytes (14,15).During hepadnavirus infection, a small fraction of hepatocytes undergo recombination with viral DNA molecules in the nucleus and acquire an integrated viral DNA that may be stably retained in that hepatocyte lineage (16)(17)(18)(1...

“…Virion-derived NCs were then purified from lysed virions by velocity gradient centrifugation. Intracellular immature and mature NCs were isolated from both the D2 and surfacenegative Dstet5 cells (33).…”

Section: Separation and Purification Of Nc Species Of Different Maturmentioning

confidence: 99%

Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids

Perlman

Berg

O’Connor

et al. 2005

Self Cite

113

172

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called ''mature'' NCs) are enveloped by the viral envelope proteins and secreted as virions; ''immature'' NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.hepatitis B virus ͉ viral maturation ͉ posttranslational modification ͉ tandem mass spectrometry ͉ vibrational cooling MALDI-Fourier transform MS H epatitis B viruses (HBVs), or hepadnaviruses, are pararetroviruses that replicate their DNA genome via an RNA intermediate (pregenomic RNA, pgRNA) by reverse transcription (1, 2). Similar to classical retroviruses, hepadnaviruses begin assembly with the formation of intracellular nucleocapsid (NC) particles that specifically package viral pgRNA. However, they undergo reverse transcription before membrane envelopment of NCs and secretion as virions, rather than subsequent to entry into new target cells, like classical retroviruses (2, 3). This replication strategy is manifested in the hepadnaviral maturation phenomenon: secreted hepadnaviral virions contain only the mature, double-stranded relaxed circular (or the mature, doublestranded linear) DNA species, whereas intracellular pools of NCs contain a mix of nucleic acid species, including the pgRNA and DNA species from all of the intermediate stages of reverse transcription (2).The maturation phenomenon implies that mature DNAcontaining NCs may be selectively enveloped and secreted. It was proposed that DNA synthesis and NC envelopment may be coupled through a maturation signal (2). Strong support for this maturation signal model has been obtained (4-6), particularly through the use of a synchronized duck HBV (DHBV) replication system (7,8)...