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

Abstract

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...

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B

“…As HBV polymerase inhibitors do not have a direct effect on cccDNA formation, the decreased intrahepatic cccDNA levels are supposed to be derived from the lack of sufficient recycling of viral nucleocapsids to the nucleus because of the strong inhibition of viral DNA synthesis in the cytoplasm and less incoming viruses from the blood. Furthermore, experiments with HBVreplicating transgenic mice 9 and chimpanzees 10,11 have shown that inflammatory cytokines can efficiently suppress viral replication through noncytolytic immune-mediated mechanisms that also contribute to diminishing the cccDNA reservoirs of infected cells. However, these processes may require a very long time, given the long half-life of both hepatocytes and cccDNA in the absence of cell division.…”

Immune selection during chronic hepadnavirus infection

“…Since hepadnavirus infection is not cytopathic, elimination of hepadnavirus-infected hepatocytes requires immune attack by virus antigen-specific CTLs, leading to the elimination of cccDNA directly by hepatocyte death or indirectly during compensatory hepatocyte proliferation [6]. Whether significant cccDNA loss occurs in the absence of hepatocyte turnover (i.e., hepatocyte death and compensatory proliferation) remains unclear [7]. We and others have reported that resolution of transient hepadnavirus infections involves immunemediated attack on large numbers of hepatocytes and significant turnover of the hepatocyte population [6,8], with turnover of up to 10% of the hepatocyte population per day [6,9,10].…”

“…As in humans, some of the foci of virus-free hepatocytes in the woodchuck liver clearly represent FAH, often considered to be preneoplastic [34][35][36][37][38], whereas virus-free hepatocytes in other foci appear to have a normal morphology [36]. In contrast, virtually all hepatocytes can be infected during a typical transient WHV infection [7,10].…”

Dynamics of hepatitis B virus clearance in chimpanzees

“…On the one hand, it has been amply demonstrated that termination of acute hepadnavirus infections in the woodchuck (7,12) and chimpanzee (9)(10)(11) systems is associated with the destruction of infected cells and hepatocellular regeneration (7)(8)(9)(10)(12)(13)(14)(15). On the other hand, it is equally clear from chimpanzee (9)(10)(11) and transgenic mouse (16,17) experiments, that HBV replication is strongly inhibited by the antiviral effects of inflammatory cytokines produced by HBVspecific CD8 ϩ T cells when they recognize antigen in the infected or transgenic liver. Furthermore, it has been shown that viral clearance in acutely infected chimpanzees is heralded by a sharp drop in the HBV DNA content of the liver, which depends on the influx of IFN-␥-producing CD8 ϩ T cells into the liver (10), but which precedes and is out of proportion to the amount of liver disease (10).…”

“…Although it is generally agreed that viral clearance and disease pathogenesis during HBV infection are mediated by the immune response (2-6), the extent to which cytopathic and noncytopathic CD8ϩ T cell antiviral functions contribute to these events is a matter of some debate (7-11). On the one hand, it has been amply demonstrated that termination of acute hepadnavirus infections in the woodchuck (7, 12) and chimpanzee (9-11) systems is associated with the destruction of infected cells and hepatocellular regeneration (7)(8)(9)(10)(12)(13)(14)(15). On the other hand, it is equally clear from chimpanzee (9-11) and transgenic mouse (16,17) experiments, that HBV replication is strongly inhibited by the antiviral effects of inflammatory cytokines produced by HBVspecific CD8 ϩ T cells when they recognize antigen in the infected or transgenic liver.…”