cccDNA Levels Research Articles

Prophylactic banding ligation of high risk esophageal varices inpatients on the waiting list for liver transplantation: an interim report

sally effective posttransplant prophylaxis. This study investigates whether this regimen may eradicate latent HBV infection posttransplant. 8 patients transplanted for HBV-cirrhosis were studied. Three were HBeAg+, 4 HBeAg neg/DNA+ and 1 HBeAg neg/DNA neg (HDV co-infection). All received pretransplant lamivudine 100 mg/day for at least 4 weeks and posttransplant lamivudine and IM HBIG 800 units/month. Serum and liver were collected at transplant and 12 months. Serum HBV DNA was detected by PCR (precore primers) and quantified by limiting dilution. Liver HBV cccDNA was detected by primers spanning the region of the HBV genome which is incomplete in non-cccDNA and quantified by real-time PCR using the LightCycler and hybridisation probes. Explant HBV cccDNA levels were higher in HBeAg+ compared to HBeAg negative patients (8.6 45.0 vs 0.03 40.01 copies/human genome equivalents (Geq); p = 0.018) and undetectable in the patient with HDV. After one year, serum in all patients was HBsAg negative, anti-HBs positive (65-1000 iu/ml) and HBV DNA negative. Liver HBV cccDNA levels were low (median 0.006 copies/GEq; range 0--0.018) compared to the explant (p = 0.04). HBV cccDNA levels were low in HBeAg negative (undetectable in 2/5) compared to HBeAg positive patients (0.002 40.002 vs. 0.013 40.004 copies/Geq; p = 0.05). Combination Lamivudine/low-dose IM HBIG suppresses HBV replication posttransplant and may eradicate latent HBV infection in HbeAg negative patients, cccDNA negative patients may be possible candidates for late HBIG withdrawal.

Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections.

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

Acute liver injury following infection with a cytopathic strain of duck hepatitis B virus.

A variant avian hepadnavirus that has been shown to destroy hepatocytes in vitro was found to be cytopathic in vivo. A single amino acid change of glycine to glutamic acid at position 133 (G133E) in the preS protein of duck hepatitis B virus (DHBV) caused an increase in the intranuclear pool of viral covalently closed circular DNA (cccDNA), resulting in a transient elevation of viral replication and eventual hepatocyte destruction. In vivo viral infection with the G133E virus was compared with infection with wild-type virus over a 72-day period. Birds were inoculated with virus at day 2 post-hatch to ensure a high percentage of infected hepatocytes and potential persistence of virus. Birds infected with the G133E virus had increased periportal cellular proliferation and numerous lysed apoptotic hepatocytes following 100% infection of hepatocytes. The liver damage within G133E virus-infected birds subsided over time, resulting in mild chronic hepatitis that was similar to that observed within wild-type virus-infected birds. The subsidence of liver damage in G133E virus-infected birds coincided with a reduction of viral cccDNA to wild-type virus levels in the liver. Our study indicates that maintenance of wild-type levels of viral cccDNA promotes persistence of virus infection by establishing a noncytopathic infection.

Competitionin Vivobetween a Cytopathic Variant and a Wild-Type Duck Hepatitis B Virus

Several examples of human hepatitis B virus strains with enhanced replicationin vitrohave been described. To understand whether this characteristic could be a cause of liver disease, we have studied a variant of the closely related duck hepatitis B virus (DHBV) that had enhanced levels of cccDNA accumulation, previously shown to be cytopathicin vitro,as a model for the pathogenesis of analogous viruses in humans.In vivoliver damage caused by this variant (G133E) occurred only during the first 2 weeks p.i., after which time cccDNA levels and liver histology returned to near normal despite continued virus replication. To determine whether recovery was due to the emergence of noncytopathic revertant, we tested whether wild-type virus would have a selective advantage in competition with the cytopathic mutant in a fully infected liver. In a mixed infection of ducklings with G133E and a small amount of wild-type virus, the wild-type virus was detected as the predominant genotype after recovery of normal liver histology. Two candidate revertant viral genomes were cloned directly from the serum virus of G133E-infected birds after recovery and tested for (i) control of cccDNA levels in primary hepatocyte cultures and (ii) their ability to compete with wild-type virus in a mixed infection. At least one noncytopathic revertant was identified by these two criteria. The results support the conclusion that the recovery from liver damage in G133E-infected ducklings was due to the emergence of spontaneous noncytopathic revertants rather than to host suppression of virus cytotoxicity. The results indicate that acute liver injury may result from infection with a cytopathic hepadnavirus but that such viruses may be rapidly replaced by noncytopathic variants during persistent infection.

Lamivudine Therapy of WHV-Infected Woodchucks

Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5–50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2′-deoxy-3′-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3–12 months, >95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9–12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.

Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.

The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.

N-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes

Hepadnaviruses cause persistent noncytopathic infections of hepatocytes in humans and other animals. Virus replication depends on the pool of viral covalently closed circular DNA (cccDNA) molecules, which serve as transcriptional templates in the nuclei of infected cells. The size of this pool of cccDNA molecules is regulated by the ability of the large envelope protein of the virus to direct newly synthesized viral DNAs into a pathway for viral secretion and thereby inhibit their utilization for viral cccDNA synthesis. In this study, we showed that single amino acid changes in the large envelope protein could cause profound changes in cccDNA levels in transfected permissive cells or in infected cultured hepatocytes. While defects in cccDNA regulation were accompanied by a decrease of enveloped virus production in transfected cells, primary hepatocytes infected by such mutant viruses transiently produced wild-type or higher levels of enveloped virus. Moreover, high levels of cccDNA were always associated with cytopathic effects in the infected hepatocytes. The results demonstrate that the large envelope protein promotes persistent noncytopathic infection of hepatocytes by acting as an overall repressor of virus replication.

Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus

We have used linker scanning and site-directed mutagenesis in an attempt to distinguish among the known functions of the duck hepatitis B virus large envelope protein, p36. We found that linker-encoded amino acid substitutions in at least one region of the pre-S envelope protein p36 produced defects in both the production of enveloped virus and the regulation of covalently closed circular DNA (cccDNA) synthesis. Most linker substitutions, typically in the 5' two-thirds of the pre-S region of the p36 gene did not affect either cccDNA regulation or enveloped virus production but did destroy the infection competence of the enveloped particles produced. Single amino acid substitutions of residues 128 and 131 demonstrated a similar correlation between defects in the ability of p36 to support enveloped virus production and to control cccDNA levels. We concluded from these studies that virus production and cccDNA regulation probably require a common activity of p36.

Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus

The envelope gene of the avian hepadnavirus, duck hepatitis B virus, was mutated in order to dissect the functions of the two major envelope proteins pre-S/S and S. Both envelope proteins were found to be required for virus particle assembly and secretion. The placement of stop codons after each of the first three AUG codons in the pre-S region allowed efficient translational initiation at downstream AUG codons to produce novel N-terminally truncated pre-S/S proteins. These proteins could substitute for pre-S/S protein in the production of enveloped virus production, but not in the production infectious virus. A mutant defective in myristylation of the pre-S/S protein produced reduced amounts of enveloped virus, and this virus was not infectious. Mutants defective in the pre-S/S protein accumulated high levels of covalently closed circular viral DNA (cccDNA) compared with the wild type or with a mutant defective in only the S protein. Hyperamplification of cccDNA resulted in high levels of viral RNA, consistent with the proposed role of cccDNA as the transcriptional template. Myristylation of the pre-S/S protein was not required for control of cccDNA amplification, and mutants that produced N-terminally truncated pre-S/S proteins displayed higher levels of cccDNA. We concluded that the pre-S/S protein, but not the S protein, is required for control of cccDNA amplification and persistent infection.

Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification

Primary duck hepatocytes were infected with a mutant duck hepatitis B virus defective in envelope protein but competent for viral DNA synthesis. Cells infected by this mutant accumulated higher levels of viral covalently closed, circular DNA (cccDNA) than those infected by wild-type virus. The accumulation of high levels of cccDNA was due to a failure of the mutant-infected cells to suppress de novo cccDNA synthesis compared with suppression by cells infected by the wild type. The envelope-defective virus failed to establish a persistent infection in vitro, possibly because of a virus-mediated cell death. Therefore, one or both viral envelope proteins are required for regulation of cccDNA synthesis and for maintenance of persistent infection in vitro.