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. 相似文献
Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.
Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses. 相似文献
Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued. 相似文献
Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision. 相似文献
The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs. 相似文献
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection. 相似文献
For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2. 相似文献
Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization. 相似文献