Initiation and termination of duck hepatitis B virus DNA synthesis during virus maturation

The template for hepadnavirus plus-strand DNA synthesis is a terminally redundant minus-strand DNA. An intramolecular template switch during plus-strand DNA synthesis, which permits plus-strand DNA elongation, has been proposed to be facilitated by this terminal redundancy, which is 7 to 9 nucleotides long. The aim of this study was to determine whether the presence of identical copies of the redundancy on the minus-strand DNA template was necessary and/or sufficient for the template switch and at what position(s) within the redundancy the switch occurs for duck hepatitis B virus. When dinucleotide insertions were placed within the copy of the redundancy at the 3 end of the minus-strand DNA template, novel sequences were copied into plus-strand DNA. The generation of these novel sequences could be explained by complete copying of the redundancy at the 5 end of the minus-strand DNA template followed by a template switch and then extension from a mismatched 3 terminus. In a second set of experiments, it was found that when one copy of the redundancy had either three or five nucleotides replaced the template switch was inhibited. When the identical, albeit mutant, sequences were restored in both copies of the redundancy, template switching was not necessarily restored. Our results indicate that the terminal redundancy on the minus-strand DNA template is necessary but not sufficient for template switching.

Section: Discussionsupporting

confidence: 91%

“…For continuation of plus-strand DNA synthesis, an intramolecular template switch must occur. It has been proposed that the 7-to 9-nucleotide terminal redundancy (called r) facilitates the template switch during synthesis of plus-strand DNA (9,17,25) (Fig. 1, part 7).…”

mentioning

confidence: 99%

Sequence identity of the terminal redundancies on the minus-strand DNA template is necessary but not sufficient for the template switch during hepadnavirus plus-strand DNA synthesis

Loeb

Gulya

Tian

1997

“…Examination of the 5' fragments of the two strands generated by EcoRI digestion (Fig. 6c and d) revealed the presence of fragments of the expected sizes for both wild-type and class II mutants (2.54 kb for the minus strand and 0.53 kb for the plus strand), indicating that initiation of both strands occurred at the proper sites (12).…”

Section: Resultsmentioning

confidence: 93%

A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly

Summers

1991

Mutations introduced into the capsid gene of duck hepatitis B virus (DHBV) were tested for their effects on viral DNA synthesis and assembly of enveloped viruses. Four classes of mutant phenotypes were observed among a series of deletions of covering the 3' end of the capsid open reading frame. Class I mutant capsids were able to support normal single-stranded and relaxed circular viral DNA synthesis; class II mutant capsids supported normal single-stranded DNA synthesis but not relaxed circular DNA synthesis; class III mutant capsids resembled class II capsids, but viral DNA synthesis was inhibited 5to 10-fold; and class IV capsids were severely restricted in their ability to support viral DNA synthesis. Class I capsids were assembled into enveloped virions, but class II, III, and IV capsids were not. Viral DNA synthesized inside class II capsids was normal with respect to minus-strand DNA initiation, plus-strand DNA initiation, and circularization of the DNA, but plus strands failed to be elongated to mature 3-kb DNA. The results suggest that a function of the capsid protein specifically required for viral DNA maturation is also required for assembly of nucleocapsids into envelopes. Thus, class II mutants appear to be defective in the appearance of the "packaging signal" for virus assembly (

“…The RNA genome intermediates (pregenomes) are transcribed from a pool of covalently closed circular viral DNA (cccDNA) in the nuclei of infected cells. Reverse transcription takes place in the cytoplasm through a process that uses a combination of template switches (3,14,15,(20)(21)(22)30) to produce relaxed circular double-stranded DNAs, which can be transported to the nucleus to form additional copies of cccDNA (29,32). Progeny cccDNAs retain the precise sequence of the parental cccDNA.We recently reported a second, minor pathway of DNA replication via reverse transcription in the avian hepadnavirus duck hepatitis B virus (DHBV).…”

mentioning

confidence: 99%

Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver

Yang

Mason

Summers

1996

Self Cite

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesiveend region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.Hepadnaviruses are small DNA-containing viruses that replicate their DNA genomes through reverse transcription of RNA (17,24). The RNA genome intermediates (pregenomes) are transcribed from a pool of covalently closed circular viral DNA (cccDNA) in the nuclei of infected cells. Reverse transcription takes place in the cytoplasm through a process that uses a combination of template switches (3,14,15,(20)(21)(22)30) to produce relaxed circular double-stranded DNAs, which can be transported to the nucleus to form additional copies of cccDNA (29,32). Progeny cccDNAs retain the precise sequence of the parental cccDNA.We recently reported a second, minor pathway of DNA replication via reverse transcription in the avian hepadnavirus duck hepatitis B virus (DHBV). In this pathway, linear doublestranded DNA produced as a result of a failure to prime second-strand (plus-strand) DNA synthesis at the correct location was efficiently converted to cccDNAs by nonhomologous recombination near the two ends of the linear DNA (33). cccDNA molecules resulting from such recombination thus acquired sequence alterations at the site of the recombination and therefore were not identical to the parental cccDNA. Some cccDNA molecules produced from linear DNA were able to produce pregenomes. However, as a result of the mutation created by nonhomologous recombination, most of the pregenomes gave rise only to linear double-stranded DNAs, which recombined to produce further successive generations of cccDNA. We called this process "illegitimate replication," because each such generation of cccDNA differed in sequence from its parent molecule at the site of nonhomologous recombination. We speculated that the low level of virus expression in cells carrying out illegitimate replication might provide such cells a survival advantage...