Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription

Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ε RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.© 2007 The WJG Press. All rights reserved. Dieter Glebe, PhD, Series EditorPO Box 2345, Beijing 100023, China World J Gastroenterol 2007 January 7; 13(1): 48-64 www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327 wjg@wjgnet.com © 2007 OVERVIEW OVER THE HEPADNAVIRAL GENOME REPLICATION CYCLEReplication of the hepadnaviral genome can broadly be divided into three phases ( Figure 1): (1) Infectious virions contain in their inner icosahedral core the genome as a partially double-stranded, circular but not covalently closed DNA of about 3.2 kb in length (relaxed circular, or RC-DNA); (2) upon infection, the RC-DNA is converted, inside the host cell nucleus, into a plasmid-like covalently closed circular DNA (cccDNA); (3) from the cccDNA, several genomic and subgenomic RNAs are transcribed by cellular RNA polymerase Ⅱ; of these, the pregenomic RNA (pgRNA) is selectively packaged into progeny capsids and is reverse transcribed by the co-packaged P protein into new RC-DNA genomes. Matured RC-DNA containing-but not immature RNA containingnucleocapsids can be used for intracellular cccDNA amplification, or be enveloped and released from the cell as progeny virions. Below we discuss these genome conversions, with emphasis on the reverse transcription step, and particularly its unique initiation mechanism. RC-DNA TO cccDNA CONVERSIONPersistent viral infections require tha...

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“…More details than is possible to show here can be found in informative schematic representations in references [144,160] .…”

Section: ( + ) -S T R a N D D N A S Y N T H E S I S A N D Circularizamentioning

confidence: 99%

“…Using quantitative genetic techniques, the Loeb laboratory has defined, mostly in DHBV, several such cis-elements on the (-)-DNA [144,[156][157][158][159][160][161] , e.g. 3E, M, and 5E, which are located at both termini and in the middle of pgRNA.…”

Section: ( + ) -S T R a N D D N A S Y N T H E S I S A N D Circularizamentioning

confidence: 99%

Hepatitis B virus replication

Beck

Nassal

2007

WJG

410

382

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“…Therefore, complementarity between the nascent minus-strand and acceptor sequence on the pgRNA is not sufficient for first-strand template switch. Studies of plusstrand DNA synthesis in duck HBV have shown that cis-acting sequences are present on minus-strand DNA that contribute to the subsequent plus-strand synthesis (11,12). It is reasonable to postulate that cis-acting sequences are present on pgRNA that facilitate minus-strand DNA synthesis in HBV.…”

mentioning

confidence: 99%

Base Pairing between the 5′ Half of ε and a cis -Acting Sequence, Φ, Makes a Contribution to the Synthesis of Minus-Strand DNA for Human Hepatitis B Virus

Abraham

Loeb

2006

J Virol

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Synthesis of minus-strand DNA of human hepatitis B virus (HBV) can be divided into three phases: initiation of DNA synthesis, the template switch, and elongation of minus-strand DNA. Although much is known about minus-strand DNA synthesis, the mechanism(s) by which this occurs has not been completely elucidated. Through a deletion analysis, we have identified a cis-acting element involved in minus-strand DNA synthesis that lies within a 27-nucleotide region between DR2 and the 3 copy of DR1. A subset of this region (termed ⌽) has been hypothesized to base pair with the 5 half of (H. Tang and A. McLachlan, Virology, 303:199-210, 2002). To test the proposed model, we used a genetic approach in which multiple sets of variants that disrupted and then restored putative base pairing between the 5 half of and ⌽ were analyzed. Primer extension analysis, using two primers simultaneously, was performed to measure encapsidated pregenomic RNA (pgRNA) and minus-strand DNA synthesized in cell culture. The efficiency of minus-strand DNA synthesis was defined as the amount of minus-strand DNA synthesized per encapsidation event. Our results indicate that base pairing between ⌽ and the 5 half of contributes to efficient minus-strand DNA synthesis. Additional results are consistent with the idea that the primary sequence of ⌽ and/or also contributes to function. How base pairing between ⌽ and contributes to minus-strand DNA synthesis is not known, but a simple speculation is that ⌽ base pairs with the 5 half of to juxtapose the donor and acceptor sites to facilitate the first-strand template switch.

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“…The final product is a partially doublestranded, relaxed circular DNA (rcDNA). Occasionally, one of the translocation steps fails (in situ priming), resulting in a double-stranded linear molecule (17,19,24,25).The C-terminal domain of the core protein plays a crucial role in viral replication. It is rich in arginine residues and contains three serine phosphorylation sites.…”

mentioning

confidence: 99%

“…Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17). Thus, both ends must be accessible to the encapsidated P protein.…”

mentioning

confidence: 99%

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

Köck

Nassal

Deres

et al. 2004

J Virol

125

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The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3 truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.Hepatitis B virus (HBV) is an important human pathogen accounting for about one million deaths each year (13). The viral genome, as present in infectious virions, is a 3.2-kbp circular, partially double-stranded DNA (dsDNA) molecule that contains overlapping reading frames encoding the core protein, a reverse transcriptase (P), three surface proteins, and the X protein (20,25).Viral replication involves reverse transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits (4-6, 30). Specific encapsidation of pgRNA occurs via binding of P protein to a 5Ј-proximal RNA stem-loop structure, termed ε. Subgenomic RNAs lack the 5Ј ε and are excluded from encapsidation. Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17). Thus, both ends must be accessible to the encapsidated P protein. The final product is a partially doublestranded, relaxed circular DNA (rcDNA). Occasionally, one of the translocation steps fails (in situ priming), resulting in a double-stranded linear molecule (17,19,24,25).The C-terminal domain of the core protein plays a crucial role in viral replication. It is rich in arginine residues and contains three serine phosphorylation sites. Though dispensable for particle assembly, it is required for pgRNA packaging (2,3,7,10). Further, the phenotypes of core pr...

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Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription

Cited by 34 publications

References 22 publications

Hepatitis B virus replication

Hepatitis B virus replication

Base Pairing between the 5′ Half of ε and a cis -Acting Sequence, Φ, Makes a Contribution to the Synthesis of Minus-Strand DNA for Human Hepatitis B Virus

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

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