Small DNA Hairpin Negatively Regulates In Situ Priming during Duck Hepatitis B Virus Reverse Transcription

2003

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Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3 end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. A small DNA hairpin has been shown to form near the 3 end of the minus-strand DNA overlapping the direct repeat 1 in avihepadnaviruses. Previously we showed that this hairpin is involved in discriminating between two mutually exclusive pathways for the initiation of plus-strand DNA synthesis. In its absence, the pathway leading to production of duplex linear DNA is favored, whereas primer translocation is favored in its presence, apparently through the inhibition of in situ priming. Circularization involves transfer of the nascent plus strand from the 5 end of the minus-strand DNA to the 3 end, where further elongation can lead to production of RC DNA. Using both genetic and biochemical approaches, we now have found that the small DNA hairpin in the duck hepatitis B virus (DHBV) makes a positive contribution to circularization. The contribution appears to be through its impact on the conformation of the acceptor site. We also identified a unique DHBV variant that can synthesize RC DNA well in the absence of the hairpin. The behavior of this variant could serve as a model for understanding the mammalian hepadnaviruses, in which an analogous hairpin does not appear to exist.Hepadnaviruses are a family of small, enveloped DNA viruses that display a narrow host range, a tropism for the liver, and are capable of both acute and chronic infections in their hosts. The human hepatitis B virus, a member of the family, is a major worldwide health problem exposing ca. 350 million chronic carriers to an increased risk of developing hepatocellular carcinoma (11). Viral replication is necessary for maintenance of the chronic carrier state; understanding this replication is critical for limiting disease in carriers. The duck hepatitis B virus (DHBV) has proven a powerful model for studying many aspects of hepadnaviral biology, including replication (reviewed in reference 3). Although divergent at the level of nucleic acid sequence, all hepadnaviruses share a similar genetic organization and general strategy of replication.Three template switches are integral to viral replication: one during synthesis of the minus-strand DNA and two during synthesis of plus-strand DNA during the production of the relaxed circular (RC) DNA genome. Minus-strand DNA synthesis occurs via reverse transcription of an RNA intermediate, the pregenomic RNA (23). Upon completion of the minusstrand DNA, there are two mutually exclusive pathways for plus-strand DNA synthesis. The predominant pathway gives rise to RC DNA (Fig. 1A, panels a to d), while the alternative pathway yields duplex linear (DL) DNA (Fig. 1A, panel e). Both pathways use the sam...

supporting

confidence: 48%

Section: Methodsmentioning

confidence: 99%

The Conformation of the 3′ End of the Minus-Strand DNA Makes Multiple Contributions to Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus

Habig

2003

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“…The primer extension reaction mixtures contained 1ϫ Thermopol buffer [10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO 4 , 0.1% Triton X-100 (New England Biolabs)], 0.2 mM (each) deoxynucleoside triphosphates, 2 U of Vent exo Ϫ DNA polymerase (New England Biolabs), ϳ0.8 pmol of end-labeled primer, and 2 ng of digested HBV plasmid DNA. Sequencing ladders were generated using individual primers and HBV plasmid DNA as the template, as described previously (8). Primer extension products were electrophoresed on a 5% denaturing polyacrylamide gel.…”

Section: Molecular Clonesmentioning

confidence: 99%

“…The reaction conditions described above were empirically determined. HBV plasmid DNA was digested with two restriction enzymes, and primer extension was performed as previously described (8). Primer extension reactions on plasmid DNA controls produced extension products from both primers.…”

Section: Molecular Clonesmentioning

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

2006

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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.

“…The chicken hepatoma cell line LMH was cultured as described previously (10). DNA transfections were performed by the calcium phosphate precipitation method.…”

Section: Methodsmentioning

confidence: 99%

Chimeras of Duck and Heron Hepatitis B Viruses Provide Evidence for Functional Interactions between Viral Components of Pregenomic RNA Encapsidation

Ostrow

2004

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Packaging of hepadnavirus pregenomic RNA (pgRNA) into capsids, or encapsidation, requires several viral components. The viral polymerase (P) and the capsid subunit (C) are necessary for pgRNA encapsidation. Previous studies of duck hepatitis B virus (DHBV) indicated that two cis-acting sequences on pgRNA are required for encapsidation: , which is near the 5 end of pgRNA, and region II, located near the middle of pgRNA. Later studies suggested that the intervening sequence between these two elements may also make a contribution. It has been demonstrated for DHBV that interacts with P to facilitate encapsidation, but it is not known how other cis-acting sequences contribute to encapsidation. We analyzed chimeras of DHBV and a related virus, heron hepatitis B virus (HHBV), to gain insight into the interactions between the various viral components during pgRNA encapsidation. We learned that having and P derived from the same virus was not sufficient for high levels of encapsidation, implying that other viral interactions contribute to encapsidation. Chimeric analysis showed that a large sequence containing region II may interact with P and/or C for efficient encapsidation. Further analysis demonstrated that possibly an RNA-RNA interaction between the intervening sequence and region II facilitates pgRNA encapsidation. Together, these results identify functional interactions among various viral components that contribute to pgRNA encapsidation.Hepadnaviruses, also known as hepatitis B viruses, are DNA viruses that replicate via reverse transcription of an RNA pregenome (pgRNA) (9). The pgRNA is transcribed from the hepadnaviral plasmid present in the nucleus, called covalently closed circular DNA (5, 28). The pgRNA can be translated to produce the viral polymerase, P, and the capsid subunit, C (8, 26). Within the cytoplasm, the pgRNA and P are packaged into capsids in a process known as RNA encapsidation (1, 11). Capsids containing pgRNA and P are competent for reverse transcription of the pgRNA (27). Once DNA synthesis is complete, capsids can be secreted from the cell to produce virions.There are several viral components important for pgRNA encapsidation (Fig. 1). The C protein is the subunit of the capsid that contains pgRNA. C protein also might contribute to pgRNA encapsidation directly (21). The P protein is required for pgRNA encapsidation, but the reverse transcriptase or DNA-priming activity of P is not required (1,11,29,30). Also, the pgRNA contains cis-acting sequences that contribute to its encapsidation (6, 12, 16). One sequence, called ε, is located near the 5Ј end of the pgRNA. ε forms a phylogenetically conserved secondary structure that is important for its function (16,23,24). For the human hepatitis B virus (HBV), ε is the only identified sequence on the pgRNA required for encapsidation (16).For the avian hepadnaviruses, a second sequence located about 900 nucleotides (nt) downstream of ε, called region II, is required for encapsidation (6,12). Analysis of duck hepatitis B virus (DHBV) region II sho...