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

ABSTRACT There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to cis-acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5′ end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3′ end of the minus-strand DNA a poor template for initiation.

Download Full-text

Mutations That Increase In Situ Priming Also Decrease Circularization for Duck Hepatitis B Virus

Journal of Virology ◽

10.1128/jvi.75.14.6492-6497.2001 ◽

2001 ◽

Vol 75 (14) ◽

pp. 6492-6497 ◽

Cited By ~ 8

Author(s):

Daniel D. Loeb ◽

Ru Tian

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Minus Strand ◽

Complementary Sequence ◽

Duck Hepatitis B Virus ◽

Circular Dna ◽

Duck Hepatitis ◽

B Virus ◽

Template Switch

ABSTRACT The process of hepadnavirus reverse transcription involves two template switches during the synthesis of plus-strand DNA. The first involves translocation of the plus-strand primer from its site of generation, the 3′ end of minus-strand DNA, to the complementary sequence DR2, located near the 5′ end of the minus-strand DNA. Plus strands initiated from DR2 are extended to the 5′ end of the minus-strand DNA. At this point, the 3′ end of the minus strand becomes the template via the second template switch, a process called circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA. Although most virions contain relaxed circular DNA, some contain duplex linear DNA. Duplex linear genomes are synthesized when the plus-strand primer is used at the site of its generation, the 3′ end of the minus-strand template. This type of synthesis is called in situ priming. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within the 3′ end of the minus-strand DNA can lead to increased levels of in situ priming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-strand synthesis and the template switch that circularizes the genome.

Download Full-text

Analysis of Duck Hepatitis B Virus Reverse Transcription Indicates a Common Mechanism for the Two Template Switches during Plus-Strand DNA Synthesis

Journal of Virology ◽

10.1128/jvi.76.6.2763-2769.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2763-2769 ◽

Cited By ~ 10

Author(s):

Michael B. Havert ◽

Lin Ji ◽

Daniel D. Loeb

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Minus Strand ◽

Duck Hepatitis B Virus ◽

Circular Dna ◽

Duck Hepatitis ◽

Donor And Acceptor ◽

B Virus ◽

Template Switches

ABSTRACT The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5′r and 3′r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5′ end, the middle, and the 3′ end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3′ of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.77.23.12401-12411.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12401-12411 ◽

Cited By ~ 6

Author(s):

Jeffrey W. Habig ◽

Daniel D. Loeb

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Acceptor Site ◽

Minus Strand ◽

Dna Hairpin ◽

Duck Hepatitis ◽

B Virus ◽

Template Switch ◽

Template Switches

ABSTRACT 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.

Download Full-text

Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis.

Journal of Virology ◽

10.1128/jvi.68.8.5232-5238.1994 ◽

1994 ◽

Vol 68 (8) ◽

pp. 5232-5238 ◽

Cited By ~ 38

Author(s):

Y Chen ◽

W S Robinson ◽

P L Marion

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Rnase H ◽

Minus Strand ◽

Rna Packaging ◽

Duck Hepatitis B Virus ◽

P Gene ◽

Duck Hepatitis ◽

B Virus

Download Full-text

Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis

Journal of General Virology ◽

10.1099/0022-1317-81-8-2059 ◽

2000 ◽

Vol 81 (8) ◽

pp. 2059-2065 ◽

Cited By ~ 6

Author(s):

Yunhao Gong ◽

Ermei Yao ◽

Melissa Stevens ◽

John E. Tavis

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Reverse Transcription ◽

Transfer Reaction ◽

Strand Transfer ◽

Minus Strand ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus

Deletion of amino acids 79–88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.

Download Full-text

Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus Are Influenced by the Base Composition of the Minus-Strand Terminal Redundancy

Journal of Virology ◽

10.1128/jvi.77.23.12412-12420.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12412-12420 ◽

Cited By ~ 6

Author(s):

Jeffrey W. Habig ◽

Daniel D. Loeb

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Base Composition ◽

Minus Strand ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus ◽

Template Switch ◽

Template Switches

ABSTRACT 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. Circularization involves transfer of the nascent 3′ end of the 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. In duck hepatitis B virus (DHBV), a small terminal redundancy (5′r and 3′r) on the ends of the minus-strand DNA has been shown to be important, but not sufficient, for circularization. We investigated what contribution, if any, the base composition of the terminal redundancy made to the circularization process. Using a genetic approach, we found a strong positive correlation between the fraction of A and T residues within the terminal redundancy and the efficiency of the circularization process in those variants. Additionally, we found that the level of in situ priming increases, at the expense of primer translocation, as the fraction of A and T residues in the 3′r decreases. Thus, a terminal redundancy rich in A and T residues is important for both plus-strand template switches in DHBV.

Download Full-text