scholarly journals cis-Acting Sequences 5E, M, and 3E Interact To Contribute to Primer Translocation and Circularization during Reverse Transcription of Avian Hepadnavirus DNA

2002 ◽  
Vol 76 (9) ◽  
pp. 4260-4266 ◽  
Author(s):  
Karlyn Mueller-Hill ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Template Switching ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
Donor And Acceptor ◽  
B Virus ◽  
Template Switches

ABSTRACT Hepadnaviral reverse transcription requires template switches for the genesis of relaxed circular (RC) DNA, the major genomic form in virions. Two template switches, primer translocation and circularization, are required during the synthesis of the second, or plus, strand of DNA. Studies of duck hepatitis B virus (DHBV) indicate that in addition to the requirement for repeated sequences at the donor and acceptor sites, template switching requires at least three other cis-acting sequences, 5E, M, and 3E. In this study we analyzed a series of variant heron hepatitis B viruses (HHBV) in which the regions of the genome that would be expected to contain 5E, M, and 3E were replaced with DHBV sequence. We found that all single and double chimeras were partially defective in the synthesis of RC DNA. In contrast, the triple chimera was able to synthesize RC DNA at a level comparable to that of unchanged HHBV. These results indicate that the three cis-acting sequences, 5E, M, and 3E, need to be compatible to contribute to RC DNA synthesis, suggesting that these sequences interact during plus-strand synthesis. Second, we found that the defect in RC DNA synthesis for several of the single and double chimeric viruses resulted from a partial defect in primer translocation/utilization and a partial defect in circularization. These findings indicate that the processes of primer translocation and circularization share a mechanism during which 5E, M, and 3E interact.

scholarly journals Base Pairing between cis-Acting Sequences Contributes to Template Switching during Plus-Strand DNA Synthesis in Human Hepatitis B Virus

2007 ◽  
Vol 81 (12) ◽  
pp. 6207-6215 ◽  
Author(s):  
Eric B. Lewellyn ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Template Switching ◽  
Base Pairing ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
Human Hepatitis ◽  
B Virus ◽  
Template Switches

ABSTRACT Hepadnaviruses utilize two template switches (primer translocation and circularization) during synthesis of plus-strand DNA to generate a relaxed-circular (RC) DNA genome. In duck hepatitis B virus (DHBV) three cis-acting sequences, 3E, M, and 5E, contribute to both template switches through base pairing, 3E with the 3′ portion of M and 5E with the 5′ portion of M. Human hepatitis B virus (HBV) also contains multiple cis-acting sequences that contribute to the accumulation of RC DNA, but the mechanisms through which these sequences contribute were previously unknown. Three of the HBV cis-acting sequences (h3E, hM, and h5E) occupy positions equivalent to those of the DHBV 3E, M, and 5E. We present evidence that h3E and hM contribute to the synthesis of RC DNA through base pairing during both primer translocation and circularization. Mutations that disrupt predicted base pairing inhibit both template switches while mutations that restore the predicted base pairing restore function. Therefore, the h3E-hM base pairing appears to be a conserved requirement for template switching during plus-strand DNA synthesis of HBV and DHBV. Also, we show that base pairing is not sufficient to explain the mechanism of h3E and hM, as mutating sequences adjacent to the base pairing regions inhibited both template switches. Finally, we did not identify predicted base pairing between h5E and the hM region, indicating a possible difference between HBV and DHBV. The significance of these similarities and differences between HBV and DHBV will be discussed.

scholarly journals Three Novel cis-Acting Elements Required for Efficient Plus-Strand DNA Synthesis of the Hepatitis B Virus Genome

2004 ◽  
Vol 78 (14) ◽  
pp. 7455-7464 ◽  
Author(s):  
Jehan Lee ◽  
Myeong-Kyun Shin ◽  
Hye-Jin Lee ◽  
Gyesoon Yoon ◽  
Wang-Shick Ryu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Virus Genome ◽  
Complex Nature ◽  
Minus Strand ◽  
Cis Acting ◽  
Donor And Acceptor ◽  
B Virus ◽  
Template Switches

ABSTRACT Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.

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

2002 ◽  
Vol 76 (6) ◽  
pp. 2763-2769 ◽  
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.

scholarly journals cis-Acting Sequences That Contribute to Synthesis of Minus-Strand DNA Are Not Conserved between Hepadnaviruses

2010 ◽  
Vol 84 (24) ◽  
pp. 12824-12831 ◽  
Author(s):  
Megan L. Maguire ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Minus Strand ◽  
Wild Type ◽  
Dna Viruses ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
B Virus ◽  
The Individual

ABSTRACT Hepadnaviruses are DNA viruses that are found in several mammalian and avian species. These viruses replicate their genome through reverse transcription of an RNA intermediate termed pregenomic RNA (pgRNA). pgRNA is reverse transcribed by the viral polymerase into a minus-strand DNA, followed by synthesis of the plus-strand DNA. There are multiple cis-acting sequences that contribute to the synthesis of minus-strand DNA for human hepatitis B virus (HBV). Less is known about the cis-acting sequences of avian hepadnaviruses that contribute to synthesis of minus-strand DNA. To identify cis-acting sequences of duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV), we analyzed variants containing 200-nucleotide (nt) deletions. Most variants of DHBV synthesized minus-strand DNA to 50 to 100% of the wild-type (WT) level, while two variants synthesized less than 50%. For HHBV, most variants synthesized minus-strand DNA to less than 50% the WT level. These results differ from those for HBV, where most of the genome can be removed with little consequence. HBV contains a sequence, φ, that contributes to the synthesis of minus-strand DNA. It has been proposed that DHBV has an analogous sequence. We determined that the proposed φ sequence of DHBV does not contribute to the synthesis of minus-strand DNA. Finally, we found that the DR2 sequence present in all hepadnaviruses is important for synthesis of minus-strand DNA in both DHBV and HHBV but not in HBV. These differences in cis-acting sequences suggest that the individual hepadnaviruses have evolved differences in their mechanisms for synthesizing minus-strand DNA, more so than for other steps in replication.

scholarly journals 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

2000 ◽  
Vol 81 (8) ◽  
pp. 2059-2065 ◽  
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.

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

2003 ◽  
Vol 77 (23) ◽  
pp. 12412-12420 ◽  
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.

scholarly journals 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

2003 ◽  
Vol 77 (23) ◽  
pp. 12401-12411 ◽  
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.

scholarly journals cis-Acting Sequences That Contribute to the Synthesis of Relaxed-Circular DNA of Human Hepatitis B Virus

2004 ◽  
Vol 78 (2) ◽  
pp. 642-649 ◽  
Author(s):  
Ning Liu ◽  
Lin Ji ◽  
Megan L. Maguire ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Analysis ◽  
Hepatitis Virus ◽  
Template Switching ◽  
Duck Hepatitis ◽  
Human Hepatitis ◽  
Study Results ◽  
Donor And Acceptor ◽  
B Virus

ABSTRACT Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of cis-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about cis-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for cis-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5′r, and 3′r were not analyzed in the study. Results from Southern blotting showed that multiple cis-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of cis-acting sequences that contribute to the synthesis of HBV RC DNA.

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