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 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 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 Inhibitory Effect of Penciclovir-Triphosphate on Duck Hepatitis B Virus Reverse Transcription

1997 ◽  
Vol 8 (1) ◽  
pp. 38-46 ◽  
Author(s):  
E Dannaoui ◽  
C Trépo ◽  
F Zoulim
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Inhibitory Effect ◽  
In Vitro Translation ◽  
Chain Elongation ◽  
Minus Strand ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

The aim of this study was to investigate the mechanism of inhibition of hepatitis B virus replication by penciclovir-triphosphate, the active metabolite of famciclovir. A recently developed in vitro translation assay for the expression of an enzymatically active duck hepatitis B virus (DHBV) reverse transcriptase was used to assess the inhibitory activity of penciclovir-triphosphate (PCV-TP) in comparison with other guanosine analogue triphosphates. Acyclovir-triphosphate (ACV-TP), the chiral triphosphates of penciclovir (PCV), ( R)-PCV-TP and ( S)-PCV-TP, and carbocyclic 2′-deoxyguanosine-TP (CDG-TP) did inhibit reproducibly minus strand DNA synthesis to different extents. CDG-TP was the most potent inhibitor of dGTP incorporation. The inhibitory effect of these compounds against the incorporation of the first nucleotide of minus strand DNA, dGMP, was similar to that observed with DNA chain elongation. 2′,3′-dideoxyguanosine-TP (ddG-TP), ACV-TP and both ( R) and ( S)-PCV-TP inhibited the incorporation of the next nucleotides in the short DNA primer, whereas CDG-TP did not. These results demonstrate that PCV-TP inhibits hepadnavirus reverse transcription by inhibiting the synthesis of the short DNA primer. The data obtained with the inhibition of the enzymatic activity of the DHBV polymerase provides a new insight into the mechanism of action of penciclovir-triphosphate on HBV replication.

scholarly journals Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis

Hepatology ◽  
1999 ◽  
Vol 30 (1) ◽  
pp. 308-315 ◽  
Author(s):  
Fritz von Weizsäcker ◽  
Josef Köck ◽  
Stefan Wieland ◽  
Wolf-Bernhard Offensperger ◽  
Hubert E. Blum
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Core Protein ◽  
Dominant Negative ◽  
Viral Dna ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

Transcripts and the putative RNA pregenome of duck hepatitis B virus: Implications for reverse transcription

Cell ◽  
1985 ◽  
Vol 40 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Marita Büscher ◽  
Walter Reiser ◽  
Hans Will ◽  
Heinz Schaller
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Duck Hepatitis B Virus Nucleocapsids Formed by N-Terminally Extended or C-Terminally Truncated Core Proteins Disintegrate during Viral DNA Maturation

1998 ◽  
Vol 72 (11) ◽  
pp. 9116-9120 ◽  
Author(s):  
Josef Köck ◽  
Stefan Wieland ◽  
Hubert E. Blum ◽  
Fritz von Weizsäcker
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Core Protein ◽  
Viral Dna ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Core Proteins ◽  
B Virus ◽  
Carboxy Terminal

ABSTRACT Hepadnaviruses are DNA viruses that replicate through reverse transcription of an RNA pregenome. Viral DNA synthesis takes place inside viral nucleocapsids, formed by core protein dimers. Previous studies have identified carboxy-terminal truncations of the core protein that affect viral DNA maturation. Here, we describe the effect of small amino-terminal insertions into the duck hepatitis B virus (DHBV) core protein on viral DNA replication. All insertion mutants formed replication-competent nucleocapsids. Elongation of viral DNA, however, appeared to be incomplete. Increasing the number of additional amino acids and introducing negatively charged residues further reduced the observed size of mature viral DNA species. Mutant core proteins did not inhibit the viral polymerase. Instead, viral DNA synthesis destabilized mutant nucleocapsids, rendering mature viral DNA selectively sensitive to nuclease action. Interestingly, the phenotype of two previously described carboxy-terminal DHBV core protein deletion mutants was found to be based on the same mechanism. These data suggest that (i) the amino- as well as the carboxy-terminal portion of the DHBV core protein plays a critical role in nucleocapsid stabilization, and (ii) the hepadnavirus polymerase can perform partial second-strand DNA synthesis in the absence of intact viral nucleocapsids.

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