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 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 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 Cellular Receptor Traffic Is Essential for Productive Duck Hepatitis B Virus Infection

2000 ◽  
Vol 74 (5) ◽  
pp. 2203-2209 ◽  
Author(s):  
Klaus M. Breiner ◽  
Heinz Schaller
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Transmembrane Protein ◽  
Cellular Receptor ◽  
Receptor Function ◽  
Wild Type ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.

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

cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA.

1991 ◽  
Vol 65 (6) ◽  
pp. 3309-3316 ◽  
Author(s):  
R C Hirsch ◽  
D D Loeb ◽  
J R Pollack ◽  
D Ganem
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Duck Hepatitis B Virus ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Characterization of the cis-Acting Contributions to Avian Hepadnavirus RNA Encapsidation

2002 ◽  
Vol 76 (18) ◽  
pp. 9087-9095 ◽  
Author(s):  
Kristin M. Ostrow ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Stem Loop ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
B Virus ◽  
Viral Components ◽  
Region Ii ◽  
Broad Region

ABSTRACT Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), ε and region II. ε, an RNA stem-loop near the 5′ end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, ε and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between ε and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between ε and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.

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