scholarly journals Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

2002 ◽  
Vol 76 (12) ◽  
pp. 5857-5865 ◽  
Author(s):  
Xingtai Wang ◽  
Jianming Hu
Keyword(s):  
Reverse Transcription ◽  
Specific Interaction ◽  
Initial Step ◽  
Covalent Attachment ◽  
Dna Polymerization ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Complete Protein ◽  
Two Stages

ABSTRACT Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed ε, which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5′ end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus that were fully active in the initial step of protein priming, i.e., the covalent attachment of the first nucleotide to the protein (RT deoxynucleotidylation), but defective in any subsequent DNA polymerization. A short sequence in the RT domain was localized that was dispensable for RT deoxynucleotidylation but essential for the subsequent DNA polymerization. These results have thus revealed two distinct stages of protein priming, i.e., the initial attachment of the first nucleotide to the RT (RT deoxynucleotidylation or initiation of protein priming) and the subsequent DNA synthesis (polymerization) to complete protein priming, with the second step entailing additional RT sequences. Two models are proposed to explain the observed differential sequence requirement for the two distinct stages of the protein priming reaction.

scholarly journals Functional and Structural Dynamics of Hepadnavirus Reverse Transcriptase during Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions

2008 ◽  
Vol 82 (12) ◽  
pp. 5703-5714 ◽  
Author(s):  
Li Lin ◽  
Fen Wan ◽  
Jianming Hu
Keyword(s):  
Reverse Transcriptase ◽  
Metal Ions ◽  
Reverse Transcription ◽  
Limited Proteolysis ◽  
Full Length ◽  
Dna And Rna ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Rt Inhibitors

ABSTRACT Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed ε. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct “priming” conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.

scholarly journals In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90

2000 ◽  
Vol 74 (24) ◽  
pp. 11447-11455 ◽  
Author(s):  
Jianming Hu ◽  
Dana Anselmo
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Host Factors ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Hsp90 Function

ABSTRACT Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.

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 Regulation of Hepadnavirus Reverse Transcription by Dynamic Nucleocapsid Phosphorylation

2006 ◽  
Vol 81 (4) ◽  
pp. 1641-1649 ◽  
Author(s):  
Suresh H. Basagoudanavar ◽  
David H. Perlman ◽  
Jianming Hu
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Core Protein ◽  
Early Stage ◽  
Phosphorylation Site ◽  
Duck Hepatitis ◽  
Double Stranded Dna ◽  
B Virus ◽  
Multistep Process

ABSTRACT Reverse transcription, an essential step in the life cycle of all retroelements, is a complex, multistep process whose regulation is not yet clearly understood. We have recently shown that reverse transcription in the pararetrovirus duck hepatitis B virus is associated with complete dephosphorylation of the viral core protein, which forms the nucleocapsid wherein reverse transcription takes place. Here we present a genetic study of the role of this dynamic nucleocapsid phosphorylation in regulating viral reverse transcription. Detailed analyses of the reverse transcription products synthesized within nucleocapsids composed of core phosphorylation site mutants revealed that alanine substitutions, mimicking the nonphosphorylated state, completely blocked reverse transcription at a very early stage. In contrast, aspartate substitutions, mimicking the phosphorylated state, allowed complete first-strand DNA synthesis but were severely defective in accumulating mature double-stranded DNA. The latter defect was due to a combination of mutant nucleocapsid instability during maturation and a block in mature second-strand DNA synthesis. Thus, the reversible phosphorylation of the nucleocapsids regulates the ordered progression of reverse transcription.

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.

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