The Unstable Part of the Apical Stem of Duck Hepatitis B Virus Epsilon Shows Enhanced Base Pair Opening but Not Pico- to Nanosecond Dynamics and Is Essential for Reverse Transcriptase Binding

Biochemistry ◽  
2009 ◽  
Vol 48 (44) ◽  
pp. 10499-10508 ◽  
Author(s):  
Kirsten A. M. Ampt ◽  
Ramon M. van der Werf ◽  
Frank H. T. Nelissen ◽  
Marco Tessari ◽  
Sybren S. Wijmenga
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Base Pair ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus ◽  
Nanosecond Dynamics ◽  
Unstable Part ◽  
Base Pair Opening

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.

scholarly journals The Majority of Duck Hepatitis B Virus Reverse Transcriptase in Cells Is Nonencapsidated and Is Bound to a Cytoplasmic Structure

2000 ◽  
Vol 74 (18) ◽  
pp. 8648-8657 ◽  
Author(s):  
Ermei Yao ◽  
Yunhao Gong ◽  
Nanhai Chen ◽  
John E. Tavis
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Core Protein ◽  
Duck Hepatitis B Virus ◽  
Viral Core ◽  
The Core ◽  
Duck Hepatitis ◽  
Cytoplasmic Structure ◽  
B Virus

ABSTRACT The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.

scholarly journals Reconstitution of a Functional Duck Hepatitis B Virus Replication Initiation Complex from Separate Reverse Transcriptase Domains Expressed in Escherichia coli

2001 ◽  
Vol 75 (16) ◽  
pp. 7410-7419 ◽  
Author(s):  
Jürgen Beck ◽  
Michael Nassal
Keyword(s):  
Escherichia Coli ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Replication Initiation ◽  
In Vitro Translation ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
P Protein ◽  
B Virus

ABSTRACT Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (ɛ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by ɛ and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of ɛ-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.

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