In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins

Jianming Hu, D. Toft, D. Anselmo, X. Wang

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.

Original languageEnglish (US)
Pages (from-to)269-279
Number of pages11
JournalJournal of virology
Volume76
Issue number1
DOIs
StatePublished - Jan 1 2002

Fingerprint

Hepadnaviridae
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase
Proteins
proteins
Duck hepatitis B virus
Duck Hepatitis B Viruses
RNA
Humulus
reverse transcription
Hepatitis B virus
In Vitro Techniques
hops
Viral RNA
Product Packaging
Heat-Shock Proteins
heat shock proteins
packaging
Reverse Transcription
adenosinetriphosphatase

All Science Journal Classification (ASJC) codes

  • Immunology

Cite this

@article{62b6ab7a3dcd4378b5692cf795d9ac0e,
title = "In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins",
abstract = "Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.",
author = "Jianming Hu and D. Toft and D. Anselmo and X. Wang",
year = "2002",
month = "1",
day = "1",
doi = "10.1128/JVI.76.1.269-279.2002",
language = "English (US)",
volume = "76",
pages = "269--279",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "1",

}

In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. / Hu, Jianming; Toft, D.; Anselmo, D.; Wang, X.

In: Journal of virology, Vol. 76, No. 1, 01.01.2002, p. 269-279.

Research output: Contribution to journalArticle

TY - JOUR

T1 - In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins

AU - Hu, Jianming

AU - Toft, D.

AU - Anselmo, D.

AU - Wang, X.

PY - 2002/1/1

Y1 - 2002/1/1

N2 - Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.

AB - Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.

UR - http://www.scopus.com/inward/record.url?scp=0036133258&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036133258&partnerID=8YFLogxK

U2 - 10.1128/JVI.76.1.269-279.2002

DO - 10.1128/JVI.76.1.269-279.2002

M3 - Article

C2 - 11739692

AN - SCOPUS:0036133258

VL - 76

SP - 269

EP - 279

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 1

ER -