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In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins
Jianming Hu1, David Toft, Dana Anselmo
1Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. jmhu@bu.edu
Journal of Virology
|December 12, 2001
Summary
Host chaperone proteins are essential for activating hepatitis B virus reverse transcriptase (RT) for RNA binding and protein priming. This study identifies a minimal set of five cellular chaperones sufficient for in vitro RT activation.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Hepadnavirus reverse transcription initiation involves viral reverse transcriptase (RT) binding to an RNA signal (epsilon) and protein priming.
- Hepatitis B virus (HBV) RT-epsilon interaction and protein priming are known to require the host heat shock protein Hsp90.
- Additional host factors involved in these crucial steps remained unidentified.
Purpose of the Study:
- To identify the minimal set of host factors required for HBV reverse transcriptase (RT) activation.
- To reconstitute HBV RT activity in vitro using identified host factors.
- To elucidate the role of cellular chaperones in HBV replication initiation.
Main Methods:
- In vitro reconstitution assays using purified duck hepatitis B virus (DHBV) RT.
- Employing a panel of cellular chaperone proteins, including Hsp90 and its cofactors.
- Assessing RT activity through epsilon binding and protein priming assays.
- Investigating the dependence of RT activation on ATP hydrolysis and Hsp90 ATPase activity.
Main Results:
- Five cellular chaperone proteins (Hsp90, Hsp70, Hsp40, Hop, and p23) were found sufficient to reconstitute DHBV RT activity in vitro.
- Four chaperones (Hsp90, Hsp70, Hsp40, Hop) were essential for RT activity, while p23 enhanced the kinetics.
- RT activation was demonstrated to be a dynamic process dependent on ATP hydrolysis and Hsp90 ATPase activity.
Conclusions:
- A minimal complement of five host chaperone proteins is necessary and sufficient for HBV RT activation in vitro.
- This reconstituted system provides a platform for future mechanistic studies on RT activation and chaperone function.
- The findings highlight the critical role of host chaperones in the life cycle of hepadnaviruses.