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Journal of Virology, December 2007, p. 13354-13364, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Chaperones Activate Hepadnavirus Reverse Transcriptase by Transiently Exposing a C-Proximal Region in the Terminal Protein Domain That Contributes to {varepsilon} RNA Binding{triangledown}

Michael Stahl, Jürgen Beck, and Michael Nassal*

University Hospital Freiburg, Internal Med. 2/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany

Received 1 June 2007/ Accepted 24 September 2007

All hepatitis B viruses replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase, P protein, that carries a unique terminal protein (TP) domain. To initiate reverse transcription, P protein must bind to a stem-loop, {varepsilon}, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an {varepsilon}-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. {varepsilon} binding requires the chaperone-dependent conversion of inactive P protein into an activated, metastable form designated P*. However, how P* differs structurally from P protein is not known. Here we used an in vitro reconstitution system for active duck hepatitis B virus P combined with limited proteolysis, site-specific antibodies, and defined P mutants to structurally compare nonactivated versus chaperone-activated versus primed P protein. The data show that Hsp70 action, under conditions identical to those required for functional activation, transiently exposes the C proximal TP region which is, probably directly, involved in {varepsilon} RNA binding. Notably, after priming and {varepsilon} RNA removal, a very similar new conformation appears stable without further chaperone activity; hence, the activation of P protein is triggered by energy-consuming chaperone action but may be completed by template RNA binding.

* Corresponding author. Mailing address: University Hospital Freiburg, Internal Med. II/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone and fax: 49-761-270 3507. E-mail: nassal2{at}ukl.uni-freiburg.de

{triangledown} Published ahead of print on 3 October 2007.

Journal of Virology, December 2007, p. 13354-13364, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Liu, Z., Yang, F., Robotham, J. M., Tang, H. (2009). Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex. J. Virol. 83: 6554-6565 [Abstract] [Full Text]  
  • Lin, L., Wan, F., Hu, J. (2008). Functional and Structural Dynamics of Hepadnavirus Reverse Transcriptase during Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions. J. Virol. 82: 5703-5714 [Abstract] [Full Text]  


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