Chaperones activate hepadnavirus reverse transcriptase by transiently exposing a C-proximal region in the terminal protein domain that contributes to epsilon RNA binding.
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, epsilon, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an epsilon-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. epsilon 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 epsilon RNA binding. Notably, after priming and epsilon 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.
Stahl
Michael
M
University Hospital Freiburg, Internal Med. II/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany.
Beck
Jürgen
J
Nassal
Michael
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D015967:Gene Expression Regulation, Viral; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D015613:Hepadnaviridae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015611:Hepatitis B Virus, Duck / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D012367:RNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D012194:RNA-Directed DNA Polymerase