ABSTRACT
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, ε, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an ε-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. ε 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 ε RNA binding. Notably, after priming and ε 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.
Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase.