Functional and Structural Dynamics of Hepadnavirus Reverse Transcriptase during Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions

Abstract

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed epsilon. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg(2+). Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn(2+), in comparison with Mg(2+), showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn(2+), indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg(2+). Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn(2+). Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn(2+). Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn(2+) but not in the presence of Mg(2+). Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn(2+) versus that of Mg(2+) and that the transition from initiation to polymerization was accompanied by RT conformational change.