In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase

Abstract

The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

Keywords: Hepatitis B virus; Polymerase; Protein priming; RNA binding; Reverse transcriptase.

Fig. 1

Protein priming and RNA binding protocol diagram. (a) The HBV polymerase can be purified (with or without epsilon RNA) by binding the FLAG-tagged polymerase to agarose beads, which are pre-bound with anti-FLAG antibodies. (b) An in vitro synthesized RNA is added to HBV polymerase (purified without epsilon) to perform the in vitro RNA binding assay. (c) The epsilon RNA which is co-purified with the polymerase can be evaluated for the in vivo RNA binding assay. (d) The purified polymerase, which is enzymatically active, can synthesize new DNA by adding nucleotides and buffer in the in vitro priming assay. Due to the protein priming activity, the polymerase itself is covalently labeled with the nucleotides and can be detected by autoradiography. (e) Alternatively, the DNA product from the priming reaction can be released from the polymerase by Tdp2 (the Tdp2 cleavage assay). *The priming assay, when using the manganese buffer, can also evaluate HBV polymerase’s terminal transferase activity. This novel activity is epsilon independent but also protein-primed. **The epsilon RNA template is copied beginning with an initial dGAA oligonucleotide. Therefore the initiation is dependent on dGTP, whereas the continued polymerization is dependent on dATP, and then other nucleotides