Disease relevance of DHBVgp4

• Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed the secondary structure of the protein-bound DHBV epsilon RNA (Depsilon) and observed a marked conformational change compared to free Depsilon RNA [1].
• Replication initiation does not involve a nucleic acid primer; instead, the hepadnavirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a short DNA primer that becomes covalently linked to the enzyme [1].
• Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the reverse transcriptase, P protein, with the cis-acting, 5' end-proximal encapsidation signal epsilon [2].
• To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL [3].
• We have therefore experimentally determined the secondary structures of D epsilon and of the H epsilon-signal of heron hepatitis B virus which differs considerably from D epsilon in primary sequence yet interacts productively with DHBV P protein [4].

High impact information on DHBVgp4

• Because P protein activity depends on cellular chaperones this complex can as yet only be generated by in vitro translation of duck hepatitis B virus P protein in rabbit reticulocyte lysate; functional interaction with its cognate RNA element Depsilon can be monitored by the covalent labeling of P protein during primer synthesis [5].
• Using the structured RNA encapsidation signal (D(epsilon)) and the reverse transcriptase (P protein) of duck hepatitis B virus (DHBV) as an example, we devised a sensitive mapping procedure that yields accurate information on the minimal RNA sequence required for interaction with a few nanograms of an RNA-binding protein [6].
• Genetic analysis of cis-acting signals required for viral replication implicates an RNA stem-loop structure in both RNA packaging and the initiation of reverse transcription, a process in which P protein also serves as the primer [7].
• This assignment was confirmed by direct biochemical analysis: tryptic peptides from the DHBV P protein, 32P labelled at the priming amino acid by the initiating dGTP and additionally labelled internally by [35S]methionine, were isolated and analyzed in parallel to reference peptides synthesized chemically and 33P labelled by a tyrosine kinase [8].
• Mutations outside the YMDD motif in the P protein can also cause DHBV resistant to Lamivudine [9].

Chemical compound and disease context of DHBVgp4

• Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451) [10].

References