Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli.
Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. 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. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
Beck
J
J
Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany. jbeck@ukl.uni-freiburg.de
Nassal
M
M
D000595:Amino Acid Sequence; D000818:Animals; D004265:DNA Helicases / Q000235:genetics*; D004268:DNA-Binding Proteins; D004926:Escherichia coli; D015611:Hepatitis B Virus, Duck / Q000502:physiology*; D008969:Molecular Sequence Data; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D012194:RNA-Directed DNA Polymerase; D004265:DNA Helicases