Hepatitis B virus reverse transcriptase and epsilon RNA sequences required for specific interaction in vitro.
Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific epsilon binding, we have defined the protein and RNA sequences required for specific HBV RT-epsilon interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for epsilon binding. With respect to the epsilon RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of epsilon, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-epsilon interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-epsilon interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some epsilon sequences by the RT may be required specifically for viral DNA synthesis.
Hu
Jianming
J
Department of Microbiology and Immunology, H107, The Penn State University College of Medicine, Hershey, 17033, USA. juh13@psu.edu
Boyer
Morgan
M
D024202:Electrophoretic Mobility Shift Assay; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048348:Reverse Transcription; D019065:Virus Assembly
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012194:RNA-Directed DNA Polymerase