title: In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase.
abstract: Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.
authors.affiliation: Department of Microbiology and Immunology, The Penn State University College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Jones
authors.forename: Rajeev
authors.initials: R
authors.lastname: Boregowda
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Spratt
authors.forename: Jianming
authors.initials: J
authors.lastname: Hu
mesh_terms: D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D015870:Gene Expression; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D014157:Transcription Factors; D012194:RNA-Directed DNA Polymerase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010727:Phosphoric Diester Hydrolases; C474866:TDP2 protein, human