Hepatitis B virus reverse transcriptase and ε RNA sequences required for specific interaction in vitro

Jianming Hu, Morgan Boyer

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) 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 ε binding, we have deiined the protein and RNA sequences required for specific HBV RT-ε 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 ε binding. With respect to the ε 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 ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.

Original languageEnglish (US)
Pages (from-to)2141-2150
Number of pages10
JournalJournal of virology
Volume80
Issue number5
DOIs
StatePublished - Mar 1 2006

Fingerprint

Hepatitis B virus
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase
nucleotide sequences
Product Packaging
RNA
stems
In Vitro Techniques
HSP90 Heat-Shock Proteins
Nucleocapsid
nucleocapsid
reverse transcription
Viral DNA
Viral RNA
Base Pairing
packaging
Reverse Transcription
amino acid sequences
Nucleotides
nucleotides

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

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abstract = "Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) 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 ε binding, we have deiined the protein and RNA sequences required for specific HBV RT-ε 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 ε binding. With respect to the ε 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 ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.",
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Hepatitis B virus reverse transcriptase and ε RNA sequences required for specific interaction in vitro. / Hu, Jianming; Boyer, Morgan.

In: Journal of virology, Vol. 80, No. 5, 01.03.2006, p. 2141-2150.

Research output: Contribution to journalArticle

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