Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: Effects of metal ions

Li Lin, Fen Wan, Jianming Hu

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed ε. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn 2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.

Original languageEnglish (US)
Pages (from-to)5703-5714
Number of pages12
JournalJournal of virology
Volume82
Issue number12
DOIs
StatePublished - Jun 1 2008

Fingerprint

Hepadnaviridae
reverse transcription
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase
metal ions
Reverse Transcription
Polymerization
Metals
polymerization
Ions
Proteins
proteins
Duck hepatitis B virus
Duck Hepatitis B Viruses
Reverse Transcriptase Inhibitors
Viral DNA
Viral RNA
DNA-Directed DNA Polymerase
DNA-Directed RNA Polymerases
pyrophosphates

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

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title = "Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: Effects of metal ions",
abstract = "Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed ε. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct {"}priming{"} conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn 2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.",
author = "Li Lin and Fen Wan and Jianming Hu",
year = "2008",
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T1 - Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription

T2 - Effects of metal ions

AU - Lin, Li

AU - Wan, Fen

AU - Hu, Jianming

PY - 2008/6/1

Y1 - 2008/6/1

N2 - Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed ε. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn 2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.

AB - Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed ε. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn 2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.

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