A specific orientation of RNA secondary structures is required for initiation of reverse transcription

Ashok Aiyar, Zheng Ge, Jonathan Leis*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

68 Scopus citations

Abstract

The 5' end of avian retrovirus RNA near the primer-binding site (PBS) forms two secondary structures, the U5-inverted repeat (U5-IR) and the U5- leader stems, and contains a 7-nucleotide sequence that anneals to the TΨC loop of the tRNA(Trp) primer. Mutations that disrupt any of these base pair interactions cause defects in initiation of reverse transcription both in vivo and in vitro (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; A. Aiyar, D. Cobrinik, Z. Ge, H.-J. Kung, and J. Leis, J. Virol. 66:2464-2472, 1992). We have now examined the effect of perturbing the non-base-paired intervening 'spacer' sequences between these secondary-structure elements. Small deletions or insertions in these intervening sequences decreased initiation of reverse transcription in vitro. In contrast, base substitutions, which maintain the spacing distances between the structures, had no detectable effect. Additionally, a small deletion at the 3' end of the PBS caused a significant decrease in initiation of reverse transcription whereas substitution mutations again had no effect. Together, these results indicate that reverse transcriptase forms a complex in which the different structural elements are maintained in a specific orientation that is required for efficient initiation of reverse transcription. Specific sequence recognition of the duplex structures by reverse transcriptase is also required since mosaic RNAs that combine the human immunodeficiency virus type 1 PBS with avian sequences is not efficiently utilized for reverse transcription even though the primer used can anneal to the substituted PBS.

Original languageEnglish (US)
Pages (from-to)611-618
Number of pages8
JournalJournal of virology
Volume68
Issue number2
DOIs
StatePublished - Feb 1994

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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