Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex

Nucleic Acids Res. 1998 Mar 1;26(5):1198-204. doi: 10.1093/nar/26.5.1198.

Abstract

Retroviruses use a specific tRNA, whose 3' end is complementary to the 18 nucleotides of the primer binding site (PBS), to prime reverse transcription. Previous work has shown that initiation of HIV-1 reverse transcription is a specific process, in contrast with the subsequent elongation phase. HIV-1 reverse transcriptase (RT) specifically recognizes the complex formed by the viral RNA and tRNA3Lys. We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing. In this model, tRNA3Lysextensively interacts with the genomic RNA. Here, we have combined site-directed mutagenesis and structural probing to test crucial aspects of this model. We found that the complex interactions between tRNA3Lysand HIV-1 RNA, and the intra-molecular rearrangements did not depend on the presence of upstream and downstream viral sequences. Indeed, a short RNA template, encompassing nucleotides 123-217 of the HIV-1 Mal genome, was able, together with the primer tRNA, to adopt the same structure as longer viral RNA fragments. This model primer/template is thus amenable to detailed structural and functional studies. The probing data obtained on the tRNA3Lys/mutant viral RNA complexes support the previously proposed model. Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA3Lysand the so-called viral 'A-rich loop' destabilizes all four helices of the extended tRNA3Lys/HIV-1 RNA interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anticodon / genetics
  • Base Sequence
  • DNA Primers / genetics
  • HIV-1 / genetics*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • RNA, Transfer, Lys / chemistry*
  • RNA, Transfer, Lys / genetics*
  • RNA, Viral / chemistry*
  • RNA, Viral / genetics*

Substances

  • Anticodon
  • DNA Primers
  • RNA, Transfer, Lys
  • RNA, Viral