In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs

  1. Lucile Sinck1,
  2. Delphine Richer1,
  3. Jane Howard2,
  4. Marina Alexander2,
  5. Damian F.J. Purcell2,
  6. Roland Marquet1, and
  7. Jean-Christophe Paillart1
  1. 1Architecture et Réactivité de l'ARN, Université Louis Pasteur, CNRS, IBMC, 67084, Strasbourg cedex, France
  2. 2Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia

Abstract

The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 5′ untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop–loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.

Keywords

Footnotes

  • Reprint requests to: Jean-Christophe Paillart, Architecture et Réactivité de l'ARN, Université Louis Pasteur, CNRS, IBMC, 67084, Strasbourg cedex, France; e-mail: jc.paillart{at}ibmc.u-strasbg.fr; fax: (+33) (0)3 88 60 22 18; or Roland Marquet, Architecture et Réactivité de l'ARN, Université Louis Pasteur, CNRS, IBMC, 67084, Strasbourg cedex, France; e-mail: r.marquet{at}ibmc.u-strasbg.fr; fax: (+33) (0)3 88 60 22 18.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.678307.

    • Received June 8, 2007.
    • Accepted August 28, 2007.
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