3'-End modification of the adenoviral VA1 gene affects its expression in human cells: Consequences for the design of chimeric VA1 RNA ribozymes

Sylvie Barcellini-Couget, David Fenard, Edouard Bertrand, Robert H. Singer, Jean Claude Lefebvre, Alain Doglio

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAδIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.

Original languageEnglish (US)
Pages (from-to)379-390
Number of pages12
JournalAntisense and Nucleic Acid Drug Development
Volume8
Issue number5
StatePublished - 1998

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Catalytic RNA
Genes
Cells
RNA
Viruses
HIV
eIF-2 Kinase
Transcription
Virus Replication
Cell Nucleus
Interferons
Assays
Cytoplasm
Derivatives
Molecules

ASJC Scopus subject areas

  • Genetics
  • Pharmacology

Cite this

3'-End modification of the adenoviral VA1 gene affects its expression in human cells : Consequences for the design of chimeric VA1 RNA ribozymes. / Barcellini-Couget, Sylvie; Fenard, David; Bertrand, Edouard; Singer, Robert H.; Lefebvre, Jean Claude; Doglio, Alain.

In: Antisense and Nucleic Acid Drug Development, Vol. 8, No. 5, 1998, p. 379-390.

Research output: Contribution to journalArticle

Barcellini-Couget, Sylvie ; Fenard, David ; Bertrand, Edouard ; Singer, Robert H. ; Lefebvre, Jean Claude ; Doglio, Alain. / 3'-End modification of the adenoviral VA1 gene affects its expression in human cells : Consequences for the design of chimeric VA1 RNA ribozymes. In: Antisense and Nucleic Acid Drug Development. 1998 ; Vol. 8, No. 5. pp. 379-390.
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