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Nucleic Acids Research, 1992, Vol. 20, No. 22 5999-6006
© 1992

MOLECULAR BIOLOGY

In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription

B. Bordier, C. Hélène1, P.J. Barr2, S. Litvak and L. Sarih-Cottin*

Institut de Biochimie Cellulaire du CNRS 1 rue Camille Saint Saëns, 33077 Bordeaux cedex 1Laboratoire de Biophysique, INSERM U.201-CNRS UA.481, Muséum National d'Histoire Naturelle 43 rue Cuvier, 75005 Paris, France 2CHIRON Corp. 4560 Horton St Emeryville, CA 94608, USA

* To whom correspondence should be addressed

Received August 7, 1992. Revised October 26, 1992. Accepted October 26, 1992.

The molecular events involved in antisense-mediated inhibition of retroviral transcription were studied by analyzing the in vitro effect of antisense oligodeoxynucleotides on reverse transcription by Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT). Oligonucleotides have been designed to be complementary to three targets located in the 5' region of the HIV-1 RNA genome : the trans-activating response element (TAR), the U5 region and a sequence contiguous to the primer binding site (PrePBS). Antisense oligodeoxynucleotides were used with their 3'-OH end either free or blocked by a dideoxynucleotide in order to avoid cDNA synthesis. Experiments with two recombinant forms of HIV RT, carrying or not RNase H activity, showed that antisense oligonucleotides can arrest reverse transcription by an RNase H-independent mechanism. The AntiTAR oligonucleotide did not affect reverse transcription. In contrast, the AntiU5 and AntiPrePBS oligonucleotides led to an efficient inhibition of both forms of HIV RT. In the case of the AntiU5, the inhibition obtained in the absence of the RNase H activity indicates that this effect can be related to features of the RNA secondary structure. The AntiPrePBS oligonucleotide did bind to its target only in the presence of PBS primer. Use of shifted oligonucleotides showed that the AntiPrePBS inhibitory effect depends on a cooperative annealing with the AntiPBS primer on the template.


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