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Nucleic Acids Research, 1991, Vol. 19, No. 4 751-757
© 1991

MOLECULAR BIOLOGY

Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNALys,3

Corinne Barat, Stuart F.J. Le Grice2,+ and Jean-Luc Daelix1,*

CRBGC du CNRS, 118 Route de Narbonne, 31 062 Toulouse 1 LaboRetro, Ecole Normale Superieure de Lyon, 46 allée d'ltalie, 69364 Lyon, France 2Central Research Units F.Hoffmann-La Roche Ltd, Basel, Switzerland

* To whom correspondence should be addressed

Received November 30, 1990. Revised January 21, 1991. Accepted January 21, 1991.

Using synthetic oligonucleotides, a gene encoding the HIV-1 replication primer, tRNALys,3, was constructed and placed downstream from a bacteriophage T7 promoter. In vitro transcription of this gene yielded a form of tRNALys,3 which lacks the modified bases characteristic of the natural species and the 3'-G-A-dinucieotide. Synthetic tRNALys,3 annealed to a pbs- HIV1 RNA template can prime cDNA synthesis catalysed by recombinant HIV-1 reverse transcriptase. Trans-DDP crosslinking indicates that this synthetic tRNA is still capable of interacting with HIV-1 RT via a 12-nucleotide portion encompassing the anticodon domain. Gel-mobility shift and competition analyses imply that the affinity of synthetic tRNA for RT is reduced. In contrast to earlier observations, synthetic tRNA is readily competed from RT by natural tRNAPro. The reduced affinity of synthetic tRNALys,3 for RT is not appreciably affected by mutations in positions within the loop of the anticodon domain. These results would imply that the overall structure of the anticodon domain of tRNALys,3 is an important factor in its recognition by HIV-1 RT. In addition, modified bases within this, although not absolutely required, would appear to make a significant contribution to the enhanced stability of the ribonucleoprotein complex.

+ Present address: Division of Infectious Diseases. Case Western Reserve University School of Medicine. 2109 Adelbert Road, Cleveland. Ohio 44105, USA


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