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Nucleic Acids Research, 1990, Vol. 18, No. 3 429-436
© 1990

ENZYMOLOGY

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNALys

Bruno Bordier, Laura Tarrago-Litvak, Marie-Line Sallafranque-Andreola, Dominique Robert, Danièle Tharaud, Michel Fournier, Philip J. Barr1, Simon Litvak* and Leila Sarih-Cottin*

Institut de Biochimie Cellulaire et Neurochimie du CNRS 1 rue Camille Saint Saëns, 33077 Bordeaux cedex, France 1Chiron Co., Horton Street, Emeryville, CA, USA

* To whom correspondence should be addressed

Received November 27, 1989. Accepted January 10, 1990.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNALys partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNALys we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase {alpha}, while avian retrovirus RT was neither affected by tRNALys nor by its specific primer tRNATrp. Although the strongest inhibition was observed with tRNALys, other tRNas like tRNAPhe and tRNATrP inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNALys with pancreatic RNase abolished the inhibition ; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNALys isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNALys was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNALys the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNALys primer, are different.


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