Nucleic Acids Research, 2000, Vol. 28, No. 16 3065-3074
© 2000 Oxford University Press
Human immunodeficiency virus type-1 reverse transcription can be inhibited in vitro by oligonucleotides that target both natural and synthetic tRNA primers
1McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755, chemin Côte-Ste-Catherine, Montréal, Québec H3T 1E2, Canada and 2Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
Reverse transcription of human immunodeficiency virus type-1 is primed by cellular tRNALys3, which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). Since cellular tRNALys3 is highly conserved, it might conceivably serve as a good target for novel antagonists to block reverse transcriptase (RT) activity. In this study, we have examined a number of antisense oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer and, therefore, may interfere with the initiation of RT-mediated DNA synthesis. We found that the stability of complexes between synthetic tRNALys3 and ODNs was significantly increased when binding occurred via sequences involved in tertiary interactions of the tRNA. In particular, ODNs with complementarity to both the variable and T
C stem–loop of tRNALys3 bound with high affinity to both free tRNALys3 as well as to the binary tRNALys3/RNA complex. As a result, the initiation of DNA synthesis was severely compromised under these conditions. Moreover, RT-associated RNase H activity recognized the tRNA within this ternary tRNALys3/RNA/ODN complex as an RNA template and initiated its degradation. Both this RNase H degradation of tRNALys3 as well as the altered structure of the tRNA/RNA complex, due to the binding of the ODN, contributed to the inhibition of synthesis of viral DNA. The initiation of RT activity was almost completely blocked when using ODNs that interfered with intermolecular tRNA/RNA interactions that involved both the PBS and sequences outside the PBS. Similar findings were obtained with natural preparation of tRNALys3.
* To whom correspondence should be addressed. Tel: +1 514 340 7536; Fax: +1 514 340 7537; Email: mgoette@ldi.jgh.mcgill.ca
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