Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 Oct;41(10):2141–2148. doi: 10.1128/aac.41.10.2141

Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells.

R El Dirani-Diab 1, L Sarih-Cottin 1, B Delord 1, B Dumon 1, S Moreau 1, J J Toulme 1, H Fleury 1, S Litvak 1
PMCID: PMC164084  PMID: 9333039

Abstract

Retroviral reverse transcriptase (RT) is involved in the selection of a specific tRNA primer which initiates proviral DNA minus-strand synthesis. Studies of the interactions between human immunodeficiency virus type 1 (HIV-1) RT and primer tRNALys3 have shown that the dihydrouridine (diHU), anticodon, and pseudouridine regions of tRNA are highly protected in the RT-tRNA complex. The CCA 3' end of tRNA is also in close contact with the enzyme during the cDNA initiation step. Using synthetic oligoribonucleotides corresponding to the anticodon and diHU regions, we have previously shown a low but significant inhibition of HIV-1 RT activity. We extend this observation and show that primer tRNA-derived oligodeoxynucleotides (ODNs) carrying a phosphorothioate (PS) modification are strong inhibitors of HIV-1 RT. The affinity of PS-ODNs for the enzyme was monitored by gel mobility shift electrophoresis. Experiments with HIV-1-infected human cells (MT-2 cells) were performed with the latter ODNs. A PS-ODN corresponding to the 3' end of tRNALys3 (acceptor stem [AS]) was able to inhibit HIV-1 replication. No effect of the other modified ODNs was observed in infected cells. The analysis of HIV-1 RNase H activity in a cell-free system strongly suggests that the inhibitory effect of the PS-AS may be mediated via both a sense and an antisense mechanism.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. APOSHIAN H. V., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. IX. The polymerase formed after T2 bacteriophage infection of Escherichia coli: a new enzyme. J Biol Chem. 1962 Feb;237:519–525. [PubMed] [Google Scholar]
  2. Alizon M., Wain-Hobson S., Montagnier L., Sonigo P. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell. 1986 Jul 4;46(1):63–74. doi: 10.1016/0092-8674(86)90860-3. [DOI] [PubMed] [Google Scholar]
  3. Arion D., Harada R., Li X., Wainberg M. A., Parniak M. A. HIV-1 reverse transcriptase shows no specificity for the binding of primer tRNA(Lys3). Biochem Biophys Res Commun. 1996 Aug 23;225(3):839–843. doi: 10.1006/bbrc.1996.1260. [DOI] [PubMed] [Google Scholar]
  4. Arts E. J., Wainberg M. A. Human immunodeficiency virus type 1 reverse transcriptase and early events in reverse transcription. Adv Virus Res. 1996;46:97–163. doi: 10.1016/s0065-3527(08)60071-8. [DOI] [PubMed] [Google Scholar]
  5. Azad R. F., Brown-Driver V., Buckheit R. W., Jr, Anderson K. P. Antiviral activity of a phosphorothioate oligonucleotide complementary to human cytomegalovirus RNA when used in combination with antiviral nucleoside analogs. Antiviral Res. 1995 Oct;28(2):101–111. doi: 10.1016/0166-3542(95)00035-k. [DOI] [PubMed] [Google Scholar]
  6. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barat C., Schatz O., Le Grice S., Darlix J. L. Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. J Mol Biol. 1993 May 20;231(2):185–190. doi: 10.1006/jmbi.1993.1273. [DOI] [PubMed] [Google Scholar]
  8. Boiziau C., Debart F., Rayner B., Imbach J. L., Toulme J. J. Chimeric alpha-beta oligonucleotides as antisense inhibitors of reverse transcription. FEBS Lett. 1995 Mar 13;361(1):41–45. doi: 10.1016/0014-5793(95)00138-y. [DOI] [PubMed] [Google Scholar]
  9. Boiziau C., Tarrago-Litvak L., Sinha N. D., Moreau S., Litvak S., Toulmé J. J. Antisense oligonucleotides inhibit in vitro cDNA synthesis by HIV-1 reverse transcriptase. Antisense Nucleic Acid Drug Dev. 1996 Summer;6(2):103–109. doi: 10.1089/oli.1.1996.6.103. [DOI] [PubMed] [Google Scholar]
  10. Boiziau C., Thuong N. T., Toulmé J. J. Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):768–772. doi: 10.1073/pnas.89.2.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bordier B., Hélène C., Barr P. J., Litvak S., Sarih-Cottin L. In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription. Nucleic Acids Res. 1992 Nov 25;20(22):5999–6006. doi: 10.1093/nar/20.22.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bordier B., Perala-Heape M., Degols G., Lebleu B., Litvak S., Sarih-Cottin L., Hélène C. Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9383–9387. doi: 10.1073/pnas.92.20.9383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brown D. A., Kang S. H., Gryaznov S. M., DeDionisio L., Heidenreich O., Sullivan S., Xu X., Nerenberg M. I. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem. 1994 Oct 28;269(43):26801–26805. [PubMed] [Google Scholar]
  14. Darlix J. L., Lapadat-Tapolsky M., de Rocquigny H., Roques B. P. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J Mol Biol. 1995 Dec 8;254(4):523–537. doi: 10.1006/jmbi.1995.0635. [DOI] [PubMed] [Google Scholar]
  15. Idriss H., Stammers D. K. Inhibition of HIV-1 reverse transcriptase by defined template/primer DNA oligonucleotides: effect of template length and binding characteristics. J Enzyme Inhib. 1994;8(2):97–112. doi: 10.3109/14756369409020193. [DOI] [PubMed] [Google Scholar]
  16. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  17. Isel C., Marquet R., Keith G., Ehresmann C., Ehresmann B. Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription. J Biol Chem. 1993 Dec 5;268(34):25269–25272. [PubMed] [Google Scholar]
  18. Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
  19. Lisziewicz J., Sun D., Metelev V., Zamecnik P., Gallo R. C., Agrawal S. Long-term treatment of human immunodeficiency virus-infected cells with antisense oligonucleotide phosphorothioates. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3860–3864. doi: 10.1073/pnas.90.9.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lisziewicz J., Sun D., Weichold F. F., Thierry A. R., Lusso P., Tang J., Gallo R. C., Agrawal S. Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7942–7946. doi: 10.1073/pnas.91.17.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Litvak S., Sarih-Cottin L., Fournier M., Andreola M., Tarrago-Litvak L. Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. Trends Biochem Sci. 1994 Mar;19(3):114–118. doi: 10.1016/0968-0004(94)90203-8. [DOI] [PubMed] [Google Scholar]
  22. Mak J., Jiang M., Wainberg M. A., Hammarskjöld M. L., Rekosh D., Kleiman L. Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol. 1994 Apr;68(4):2065–2072. doi: 10.1128/jvi.68.4.2065-2072.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marshall W. S., Caruthers M. H. Phosphorodithioate DNA as a potential therapeutic drug. Science. 1993 Mar 12;259(5101):1564–1570. doi: 10.1126/science.7681216. [DOI] [PubMed] [Google Scholar]
  24. Mishima Y., Steitz J. A. Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. EMBO J. 1995 Jun 1;14(11):2679–2687. doi: 10.1002/j.1460-2075.1995.tb07266.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oude Essink B. B., Das A. T., Berkhout B. Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1. J Biol Chem. 1995 Oct 6;270(40):23867–23874. doi: 10.1074/jbc.270.40.23867. [DOI] [PubMed] [Google Scholar]
  26. Pauwels R., Balzarini J., Baba M., Snoeck R., Schols D., Herdewijn P., Desmyter J., De Clercq E. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods. 1988 Aug;20(4):309–321. doi: 10.1016/0166-0934(88)90134-6. [DOI] [PubMed] [Google Scholar]
  27. Raba M., Limburg K., Burghagen M., Katze J. R., Simsek M., Heckman J. E., Rajbhandary U. L., Gross H. J. Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts. Eur J Biochem. 1979 Jun;97(1):305–318. doi: 10.1111/j.1432-1033.1979.tb13115.x. [DOI] [PubMed] [Google Scholar]
  28. Ramazeilles C., Mishra R. K., Moreau S., Pascolo E., Toulmé J. J. Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7859–7863. doi: 10.1073/pnas.91.17.7859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Robert D., Sallafranque-Andreola M. L., Bordier B., Sarih-Cottin L., Tarrago-Litvak L., Graves P. V., Barr P. J., Fournier M., Litvak S. Interactions with tRNA(Lys) induce important structural changes in human immunodeficiency virus reverse transcriptase. FEBS Lett. 1990 Dec 17;277(1-2):239–242. doi: 10.1016/0014-5793(90)80855-d. [DOI] [PubMed] [Google Scholar]
  30. Sallafranque-Andreola M. L., Robert D., Barr P. J., Fournier M., Litvak S., Sarih-Cottin L., Tarrago-Litvak L. Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys. Eur J Biochem. 1989 Sep 15;184(2):367–374. doi: 10.1111/j.1432-1033.1989.tb15028.x. [DOI] [PubMed] [Google Scholar]
  31. Sarih-Cottin L., Bordier B., Musier-Forsyth K., Andreola M. L., Barr P. J., Litvak S. Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides. J Mol Biol. 1992 Jul 5;226(1):1–6. doi: 10.1016/0022-2836(92)90117-3. [DOI] [PubMed] [Google Scholar]
  32. Schwartz O., Henin Y., Marechal V., Montagnier L. A rapid and simple colorimetric test for the study of anti-HIV agents. AIDS Res Hum Retroviruses. 1988 Dec;4(6):441–448. doi: 10.1089/aid.1988.4.441. [DOI] [PubMed] [Google Scholar]
  33. Stein C. A., Cleary A. M., Yakubov L., Lederman S. Phosphorothioate oligodeoxynucleotides bind to the third variable loop domain (v3) of human immunodeficiency virus type 1 gp120. Antisense Res Dev. 1993 Spring;3(1):19–31. doi: 10.1089/ard.1993.3.19. [DOI] [PubMed] [Google Scholar]
  34. Stein C. A., Neckers L. M., Nair B. C., Mumbauer S., Hoke G., Pal R. Phosphorothioate oligodeoxycytidine interferes with binding of HIV-1 gp120 to CD4. J Acquir Immune Defic Syndr. 1991;4(7):686–693. [PubMed] [Google Scholar]
  35. Tanaka H., Vickart P., Bertrand J. R., Rayner B., Morvan F., Imbach J. L., Paulin D., Malvy C. Sequence-specific interaction of alpha-beta-anomeric double-stranded DNA with the p50 subunit of NF kappa B: application to the decoy approach. Nucleic Acids Res. 1994 Aug 11;22(15):3069–3074. doi: 10.1093/nar/22.15.3069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tarrago-Litvak L., Andréola M. L., Nevinsky G. A., Sarih-Cottin L., Litvak S. The reverse transcriptase of HIV-1: from enzymology to therapeutic intervention. FASEB J. 1994 May;8(8):497–503. doi: 10.1096/fasebj.8.8.7514143. [DOI] [PubMed] [Google Scholar]
  37. Thrall S. H., Reinstein J., Wöhrl B. M., Goody R. S. Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding. Biochemistry. 1996 Apr 9;35(14):4609–4618. doi: 10.1021/bi9526387. [DOI] [PubMed] [Google Scholar]
  38. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  39. Wöhrl B. M., Ehresmann B., Keith G., Le Grice S. F. Nuclease footprinting of human immunodeficiency virus reverse transcriptase/tRNA(Lys-3) complexes. J Biol Chem. 1993 Jun 25;268(18):13617–13624. [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES