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. 1996 Feb 15;15(4):917–924.

Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys.

C Isel 1, J M Lanchy 1, S F Le Grice 1, C Ehresmann 1, B Ehresmann 1, R Marquet 1
PMCID: PMC450289  PMID: 8631312

Abstract

Initiation of RNA-dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV-1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV-1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo- and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV-1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV-1 RNA, HIV-1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer-template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.

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Selected References

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

  1. Aiyar A., Cobrinik D., Ge Z., Kung H. J., Leis J. Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription. J Virol. 1992 Apr;66(4):2464–2472. doi: 10.1128/jvi.66.4.2464-2472.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiyar A., Ge Z., Leis J. A specific orientation of RNA secondary structures is required for initiation of reverse transcription. J Virol. 1994 Feb;68(2):611–618. doi: 10.1128/jvi.68.2.611-618.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arts E. J., Li X., Gu Z., Kleiman L., Parniak M. A., Wainberg M. A. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J Biol Chem. 1994 May 20;269(20):14672–14680. [PubMed] [Google Scholar]
  4. Arts E. J., Wainberg M. A. Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription. Antimicrob Agents Chemother. 1994 May;38(5):1008–1016. doi: 10.1128/aac.38.5.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
  6. Barat C., Le Grice S. F., Darlix J. L. Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3). Nucleic Acids Res. 1991 Feb 25;19(4):751–757. doi: 10.1093/nar/19.4.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cordell B., Swanstrom R., Goodman H. M., Bishop J. M. tRNATrp as primer for RNA-directed DNA polymerase: structural determinants of function. J Biol Chem. 1979 Mar 25;254(6):1866–1874. [PubMed] [Google Scholar]
  9. Das A. T., Klaver B., Berkhout B. Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). J Virol. 1995 May;69(5):3090–3097. doi: 10.1128/jvi.69.5.3090-3097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Götte M., Fackler S., Hermann T., Perola E., Cellai L., Gross H. J., Le Grice S. F., Heumann H. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA. EMBO J. 1995 Feb 15;14(4):833–841. doi: 10.1002/j.1460-2075.1995.tb07061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harada F., Peters G. G., Dahlberg J. E. The primer tRNA for Moloney murine leukemia virus DNA synthesis. Nucleotide sequence and aminoacylation of tRNAPro. J Biol Chem. 1979 Nov 10;254(21):10979–10985. [PubMed] [Google Scholar]
  12. Harada F., Sawyer R. C., Dahlberg J. E. A primer ribonucleic acid for initiation of in vitro Rous sarcarcoma virus deoxyribonucleic acid synthesis. J Biol Chem. 1975 May 10;250(9):3487–3497. [PubMed] [Google Scholar]
  13. Hizi A., Leis J. P., Joklik W. K. The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme. J Biol Chem. 1977 Oct 10;252(19):6878–6884. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Jäger J., Smerdon S. J., Wang J., Boisvert D. C., Steitz T. A. Comparison of three different crystal forms shows HIV-1 reverse transcriptase displays an internal swivel motion. Structure. 1994 Sep 15;2(9):869–876. doi: 10.1016/s0969-2126(94)00087-5. [DOI] [PubMed] [Google Scholar]
  17. Kati W. M., Johnson K. A., Jerva L. F., Anderson K. S. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed] [Google Scholar]
  18. Kohlstaedt L. A., Steitz T. A. Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9652–9656. doi: 10.1073/pnas.89.20.9652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  20. Lazcano A., Valverde V., Hernández G., Gariglio P., Fox G. E., Oró J. On the early emergence of reverse transcription: theoretical basis and experimental evidence. J Mol Evol. 1992 Dec;35(6):524–536. doi: 10.1007/BF00160213. [DOI] [PubMed] [Google Scholar]
  21. Le Grice S. F., Grüninger-Leitch F. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J Biochem. 1990 Jan 26;187(2):307–314. doi: 10.1111/j.1432-1033.1990.tb15306.x. [DOI] [PubMed] [Google Scholar]
  22. Li X., Mak J., Arts E. J., Gu Z., Kleiman L., Wainberg M. A., Parniak M. A. Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication. J Virol. 1994 Oct;68(10):6198–6206. doi: 10.1128/jvi.68.10.6198-6206.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Marquet R., Baudin F., Gabus C., Darlix J. L., Mougel M., Ehresmann C., Ehresmann B. Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism. Nucleic Acids Res. 1991 May 11;19(9):2349–2357. doi: 10.1093/nar/19.9.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marquet R., Isel C., Ehresmann C., Ehresmann B. tRNAs as primer of reverse transcriptases. Biochimie. 1995;77(1-2):113–124. doi: 10.1016/0300-9084(96)88114-4. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Panet A., Haseltine W. A., Baltimore D., Peters G., Harada F., Dahlberg J. E. Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase). Proc Natl Acad Sci U S A. 1975 Jul;72(7):2535–2539. doi: 10.1073/pnas.72.7.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Patel P. H., Jacobo-Molina A., Ding J., Tantillo C., Clark A. D., Jr, Raag R., Nanni R. G., Hughes S. H., Arnold E. Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. Biochemistry. 1995 Apr 25;34(16):5351–5363. doi: 10.1021/bi00016a006. [DOI] [PubMed] [Google Scholar]
  29. Peters G. G., Hu J. Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles. J Virol. 1980 Dec;36(3):692–700. doi: 10.1128/jvi.36.3.692-700.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  31. Reardon J. E. Human immunodeficiency virus reverse transcriptase. A kinetic analysis of RNA-dependent and DNA-dependent DNA polymerization. J Biol Chem. 1993 Apr 25;268(12):8743–8751. [PubMed] [Google Scholar]
  32. Spence R. A., Kati W. M., Anderson K. S., Johnson K. A. Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors. Science. 1995 Feb 17;267(5200):988–993. doi: 10.1126/science.7532321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Zhan X., Tan C. K., Scott W. A., Mian A. M., Downey K. M., So A. G. Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide. Biochemistry. 1994 Feb 15;33(6):1366–1372. doi: 10.1021/bi00172a012. [DOI] [PubMed] [Google Scholar]

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