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. 1994 Mar;68(3):1605–1614. doi: 10.1128/jvi.68.3.1605-1614.1994

Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.

J K Wakefield 1, H Rhim 1, C D Morrow 1
PMCID: PMC236618  PMID: 7508999

Abstract

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)

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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. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Bordier B., Tarrago-Litvak L., Sallafranque-Andreola M. L., Robert D., Tharaud D., Fournier M., Barr P. J., Litvak S., Sarih-Cottin L. Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). Nucleic Acids Res. 1990 Feb 11;18(3):429–436. doi: 10.1093/nar/18.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chapman K. B., Byström A. S., Boeke J. D. Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3236–3240. doi: 10.1073/pnas.89.8.3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charneau P., Clavel F. A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J Virol. 1991 May;65(5):2415–2421. doi: 10.1128/jvi.65.5.2415-2421.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cobrinik D., Aiyar A., Ge Z., Katzman M., Huang H., Leis J. Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription. J Virol. 1991 Jul;65(7):3864–3872. doi: 10.1128/jvi.65.7.3864-3872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cobrinik D., Soskey L., Leis J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J Virol. 1988 Oct;62(10):3622–3630. doi: 10.1128/jvi.62.10.3622-3630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Colicelli J., Goff S. P. Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J Virol. 1986 Jan;57(1):37–45. doi: 10.1128/jvi.57.1.37-45.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Darlix J. L., Zuker M., Spahr P. F. Structure-function relationship of Rous sarcoma virus leader RNA. Nucleic Acids Res. 1982 Sep 11;10(17):5183–5196. doi: 10.1093/nar/10.17.5183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  15. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  16. Hungnes O., Tjøtta E., Grinde B. Mutations in the central polypurine tract of HIV-1 result in delayed replication. Virology. 1992 Sep;190(1):440–442. doi: 10.1016/0042-6822(92)91230-r. [DOI] [PubMed] [Google Scholar]
  17. Jiang M., Mak J., Ladha A., Cohen E., Klein M., Rovinski B., Kleiman L. Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. J Virol. 1993 Jun;67(6):3246–3253. doi: 10.1128/jvi.67.6.3246-3253.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kikuchi Y., Ando Y., Shiba T. Unusual priming mechanism of RNA-directed DNA synthesis in copia retrovirus-like particles of Drosophila. 1986 Oct 30-Nov 5Nature. 323(6091):824–826. doi: 10.1038/323824a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lever A., Gottlinger H., Haseltine W., Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. doi: 10.1128/jvi.63.9.4085-4087.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levin J. G., Seidman J. G. Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. J Virol. 1981 Apr;38(1):403–408. doi: 10.1128/jvi.38.1.403-408.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levin J. G., Seidman J. G. Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA. J Virol. 1979 Jan;29(1):328–335. doi: 10.1128/jvi.29.1.328-335.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li P., Stephenson A. J., Kuiper L. J., Burrell C. J. Double-stranded strong-stop DNA and the second template switch in human immunodeficiency virus (HIV) DNA synthesis. Virology. 1993 May;194(1):82–88. doi: 10.1006/viro.1993.1237. [DOI] [PubMed] [Google Scholar]
  24. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luo G. X., Sharmeen L., Taylor J. Specificities involved in the initiation of retroviral plus-strand DNA. J Virol. 1990 Feb;64(2):592–597. doi: 10.1128/jvi.64.2.592-597.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  27. Nagashunmugam T., Velpandi A., Goldsmith C. S., Zaki S. R., Kalyanaraman V. S., Srinivasan A. Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4114–4118. doi: 10.1073/pnas.89.9.4114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Panet A., Berliner H. Binding of tRNA to reverse transcriptase of RNA tumor viruses. J Virol. 1978 May;26(2):214–220. doi: 10.1128/jvi.26.2.214-220.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Panganiban A. T., Fiore D. Ordered interstrand and intrastrand DNA transfer during reverse transcription. Science. 1988 Aug 26;241(4869):1064–1069. doi: 10.1126/science.2457948. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Peters G., Dahlberg J. E. RNA-directed DNA synthesis in Moloney murine leukemia virus: interaction between the primer tRNA and the genome RNA. J Virol. 1979 Aug;31(2):398–407. doi: 10.1128/jvi.31.2.398-407.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pullen K. A., Champoux J. J. Plus-strand origin for human immunodeficiency virus type 1: implications for integration. J Virol. 1990 Dec;64(12):6274–6277. doi: 10.1128/jvi.64.12.6274-6277.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rhim H., Park J., Morrow C. D. Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. J Virol. 1991 Sep;65(9):4555–4564. doi: 10.1128/jvi.65.9.4555-4564.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  38. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taylor J. M. An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes. Biochim Biophys Acta. 1977 Mar 21;473(1):57–71. doi: 10.1016/0304-419x(77)90007-5. [DOI] [PubMed] [Google Scholar]
  41. Taylor J. M., Hsu T. W. Reverse transcription of avian sarcoma virus RNA into DNA might involve copying of the tRNA primer. J Virol. 1980 Jan;33(1):531–534. doi: 10.1128/jvi.33.1.531-534.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Temin H. M. Structure, variation and synthesis of retrovirus long terminal repeat. Cell. 1981 Nov;27(1 Pt 2):1–3. doi: 10.1016/0092-8674(81)90353-6. [DOI] [PubMed] [Google Scholar]
  43. Varmus H. E. Form and function of retroviral proviruses. Science. 1982 May 21;216(4548):812–820. doi: 10.1126/science.6177038. [DOI] [PubMed] [Google Scholar]
  44. Vzorov A. N., Bukrinsky M. I., Grigoriev V. B., Tentsov YYu, Bukrinskaya A. G. Highly immunogenic human immunodeficiency viruslike particles are produced by recombinant vaccinia virus-infected cells. AIDS Res Hum Retroviruses. 1991 Jan;7(1):29–36. doi: 10.1089/aid.1991.7.29. [DOI] [PubMed] [Google Scholar]
  45. Wakefield J. K., Jablonski S. A., Morrow C. D. In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. J Virol. 1992 Nov;66(11):6806–6812. doi: 10.1128/jvi.66.11.6806-6812.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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