Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1991 Sep;65(9):4555–4564. doi: 10.1128/jvi.65.9.4555-4564.1991

Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription.

H Rhim 1, J Park 1, C D Morrow 1
PMCID: PMC248909  PMID: 1714513

Abstract

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA 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 region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)

Full text

PDF
4555

Images in this article

4558Image
on p.4558

Selected References

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

  1. 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]
  2. Araya A., Sarih L., Litvak S. Reverse transcriptase mediated binding of primer tRNA to the viral genome. Nucleic Acids Res. 1979 Aug 24;6(12):3831–3843. doi: 10.1093/nar/6.12.3831. [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., 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]
  5. Cann A. J., Karn J. Molecular biology of HIV: new insights into the virus life-cycle. AIDS. 1989;3 (Suppl 1):S19–S34. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Hong T., Drlica K., Pinter A., Murphy E. Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences. J Virol. 1991 Jan;65(1):551–555. doi: 10.1128/jvi.65.1.551-555.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Kikuchi Y., Sasaki N., Ando-Yamagami Y. Cleavage of tRNA within the mature tRNA sequence by the catalytic RNA of RNase P: implication for the formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8105–8109. doi: 10.1073/pnas.87.20.8105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6024–6028. doi: 10.1073/pnas.87.16.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6019–6023. doi: 10.1073/pnas.87.16.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Roberts J. D., Bebenek K., Kunkel T. A. The accuracy of reverse transcriptase from HIV-1. Science. 1988 Nov 25;242(4882):1171–1173. doi: 10.1126/science.2460925. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Smith J. S., Kim S. Y., Roth M. J. Analysis of long terminal repeat circle junctions of human immunodeficiency virus type 1. J Virol. 1990 Dec;64(12):6286–6290. doi: 10.1128/jvi.64.12.6286-6290.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Tindall K. R., Kunkel T. A. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. doi: 10.1021/bi00416a027. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Whitcomb J. M., Kumar R., Hughes S. H. Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration. J Virol. 1990 Oct;64(10):4903–4906. doi: 10.1128/jvi.64.10.4903-4906.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  36. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES