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. 1993 Jun;67(6):3246–3253. doi: 10.1128/jvi.67.6.3246-3253.1993

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1.

M Jiang 1, J Mak 1, A Ladha 1, E Cohen 1, M Klein 1, B Rovinski 1, L Kleiman 1
PMCID: PMC237665  PMID: 8497049

Abstract

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

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Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Bruce A. G., Uhlenbeck O. C. Reactions at the termini of tRNA with T4 RNA ligase. Nucleic Acids Res. 1978 Oct;5(10):3665–3677. doi: 10.1093/nar/5.10.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Crawford S., Goff S. P. A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J Virol. 1985 Mar;53(3):899–907. doi: 10.1128/jvi.53.3.899-907.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Erikson E., Erikson R. L. Association of 4S ribonucleic acid with oncornavirus ribonucleic acids. J Virol. 1971 Aug;8(2):254–256. doi: 10.1128/jvi.8.2.254-256.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Faras A. J., Dibble N. A. RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells. Proc Natl Acad Sci U S A. 1975 Mar;72(3):859–863. doi: 10.1073/pnas.72.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Faras A. J., Garapin A. C., Levinson W. E., Bishop J. M., Goodman H. M. Characterization of the low-molecular-weight RNAs associated with the 70S RNA of Rous sarcoma virus. J Virol. 1973 Aug;12(2):334–342. doi: 10.1128/jvi.12.2.334-342.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorelick R. J., Nigida S. M., Jr, Bess J. W., Jr, Arthur L. O., Henderson L. E., Rein A. Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol. 1990 Jul;64(7):3207–3211. doi: 10.1128/jvi.64.7.3207-3211.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Göttlinger H. G., Sodroski J. G., Haseltine W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781–5785. doi: 10.1073/pnas.86.15.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Haynes J. R., Cao S. X., Rovinski B., Sia C., James O., Dekaban G. A., Klein M. H. Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines. AIDS Res Hum Retroviruses. 1991 Jan;7(1):17–27. doi: 10.1089/aid.1991.7.17. [DOI] [PubMed] [Google Scholar]
  16. Jiang M., Mak J., Wainberg M. A., Parniak M. A., Cohen E., Kleiman L. Variable tRNA content in HIV-1IIIB. Biochem Biophys Res Commun. 1992 Jun 30;185(3):1005–1015. doi: 10.1016/0006-291x(92)91727-8. [DOI] [PubMed] [Google Scholar]
  17. Jiang M., Parniak M. A., Kleiman L. Isolation and fractionation of retroviral tRNAs. J Virol Methods. 1992 Aug;38(2):205–213. doi: 10.1016/0166-0934(92)90111-p. [DOI] [PubMed] [Google Scholar]
  18. Keith G., Heyman T. Heterogeneities in vertebrate tRNAs(Trp) avian retroviruses package only as a primer the tRNA(Trp) lacking modified m2G in position 7. Nucleic Acids Res. 1990 Feb 25;18(4):703–710. doi: 10.1093/nar/18.4.703. [DOI] [PMC free article] [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. Kuchino Y., Nishimura S. Enzymatic RNA sequencing. Methods Enzymol. 1989;180:154–163. doi: 10.1016/0076-6879(89)80099-0. [DOI] [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. 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]
  24. Peters G., Glover C. tRNA's and priming of RNA-directed DNA synthesis in mouse mammary tumor virus. J Virol. 1980 Jul;35(1):31–40. doi: 10.1128/jvi.35.1.31-40.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peters G., Harada F., Dahlberg J. E., Panet A., Haseltine W. A., Baltimore D. Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis. J Virol. 1977 Mar;21(3):1031–1041. doi: 10.1128/jvi.21.3.1031-1041.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Roe B. A. Studies on human tRNA. I. The rapid, large scale isolation and partial fractionation of placenta and liver tRNA. Nucleic Acids Res. 1975 Jan;2(1):21–42. doi: 10.1093/nar/2.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rosenthal L. J., Zamecnik P. C. Amino-acid acceptor activity of the "70S-associated" 4S RNA from avian myeloblastosis virus. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1184–1185. doi: 10.1073/pnas.70.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Sawyer R. C., Dahlberg J. E. Small RNAs of Rous sarcoma virus: characterization by two-dimensional polyacrylamide gel electrophoresis and fingerprint analysis. J Virol. 1973 Dec;12(6):1226–1237. doi: 10.1128/jvi.12.6.1226-1237.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sawyer R. C., Harada F., Dahlberg J. E. Virion-associated RNA primer for Rous sarcoma virus DNA synthesis: isolation from uninfected cells. J Virol. 1974 Jun;13(6):1302–1311. doi: 10.1128/jvi.13.6.1302-1311.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Waters L. C. Lysine tRNA is the predominant tRNA in murine mammary tumor virus. Biochem Biophys Res Commun. 1978 Apr 14;81(3):822–827. doi: 10.1016/0006-291x(78)91425-0. [DOI] [PubMed] [Google Scholar]
  36. Waters L. C., Mullin B. C., Ho T., Yang W. K. Ability of tryptophan tRNA to hybridize with 35S RNA of avian myeloblastosis virus and to prime reverse transcription in vitro. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2155–2159. doi: 10.1073/pnas.72.6.2155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Waters L. C., Mullin B. C. Transfer RNA into RNA tumor viruses. Prog Nucleic Acid Res Mol Biol. 1977;20:131–160. doi: 10.1016/s0079-6603(08)60471-7. [DOI] [PubMed] [Google Scholar]
  38. Witte O. N., Baltimore D. Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants. J Virol. 1978 Jun;26(3):750–761. doi: 10.1128/jvi.26.3.750-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yao X. J., Göttlinger H., Haseltine W. A., Cohen E. A. Envelope glycoprotein and CD4 independence of vpu-facilitated human immunodeficiency virus type 1 capsid export. J Virol. 1992 Aug;66(8):5119–5126. doi: 10.1128/jvi.66.8.5119-5126.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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