Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1994 Oct;68(10):6198–6206. doi: 10.1128/jvi.68.10.6198-6206.1994

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication.

X Li 1, J Mak 1, E J Arts 1, Z Gu 1, L Kleiman 1, M A Wainberg 1, M A Parniak 1
PMCID: PMC237039  PMID: 7521916

Abstract

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).

Full text

PDF
6198

Images in this article

6202Image
on p.6202
6203Image
on p.6203
6203Image
on p.6203
6204Image
on p.6204

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. 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. Boulerice F., Bour S., Geleziunas R., Lvovich A., Wainberg M. A. High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells. J Virol. 1990 Apr;64(4):1745–1755. doi: 10.1128/jvi.64.4.1745-1755.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Dahlberg J. E., Sawyer R. C., Taylor J. M., Faras A. J., Levinson W. E., Goodman H. M., Bishop J. M. Transcription of DNA from the 70S RNA of Rous sarcoma virus. I. Identification of a specific 4S RNA which serves as primer. J Virol. 1974 May;13(5):1126–1133. doi: 10.1128/jvi.13.5.1126-1133.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeStefano J. J., Bambara R. A., Fay P. J. The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates. J Biol Chem. 1994 Jan 7;269(1):161–168. [PubMed] [Google Scholar]
  12. 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]
  13. Goff S. P. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed] [Google Scholar]
  14. Gu Z., Gao Q., Li X., Parniak M. A., Wainberg M. A. Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. J Virol. 1992 Dec;66(12):7128–7135. doi: 10.1128/jvi.66.12.7128-7135.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hu J. C., Dahlberg J. E. Structural features required for the binding of tRNATrp to avian myeloblastosis virus reverse transcriptase. Nucleic Acids Res. 1983 Jul 25;11(14):4823–4833. doi: 10.1093/nar/11.14.4823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Huber H. E., Richardson C. C. Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. J Biol Chem. 1990 Jun 25;265(18):10565–10573. [PubMed] [Google Scholar]
  19. 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]
  20. Jacques P. S., Wöhrl B. M., Howard K. J., Le Grice S. F. Modulation of HIV-1 reverse transcriptase function in "selectively deleted" p66/p51 heterodimers. J Biol Chem. 1994 Jan 14;269(2):1388–1393. [PubMed] [Google Scholar]
  21. 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]
  22. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  23. Klarmann G. J., Schauber C. A., Preston B. D. Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro. J Biol Chem. 1993 May 5;268(13):9793–9802. [PubMed] [Google Scholar]
  24. Lee Y. M., Coffin J. M. Relationship of avian retrovirus DNA synthesis to integration in vitro. Mol Cell Biol. 1991 Mar;11(3):1419–1430. doi: 10.1128/mcb.11.3.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lobel L. I., Goff S. P. Reverse transcription of retroviral genomes: mutations in the terminal repeat sequences. J Virol. 1985 Feb;53(2):447–455. doi: 10.1128/jvi.53.2.447-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lund A. H., Duch M., Lovmand J., Jørgensen P., Pedersen F. S. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication. J Virol. 1993 Dec;67(12):7125–7130. doi: 10.1128/jvi.67.12.7125-7130.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Olsen J. C., Bova-Hill C., Grandgenett D. P., Quinn T. P., Manfredi J. P., Swanstrom R. Rearrangements in unintegrated retroviral DNA are complex and are the result of multiple genetic determinants. J Virol. 1990 Nov;64(11):5475–5484. doi: 10.1128/jvi.64.11.5475-5484.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Omer C. A., Faras A. J. Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell. 1982 Oct;30(3):797–805. doi: 10.1016/0092-8674(82)90284-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Polacino P. S., Liang H. A., Firpo E. J., Clark E. A. T-cell activation influences initial DNA synthesis of simian immunodeficiency virus in resting T lymphocytes from macaques. J Virol. 1993 Dec;67(12):7008–7016. doi: 10.1128/jvi.67.12.7008-7016.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Richter-Cook N. J., Howard K. J., Cirino N. M., Wöhrl B. M., Le Grice S. F. Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase. J Biol Chem. 1992 Aug 5;267(22):15952–15957. [PubMed] [Google Scholar]
  39. 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]
  40. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  41. Swanstrom R., Bishop J. M., Varmus H. E. Structure of a replication intermediate in the synthesis of Rous sarcoma virus DNA in vivo. J Virol. 1982 Apr;42(1):337–341. doi: 10.1128/jvi.42.1.337-341.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wakefield J. K., Rhim H., Morrow C. D. Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site. J Virol. 1994 Mar;68(3):1605–1614. doi: 10.1128/jvi.68.3.1605-1614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]

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

RESOURCES