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. 1994 May 24;91(11):4945–4949. doi: 10.1073/pnas.91.11.4945

Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.

E Skripkin 1, J C Paillart 1, R Marquet 1, B Ehresmann 1, C Ehresmann 1
PMCID: PMC43906  PMID: 8197162

Abstract

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides 1-707 of HIV-1 RNA. These nucleotides map exclusively in a restricted area upstream of the splice donor site and downstream of the primer binding site. They are centered around a palindromic sequence (GUGCAC279) located in a hairpin loop. Our results support a model in which dimer formation is initiated by the annealing of the palindromic sequences, possibly by a loop-loop interaction between the two monomers. Further experiments show that the deletion of the stem-loop or base substitutions in the loop abolish dimerization, despite the presence of the previously postulated dimer linkage structure. On the other hand, deletions of the purine tracts downstream of the splice donor site do not prevent dimerization. Therefore, we conclude that the palindromic region represents the dimerization initiation site of genomic RNA.

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Selected References

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  1. Alford R. L., Honda S., Lawrence C. B., Belmont J. W. RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus. Virology. 1991 Aug;183(2):611–619. doi: 10.1016/0042-6822(91)90990-s. [DOI] [PubMed] [Google Scholar]
  2. Awang G., Sen D. Mode of dimerization of HIV-1 genomic RNA. Biochemistry. 1993 Oct 26;32(42):11453–11457. doi: 10.1021/bi00093a024. [DOI] [PubMed] [Google Scholar]
  3. Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
  4. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bender W., Chien Y. H., Chattopadhyay S., Vogt P. K., Gardner M. B., Davidson N. High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures. J Virol. 1978 Mar;25(3):888–896. doi: 10.1128/jvi.25.3.888-896.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bender W., Davidson N. Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules. Cell. 1976 Apr;7(4):595–607. doi: 10.1016/0092-8674(76)90210-5. [DOI] [PubMed] [Google Scholar]
  7. Berkhout B., Essink B. B., Schoneveld I. In vitro dimerization of HIV-2 leader RNA in the absence of PuGGAPuA motifs. FASEB J. 1993 Jan;7(1):181–187. doi: 10.1096/fasebj.7.1.8422965. [DOI] [PubMed] [Google Scholar]
  8. Bieth E., Gabus C., Darlix J. L. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 1990 Jan 11;18(1):119–127. doi: 10.1093/nar/18.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clavel F., Orenstein J. M. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. doi: 10.1128/jvi.64.10.5230-5234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Darlix J. L., Gabus C., Allain B. Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro. J Virol. 1992 Dec;66(12):7245–7252. doi: 10.1128/jvi.66.12.7245-7252.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
  12. Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hayashi T., Shioda T., Iwakura Y., Shibuta H. RNA packaging signal of human immunodeficiency virus type 1. Virology. 1992 Jun;188(2):590–599. doi: 10.1016/0042-6822(92)90513-o. [DOI] [PubMed] [Google Scholar]
  14. Hu W. S., Temin H. M. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1556–1560. doi: 10.1073/pnas.87.4.1556. [DOI] [PMC free article] [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. Katoh I., Yasunaga T., Yoshinaka Y. Bovine leukemia virus RNA sequences involved in dimerization and specific gag protein binding: close relation to the packaging sites of avian, murine, and human retroviruses. J Virol. 1993 Apr;67(4):1830–1839. doi: 10.1128/jvi.67.4.1830-1839.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kung H. J., Hu S., Bender W., Bailey J. M., Davidson N., Nicolson M. O., McAllister R. M. RD-114, baboon, and woolly monkey viral RNA's compared in size and structure. Cell. 1976 Apr;7(4):609–620. doi: 10.1016/0092-8674(76)90211-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Luo G. X., Taylor J. Template switching by reverse transcriptase during DNA synthesis. J Virol. 1990 Sep;64(9):4321–4328. doi: 10.1128/jvi.64.9.4321-4328.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Marquet R., Paillart J. C., Skripkin E., Ehresmann C., Ehresmann B. Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site. Nucleic Acids Res. 1994 Jan 25;22(2):145–151. doi: 10.1093/nar/22.2.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mougel M., Tounekti N., Darlix J. L., Paoletti J., Ehresmann B., Ehresmann C. Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization. Nucleic Acids Res. 1993 Oct 11;21(20):4677–4684. doi: 10.1093/nar/21.20.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murti K. G., Bondurant M., Tereba A. Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy. J Virol. 1981 Jan;37(1):411–419. doi: 10.1128/jvi.37.1.411-419.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Persson C., Wagner E. G., Nordström K. Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT. EMBO J. 1990 Nov;9(11):3767–3775. doi: 10.1002/j.1460-2075.1990.tb07590.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol. 1990 Feb;64(2):774–783. doi: 10.1128/jvi.64.2.774-783.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Richardson J. H., Child L. A., Lever A. M. Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region. J Virol. 1993 Jul;67(7):3997–4005. doi: 10.1128/jvi.67.7.3997-4005.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roy C., Tounekti N., Mougel M., Darlix J. L., Paoletti C., Ehresmann C., Ehresmann B., Paoletti J. An analytical study of the dimerization of in vitro generated RNA of Moloney murine leukemia virus MoMuLV. Nucleic Acids Res. 1990 Dec 25;18(24):7287–7292. doi: 10.1093/nar/18.24.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sakaguchi K., Zambrano N., Baldwin E. T., Shapiro B. A., Erickson J. W., Omichinski J. G., Clore G. M., Gronenborn A. M., Appella E. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5219–5223. doi: 10.1073/pnas.90.11.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stuhlmann H., Berg P. Homologous recombination of copackaged retrovirus RNAs during reverse transcription. J Virol. 1992 Apr;66(4):2378–2388. doi: 10.1128/jvi.66.4.2378-2388.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sundquist W. I., Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3393–3397. doi: 10.1073/pnas.90.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Temin H. M. Sex and recombination in retroviruses. Trends Genet. 1991 Mar;7(3):71–74. doi: 10.1016/0168-9525(91)90272-R. [DOI] [PubMed] [Google Scholar]
  33. Tounekti N., Mougel M., Roy C., Marquet R., Darlix J. L., Paoletti J., Ehresmann B., Ehresmann C. Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. J Mol Biol. 1992 Jan 5;223(1):205–220. doi: 10.1016/0022-2836(92)90726-z. [DOI] [PubMed] [Google Scholar]
  34. Weiss S., Häusl G., Famulok M., König B. The multimerization state of retroviral RNA is modulated by ammonium ions and affects HIV-1 full-length cDNA synthesis in vitro. Nucleic Acids Res. 1993 Oct 25;21(21):4879–4885. doi: 10.1093/nar/21.21.4879. [DOI] [PMC free article] [PubMed] [Google Scholar]

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