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. 1994 Aug;68(8):5013–5018. doi: 10.1128/jvi.68.8.5013-5018.1994

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

W Fu 1, R J Gorelick 1, A Rein 1
PMCID: PMC236443  PMID: 8035501

Abstract

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.

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Selected References

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  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. 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. Bader J. P., Steck T. L. Analysis of the ribonucleic acid of murine leukemia virus. J Virol. 1969 Oct;4(4):454–459. doi: 10.1128/jvi.4.4.454-459.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Bowles N. E., Damay P., Spahr P. F. Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein. J Virol. 1993 Feb;67(2):623–631. doi: 10.1128/jvi.67.2.623-631.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Canaani E., Duesberg P. Role of subunits of 60 to 70S avian tumor virus ribonucleic acid in its template activity for the viral deoxyribonucleic acid polymerase. J Virol. 1972 Jul;10(1):23–31. doi: 10.1128/jvi.10.1.23-31.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheung K. S., Smith R. E., Stone M. P., Joklik W. K. Comparison of immature (rapid harvest) and mature Rous sarcoma virus particles. Virology. 1972 Dec;50(3):851–864. doi: 10.1016/0042-6822(72)90439-4. [DOI] [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. Duesberg P. H. Physical properties of Rous Sarcoma Virus RNA. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1511–1518. doi: 10.1073/pnas.60.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dupraz P., Oertle S., Meric C., Damay P., Spahr P. F. Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. J Virol. 1990 Oct;64(10):4978–4987. doi: 10.1128/jvi.64.10.4978-4987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fu W., Rein A. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J Virol. 1993 Sep;67(9):5443–5449. doi: 10.1128/jvi.67.9.5443-5449.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gorelick R. J., Henderson L. E., Hanser J. P., Rein A. Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8420–8424. doi: 10.1073/pnas.85.22.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  18. Holbrook S. R., Sussman J. L., Warrant R. W., Church G. M., Kim S. H. RNA-ligant interactions. (I) Magnesium binding sites in yeast tRNAPhe. Nucleic Acids Res. 1977 Aug;4(8):2811–2820. doi: 10.1093/nar/4.8.2811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khandjian E. W., Méric C. A procedure for Northern blot analysis of native RNA. Anal Biochem. 1986 Nov 15;159(1):227–232. doi: 10.1016/0003-2697(86)90332-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Levin J. G., Grimley P. M., Ramseur J. M., Berezesky I. K. Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J Virol. 1974 Jul;14(1):152–161. doi: 10.1128/jvi.14.1.152-161.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Méric C., Goff S. P. Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol. 1989 Apr;63(4):1558–1568. doi: 10.1128/jvi.63.4.1558-1568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Méric C., Spahr P. F. Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging. J Virol. 1986 Nov;60(2):450–459. doi: 10.1128/jvi.60.2.450-459.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oertle S., Spahr P. F. Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA. J Virol. 1990 Dec;64(12):5757–5763. doi: 10.1128/jvi.64.12.5757-5763.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Stein A., Crothers D. M. Conformational changes of transfer RNA. The role of magnesium(II). Biochemistry. 1976 Jan 13;15(1):160–168. doi: 10.1021/bi00646a025. [DOI] [PubMed] [Google Scholar]
  32. Stewart L., Schatz G., Vogt V. M. Properties of avian retrovirus particles defective in viral protease. J Virol. 1990 Oct;64(10):5076–5092. doi: 10.1128/jvi.64.10.5076-5092.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stoltzfus C. M., Snyder P. N. Structure of B77 sarcoma virus RNA: stabilization of RNA after packaging. J Virol. 1975 Nov;16(5):1161–1170. doi: 10.1128/jvi.16.5.1161-1170.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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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