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. 1997 Mar;71(3):2050–2058. doi: 10.1128/jvi.71.3.2050-2058.1997

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

M S McBride 1, A T Panganiban 1
PMCID: PMC191293  PMID: 9032337

Abstract

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.

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Selected References

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  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. Aronoff R., Hajjar A. M., Linial M. L. Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs. J Virol. 1993 Jan;67(1):178–188. doi: 10.1128/jvi.67.1.178-188.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Berkhout B., van Wamel J. L. Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J Virol. 1996 Oct;70(10):6723–6732. doi: 10.1128/jvi.70.10.6723-6732.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berlioz C., Darlix J. L. An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors. J Virol. 1995 Apr;69(4):2214–2222. doi: 10.1128/jvi.69.4.2214-2222.1995. [DOI] [PMC free article] [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. Buchschacher G. L., Jr, Panganiban A. T. Human immunodeficiency virus vectors for inducible expression of foreign genes. J Virol. 1992 May;66(5):2731–2739. doi: 10.1128/jvi.66.5.2731-2739.1992. [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. Clever J. L., Wong M. L., Parslow T. G. Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA. J Virol. 1996 Sep;70(9):5902–5908. doi: 10.1128/jvi.70.9.5902-5908.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clever J., Sassetti C., Parslow T. G. RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. J Virol. 1995 Apr;69(4):2101–2109. doi: 10.1128/jvi.69.4.2101-2109.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Garzino-Demo A., Gallo R. C., Arya S. K. Human immunodeficiency virus type 2 (HIV-2): packaging signal and associated negative regulatory element. Hum Gene Ther. 1995 Feb;6(2):177–184. doi: 10.1089/hum.1995.6.2-177. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Jang S. K., Pestova T. V., Hellen C. U., Witherell G. W., Wimmer E. Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site. Enzyme. 1990;44(1-4):292–309. doi: 10.1159/000468766. [DOI] [PubMed] [Google Scholar]
  17. Kaye J. F., Richardson J. H., Lever A. M. cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging. J Virol. 1995 Oct;69(10):6588–6592. doi: 10.1128/jvi.69.10.6588-6592.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim H. J., Lee K., O'Rear J. J. A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA. Virology. 1994 Jan;198(1):336–340. doi: 10.1006/viro.1994.1037. [DOI] [PubMed] [Google Scholar]
  19. Konings D. A., Nash M. A., Maizel J. V., Arlinghaus R. B. Novel GACG-hairpin pair motif in the 5' untranslated region of type C retroviruses related to murine leukemia virus. J Virol. 1992 Feb;66(2):632–640. doi: 10.1128/jvi.66.2.632-640.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laughrea M., Jetté L. A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA. Biochemistry. 1994 Nov 15;33(45):13464–13474. doi: 10.1021/bi00249a035. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Luban J., Goff S. P. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994 Jun;68(6):3784–3793. doi: 10.1128/jvi.68.6.3784-3793.1994. [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. McBride M. S., Panganiban A. T. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol. 1996 May;70(5):2963–2973. doi: 10.1128/jvi.70.5.2963-2973.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miele G., Mouland A., Harrison G. P., Cohen E., Lever A. M. The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. J Virol. 1996 Feb;70(2):944–951. doi: 10.1128/jvi.70.2.944-951.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Muriaux D., Girard P. M., Bonnet-Mathonière B., Paoletti J. Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide. J Biol Chem. 1995 Apr 7;270(14):8209–8216. doi: 10.1074/jbc.270.14.8209. [DOI] [PubMed] [Google Scholar]
  27. Paillart J. C., Berthoux L., Ottmann M., Darlix J. L., Marquet R., Ehresmann B., Ehresmann C. A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis. J Virol. 1996 Dec;70(12):8348–8354. doi: 10.1128/jvi.70.12.8348-8354.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Paillart J. C., Marquet R., Skripkin E., Ehresmann B., Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. J Biol Chem. 1994 Nov 4;269(44):27486–27493. [PubMed] [Google Scholar]
  29. Parolin C., Dorfman T., Palú G., Göttlinger H., Sodroski J. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes. J Virol. 1994 Jun;68(6):3888–3895. doi: 10.1128/jvi.68.6.3888-3895.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Skripkin E., Paillart J. C., Marquet R., Ehresmann B., Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4945–4949. doi: 10.1073/pnas.91.11.4945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsukiyama-Kohara K., Iizuka N., Kohara M., Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol. 1992 Mar;66(3):1476–1483. doi: 10.1128/jvi.66.3.1476-1483.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vagner S., Waysbort A., Marenda M., Gensac M. C., Amalric F., Prats A. C. Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor. J Biol Chem. 1995 Sep 1;270(35):20376–20383. doi: 10.1074/jbc.270.35.20376. [DOI] [PubMed] [Google Scholar]
  34. Yang S., Temin H. M. A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA. EMBO J. 1994 Feb 1;13(3):713–726. doi: 10.1002/j.1460-2075.1994.tb06311.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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