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. 1997 Mar;71(3):2346–2356. doi: 10.1128/jvi.71.3.2346-2356.1997

A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication.

A T Das 1, B Klaver 1, B I Klasens 1, J L van Wamel 1, B Berkhout 1
PMCID: PMC191344  PMID: 9032371

Abstract

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple hairpin motifs. The repeat region of the leader, which is reiterated at the 3' end of the RNA molecule, encodes the well-known TAR hairpin and a second hairpin structure with the polyadenylation signal AAUAAA in the single-stranded loop [the poly(A) hairpin]. The fact that this poly(A) stem-loop structure and its thermodynamic stability are well conserved among HIV and simian immunodeficiency virus isolates, despite considerable divergence in sequence, suggests a biological function for this RNA motif in viral replication. Consistent with this idea, we demonstrate that mutations that alter the stability of the stem region or delete the upper part of the hairpin do severely inhibit replication of HIV type 1. Whereas destabilizing mutations in either the left- or right-hand side of the base-paired stem interfere with virus replication, the double mutant, which allows the formation of new base pairs, replicates more rapidly than the two individual virus mutants. Upon prolonged culturing of viruses with an altered hairpin stability, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the poly(A) hairpin. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and reverse transcription of the genomic RNAs are not affected by mutation of the 5' poly(A) hairpin. We show that the genomic RNA content of the virions is reduced by destabilization of this poly(A) hairpin but not by stabilization or truncation of this structure. These results suggest that the formation of the poly(A) hairpin structure at the 5' end of the genomic RNA molecule is necessary for packaging of viral genomes into virions and/or stability of the virion RNA.

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Selected References

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  1. AUERSPERG N. LONG-TERM CULTIVATION OF HYPODIPLOID HUMAN TUMOR CELLS. J Natl Cancer Inst. 1964 Jan;32:135–163. [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. 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]
  4. Ashe M. P., Griffin P., James W., Proudfoot N. J. Poly(A) site selection in the HIV-1 provirus: inhibition of promoter-proximal polyadenylation by the downstream major splice donor site. Genes Dev. 1995 Dec 1;9(23):3008–3025. doi: 10.1101/gad.9.23.3008. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Berkhout B., Klaver B., Das A. T. A conserved hairpin structure predicted for the poly(A) signal of human and simian immunodeficiency viruses. Virology. 1995 Feb 20;207(1):276–281. doi: 10.1006/viro.1995.1077. [DOI] [PubMed] [Google Scholar]
  7. Berkhout B., Klaver B. In vivo selection of randomly mutated retroviral genomes. Nucleic Acids Res. 1993 Nov 11;21(22):5020–5024. doi: 10.1093/nar/21.22.5020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berkhout B., Klaver B. Revertants and pseudo-revertants of human immunodeficiency virus type 1 viruses mutated in the long terminal repeat promoter region. J Gen Virol. 1995 Apr;76(Pt 4):845–853. doi: 10.1099/0022-1317-76-4-845. [DOI] [PubMed] [Google Scholar]
  9. Berkhout B., Schoneveld I. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Res. 1993 Mar 11;21(5):1171–1178. doi: 10.1093/nar/21.5.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Berkhout B. Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis. Nucleic Acids Res. 1992 Jan 11;20(1):27–31. doi: 10.1093/nar/20.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Berkhout B. Structure and function of the human immunodeficiency virus leader RNA. Prog Nucleic Acid Res Mol Biol. 1996;54:1–34. doi: 10.1016/s0079-6603(08)60359-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Berkhout B., van Wamel J., Klaver B. Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions. J Mol Biol. 1995 Sep 8;252(1):59–69. doi: 10.1006/jmbi.1994.0475. [DOI] [PubMed] [Google Scholar]
  14. Brown P. H., Tiley L. S., Cullen B. R. Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences. J Virol. 1991 Jun;65(6):3340–3343. doi: 10.1128/jvi.65.6.3340-3343.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Böhnlein S., Hauber J., Cullen B. R. Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat. J Virol. 1989 Jan;63(1):421–424. doi: 10.1128/jvi.63.1.421-424.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cherrington J., Ganem D. Regulation of polyadenylation in human immunodeficiency virus (HIV): contributions of promoter proximity and upstream sequences. EMBO J. 1992 Apr;11(4):1513–1524. doi: 10.1002/j.1460-2075.1992.tb05196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Das A. T., Salvadó J., Boon L., Biharie G., Moorman A. F., Lamers W. H. Regulation of glutamate dehydrogenase expression in the developing rat liver: control at different levels in the prenatal period. Eur J Biochem. 1996 Feb 1;235(3):677–682. doi: 10.1111/j.1432-1033.1996.00677.x. [DOI] [PubMed] [Google Scholar]
  20. DeZazzo J. D., Kilpatrick J. E., Imperiale M. J. Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation. Mol Cell Biol. 1991 Mar;11(3):1624–1630. doi: 10.1128/mcb.11.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fu W., Gorelick R. J., Rein A. Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions. J Virol. 1994 Aug;68(8):5013–5018. doi: 10.1128/jvi.68.8.5013-5018.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Garcia J. A., Gaynor R. B. The human immunodeficiency virus type-1 long terminal repeat and its role in gene expression. Prog Nucleic Acid Res Mol Biol. 1994;49:157–196. doi: 10.1016/s0079-6603(08)60050-1. [DOI] [PubMed] [Google Scholar]
  24. Gilmartin G. M., Fleming E. S., Oetjen J. Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR. EMBO J. 1992 Dec;11(12):4419–4428. doi: 10.1002/j.1460-2075.1992.tb05542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gilmartin G. M., Fleming E. S., Oetjen J., Graveley B. R. CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition. Genes Dev. 1995 Jan 1;9(1):72–83. doi: 10.1101/gad.9.1.72. [DOI] [PubMed] [Google Scholar]
  26. Harrich D., Mavankal G., Mette-Snider A., Gaynor R. B. Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication. J Virol. 1995 Aug;69(8):4906–4913. doi: 10.1128/jvi.69.8.4906-4913.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Harrich D., Ulich C., Gaynor R. B. A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription. J Virol. 1996 Jun;70(6):4017–4027. doi: 10.1128/jvi.70.6.4017-4027.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Hayashi T., Ueno Y., Okamoto T. Elucidation of a conserved RNA stem-loop structure in the packaging signal of human immunodeficiency virus type 1. FEBS Lett. 1993 Jul 26;327(2):213–218. doi: 10.1016/0014-5793(93)80172-q. [DOI] [PubMed] [Google Scholar]
  31. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  32. Jones K. A., Peterlin B. M. Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem. 1994;63:717–743. doi: 10.1146/annurev.bi.63.070194.003441. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Klaver B., Berkhout B. Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus. EMBO J. 1994 Jun 1;13(11):2650–2659. doi: 10.1002/j.1460-2075.1994.tb06555.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Klaver B., Berkhout B. Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis. Nucleic Acids Res. 1994 Jan 25;22(2):137–144. doi: 10.1093/nar/22.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Pathak V. K., Temin H. M. 5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate. J Virol. 1992 May;66(5):3093–3100. doi: 10.1128/jvi.66.5.3093-3100.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peden K., Emerman M., Montagnier L. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology. 1991 Dec;185(2):661–672. doi: 10.1016/0042-6822(91)90537-l. [DOI] [PubMed] [Google Scholar]
  45. Rounseville M. P., Lin H. C., Agbottah E., Shukla R. R., Rabson A. B., Kumar A. Inhibition of HIV-1 replication in viral mutants with altered TAR RNA stem structures. Virology. 1996 Feb 15;216(2):411–417. doi: 10.1006/viro.1996.0077. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sakuragi J., Fukasawa M., Shibata R., Sakai H., Kawamura M., Akari H., Kiyomasu T., Ishimoto A., Hayami M., Adachi A. Functional analysis of long terminal repeats derived from four strains of simian immunodeficiency virus SIVAGM in relation to other primate lentiviruses. Virology. 1991 Nov;185(1):455–459. doi: 10.1016/0042-6822(91)90798-g. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Swain A., Coffin J. M. Influence of sequences in the long terminal repeat and flanking cell DNA on polyadenylation of retroviral transcripts. J Virol. 1993 Oct;67(10):6265–6269. doi: 10.1128/jvi.67.10.6265-6269.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Valsamakis A., Schek N., Alwine J. C. Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro. Mol Cell Biol. 1992 Sep;12(9):3699–3705. doi: 10.1128/mcb.12.9.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Valsamakis A., Zeichner S., Carswell S., Alwine J. C. The human immunodeficiency virus type 1 polyadenylylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylylation. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2108–2112. doi: 10.1073/pnas.88.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vicenzi E., Dimitrov D. S., Engelman A., Migone T. S., Purcell D. F., Leonard J., Englund G., Martin M. A. An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. J Virol. 1994 Dec;68(12):7879–7890. doi: 10.1128/jvi.68.12.7879-7890.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wakefield J. K., Kang S. M., Morrow C. D. Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His). J Virol. 1996 Feb;70(2):966–975. doi: 10.1128/jvi.70.2.966-975.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weichs an der Glon C., Ashe M., Eggermont J., Proudfoot N. J. Tat-dependent occlusion of the HIV poly(A) site. EMBO J. 1993 May;12(5):2119–2128. doi: 10.1002/j.1460-2075.1993.tb05860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weichs an der Glon C., Monks J., Proudfoot N. J. Occlusion of the HIV poly(A) site. Genes Dev. 1991 Feb;5(2):244–253. doi: 10.1101/gad.5.2.244. [DOI] [PubMed] [Google Scholar]
  56. Willey R. L., Smith D. H., Lasky L. A., Theodore T. S., Earl P. L., Moss B., Capon D. J., Martin M. A. In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol. 1988 Jan;62(1):139–147. doi: 10.1128/jvi.62.1.139-147.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]

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