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. 1997 Mar 1;25(5):940–947. doi: 10.1093/nar/25.5.940

Forced evolution of a regulatory RNA helix in the HIV-1 genome.

B Berkhout 1, B Klaver 1, A T Das 1
PMCID: PMC146548  PMID: 9023102

Abstract

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Brown M. D., DeYoung K. L., Hall D. H. A non-directed, hydroxylamine-generated suppressor mutation in the P3 pairing region of the bacteriophage T4 td intron partially restores self-splicing capability. Mol Microbiol. 1994 Jul;13(1):89–95. doi: 10.1111/j.1365-2958.1994.tb00404.x. [DOI] [PubMed] [Google Scholar]
  10. Cao X., Wimmer E. Genetic variation of the poliovirus genome with two VPg coding units. EMBO J. 1996 Jan 2;15(1):23–33. [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Das A. T., Klaver B., Berkhout B. Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). J Virol. 1995 May;69(5):3090–3097. doi: 10.1128/jvi.69.5.3090-3097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Das A. T., Klaver B., Klasens B. I., van Wamel J. L., Berkhout B. A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication. J Virol. 1997 Mar;71(3):2346–2356. doi: 10.1128/jvi.71.3.2346-2356.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Domingo E., Escarmís C., Sevilla N., Moya A., Elena S. F., Quer J., Novella I. S., Holland J. J. Basic concepts in RNA virus evolution. FASEB J. 1996 Jun;10(8):859–864. doi: 10.1096/fasebj.10.8.8666162. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Gmyl A. P., Pilipenko E. V., Maslova S. V., Belov G. A., Agol V. I. Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants. J Virol. 1993 Oct;67(10):6309–6316. doi: 10.1128/jvi.67.10.6309-6316.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hertz J. M., Huang H. V. Evolution of the Sindbis virus subgenomic mRNA promoter in cultured cells. J Virol. 1995 Dec;69(12):7768–7774. doi: 10.1128/jvi.69.12.7768-7774.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoffman M. A., Palmenberg A. C. Revertant analysis of J-K mutations in the encephalomyocarditis virus internal ribosomal entry site detects an altered leader protein. J Virol. 1996 Sep;70(9):6425–6430. doi: 10.1128/jvi.70.9.6425-6430.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huynen M. A., Stadler P. F., Fontana W. Smoothness within ruggedness: the role of neutrality in adaptation. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):397–401. doi: 10.1073/pnas.93.1.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobson S. J., Konings D. A., Sarnow P. Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification. J Virol. 1993 Jun;67(6):2961–2971. doi: 10.1128/jvi.67.6.2961-2971.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Keulen W., Boucher C., Berkhout B. Nucleotide substitution patterns can predict the requirements for drug-resistance of HIV-1 proteins. Antiviral Res. 1996 Jun;31(1-2):45–57. doi: 10.1016/0166-3542(96)00944-8. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Macadam A. J., Ferguson G., Burlison J., Stone D., Skuce R., Almond J. W., Minor P. D. Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture. Virology. 1992 Aug;189(2):415–422. doi: 10.1016/0042-6822(92)90565-7. [DOI] [PubMed] [Google Scholar]
  29. Olsthoorn R. C., Licis N., van Duin J. Leeway and constraints in the forced evolution of a regulatory RNA helix. EMBO J. 1994 Jun 1;13(11):2660–2668. doi: 10.1002/j.1460-2075.1994.tb06556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  33. Qu F., Heinrich C., Loss P., Steger G., Tien P., Riesner D. Multiple pathways of reversion in viroids for conservation of structural elements. EMBO J. 1993 May;12(5):2129–2139. doi: 10.1002/j.1460-2075.1993.tb05861.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rieger A., Nassal M. Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system. Nucleic Acids Res. 1995 Oct 11;23(19):3909–3915. doi: 10.1093/nar/23.19.3909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Vartanian J. P., Meyerhans A., Asjö B., Wain-Hobson S. Selection, recombination, and G----A hypermutation of human immunodeficiency virus type 1 genomes. J Virol. 1991 Apr;65(4):1779–1788. doi: 10.1128/jvi.65.4.1779-1788.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Zhang C., Tellinghuisen T., Guo P. Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29. RNA. 1995 Dec;1(10):1041–1050. [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. de la Torre J. C., Giachetti C., Semler B. L., Holland J. J. High frequency of single-base transitions and extreme frequency of precise multiple-base reversion mutations in poliovirus. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2531–2535. doi: 10.1073/pnas.89.7.2531. [DOI] [PMC free article] [PubMed] [Google Scholar]

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