Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Silencing of HIV-1 by AgoshRNA molecules

Gene Therapy volume 24, pages 453–461 (2017)Cite this article

Subjects

Abstract

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by the expression of a short hairpin RNA (shRNA). shRNA molecules enter the RNAi pathway at the Dicer processing step. Recent studies indicated that the cellular microRNA miR-451 is not recognized by Dicer, but that it is processed instead by the Argonaute 2 (Ago2) protein. Subsequently, Dicer-independent shRNAs were described that rely on Ago2 for processing, as well as the subsequent silencing step. We called these AgoshRNA molecules because they depend on Ago2 both for maturation and activation. Processing of an AgoshRNA yields only a single active RNA strand, thus reducing the chance of adverse off-target effects induced by the passenger strand of regular shRNAs. In this study, we converted several anti-HIV-1 shRNAs into AgoshRNAs. Seven of the 21 designed AgoshRNAs were potent anti-HIV molecules, although their RNAi activity is generally somewhat reduced compared with the matching shRNAs. The AgoshRNA candidates revealed no cellular toxicity. This may relate to the absence of passenger strand expression, which was verified for these AgoshRNA candidates. Furthermore, we demonstrate that a toxic shRNA can be converted into a non-toxic AgoshRNA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Bartel DP . MicroRNAs: target recognition and regulatory functions. Cell 2009; 136: 215–233.

    Article  CAS  Google Scholar 

  2. Brummelkamp TR, Bernards R, Agami R . A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–553.

    Article  CAS  Google Scholar 

  3. Carthew RW, Sontheimer EJ . Origins and mechanisms of miRNAs and siRNAs. Cell 2009; 136: 642–655.

    Article  CAS  Google Scholar 

  4. Berkhout B . Toward a durable anti-HIV gene therapy based on RNA interference. Ann N Y Acad Sci 2009; 1175: 3–14.

    Article  CAS  Google Scholar 

  5. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005; 436: 740–744.

    Article  CAS  Google Scholar 

  6. Jaskiewicz L, Filipowicz W . Role of Dicer in posttranscriptional RNA silencing. Curr Top Microbiol Immunol 2008; 320: 77–97.

    CAS  PubMed  Google Scholar 

  7. Siomi H, Siomi MC . Posttranscriptional regulation of microRNA biogenesis in animals. Mol Cell 2010; 38: 323–332.

    Article  CAS  Google Scholar 

  8. Yang JS, Maurin T, Robine N, Rasmussen KD, Jeffrey KL, Chandwani R et al. Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis. Proc Natl Acad Sci USA 2010; 107: 15163–15168.

    Article  CAS  Google Scholar 

  9. Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, Cheloufi S et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 2010; 328: 1694–1698.

    Article  CAS  Google Scholar 

  10. Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ . A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 2010; 465: 584–589.

    Article  CAS  Google Scholar 

  11. Yoda M, Cifuentes D, Izumi N, Sakaguchi Y, Suzuki T, Giraldez AJ et al. Poly(A)-specific ribonuclease mediates 3'-end trimming of Argonaute2-cleaved precursor microRNAs. Cell Rep 2013; 5: 715–726.

    Article  CAS  Google Scholar 

  12. Herrera-Carrillo E, Harwig A, Liu YP, Berkhout B . Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity. RNA 2014; 20: 1410–1418.

    Article  CAS  Google Scholar 

  13. Herrera-Carrillo E, Harwig A, Berkhout B . Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs. RNA Biol 2015; 12: 447–456.

    Article  Google Scholar 

  14. Harwig A, Herrera-Carrillo E, Jongejan A, van Kampen AH, Berkhout B . Deep sequence analysis of AgoshRNA processing reveals 3' A addition and trimming. Mol Ther Nucleic Acids 2015; 4: e247.

    Article  CAS  Google Scholar 

  15. Liu YP, Schopman NC, Berkhout B . Dicer-independent processing of short hairpin RNAs. Nucleic Acids Res 2013; 41: 3723–3733.

    Article  CAS  Google Scholar 

  16. Dallas A, Ilves H, Ge Q, Kumar P, Shorenstein J, Kazakov SA et al. Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing. Nucleic Acids Res 2012; 40: 9255–9271.

    Article  CAS  Google Scholar 

  17. Sun G, Yeh SY, Yuan CW, Chiu MJ, Yung BS, Yen Y . Molecular properties, functional mechanisms, and applications of sliced siRNA. Mol Ther Nucleic Acids 2015; 4: e221.

    Article  CAS  Google Scholar 

  18. Ma H, Zhang J, Wu H . Designing Ago2-specific siRNA/shRNA to avoid competition with endogenous miRNAs. Mol Ther Nucleic Acids 2014; 3: e176.

    Article  CAS  Google Scholar 

  19. Herrera-Carrillo E, Zong-liang G, Harwig A, Heemskerk MT, Berkhout B . The influence of the 5’-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site. Nucleic Acids Res 2017; 45: 4036–4050.

    Article  CAS  Google Scholar 

  20. Ma H, Wu Y, Dang Y, Choi JG, Zhang J, Wu H . Pol III promoters to express small RNAs: delineation of transcription initiation. Mol Ther Nucleic Acids 2014; 3: e161.

    Article  CAS  Google Scholar 

  21. Berkhout B, Liu YP . Towards improved shRNA and miRNA reagents as inhibitors of HIV-1 replication. Future Microbiol 2014; 9: 561–571.

    Article  CAS  Google Scholar 

  22. Coley W, Van Duyne R, Carpio L, Guendel I, Kehn-Hall K, Chevalier S et al. Absence of DICER in monocytes and its regulation by HIV-1. J Biol Chem 2010; 285: 31930–31943.

    Article  CAS  Google Scholar 

  23. Frank F, Sonenberg N, Nagar B . Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 2010; 465: 818–822.

    Article  CAS  Google Scholar 

  24. Hu HY, Yan Z, Xu Y, Hu H, Menzel C, Zhou YH et al. Sequence features associated with microRNA strand selection in humans and flies. BMC Genomics 2009; 10: 413.

    Article  Google Scholar 

  25. Liu YP, Westerink JT, Ter Brake O, Berkhout B . RNAi-inducing lentiviral vectors for anti-HIV-1 gene therapy. Methods Mol Biol 2011; 721: 293–311.

    Article  CAS  Google Scholar 

  26. Liu YP, Vink MA, Westerink JT, Ramirez de Arellano E, Konstantinova P, Ter Brake O et al. Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies. RNA 2010; 16: 1328–1339.

    Article  CAS  Google Scholar 

  27. Ter Brake O, Konstantinova P, Ceylan M, Berkhout B . Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther 2006; 14: 883–892.

    Article  CAS  Google Scholar 

  28. Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R et al. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 2004; 78: 2601–2605.

    Article  CAS  Google Scholar 

  29. 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; 185: 661–672.

    Article  CAS  Google Scholar 

  30. Centlivre M, Legrand N, Klamer S, Liu YP, Eije KJ, Bohne M et al. Preclinical in vivo evaluation of the safety of a multi-shRNA-based gene therapy against HIV-1. Mol Ther Nucleic Acids 2013; 2: e120.

    Article  Google Scholar 

  31. Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B . HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 2005; 33: 796–804.

    Article  CAS  Google Scholar 

  32. von Eije KJ, Ter Brake O, Berkhout B . Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference. J Virol 2008; 82: 2895–2903.

    Article  CAS  Google Scholar 

  33. Auwerx J . The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia 1991; 47: 22–31.

    Article  CAS  Google Scholar 

  34. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K . Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26: 171–176.

    Article  CAS  Google Scholar 

  35. Kitano K, Baldwin GC, Raines MA, Golde DW . Differentiating agents facilitate infection of myeloid leukemia cell lines by monocytotropic HIV-1 strains. Blood 1990; 76: 1980–1988.

    CAS  PubMed  Google Scholar 

  36. Ushijima H, Dairaku M, Honma H, Yamaguchi K, Shimizu H, Tsuchie H et al. Human immunodeficiency virus infection in cells of myeloid-monocytic lineage. Microbiol Immunol 1991; 35: 487–492.

    Article  CAS  Google Scholar 

  37. Ushijima H, Kunisada T, Ami Y, Tsuchie H, Takahashi I, Schacke H et al. Characterization of cells of the myeloid-monocytic lineage (ML-1, HL-60, THP-1, U-937) chronically infected with the human immunodeficiency virus-1. Pathobiology 1993; 61: 145–153.

    Article  CAS  Google Scholar 

  38. Su J, Palm A, Wu Y, Sandin S, Hoglund S, Vahlne A . Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells. AIDS Res Hum Retroviruses 2000; 16: 37–48.

    Article  CAS  Google Scholar 

  39. Khvorova A, Reynolds A, Jayasena SD . Functional siRNAs and miRNAs exhibit strand bias. Cell 2003; 115: 209–216.

    Article  CAS  Google Scholar 

  40. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD . Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115: 199–208.

    Article  CAS  Google Scholar 

  41. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A . Rational siRNA design for RNA interference. Nat Biotechnol 2004; 22: 326–330.

    Article  CAS  Google Scholar 

  42. Tomari Y, Zamore PD . Perspective: machines for RNAi. Genes Dev 2005; 19: 517–529.

    Article  CAS  Google Scholar 

  43. Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B . HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 2005; 33: 796–804.

    Article  CAS  Google Scholar 

  44. Westerhout EM, ter Brake O, Berkhout B . The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference. Retrovirology 2006; 3: 57.

    Article  Google Scholar 

  45. Westerhout EM, Berkhout B . A systematic analysis of the effect of target RNA structure on RNA interference. Nucleic Acids Res 2007; 35: 4322–4330.

    Article  CAS  Google Scholar 

  46. Low JT, Knoepfel SA, Watts JM, ter Brake O, Berkhout B, Weeks KM . SHAPE-directed discovery of potent shRNA inhibitors of HIV-1. Mol Ther 2012; 20: 820–828.

    Article  CAS  Google Scholar 

  47. Ge Q, Ilves H, Dallas A, Kumar P, Shorenstein J, Kazakov SA et al. Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity. RNA 2010; 16: 106–117.

    Article  CAS  Google Scholar 

  48. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R . Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003; 34: 263–264.

    Article  CAS  Google Scholar 

  49. Dueck A, Ziegler C, Eichner A, Berezikov E, Meister G . microRNAs associated with the different human Argonaute proteins. Nucleic Acids Res 2012; 40: 9850–9862.

    Article  CAS  Google Scholar 

  50. Petri S, Dueck A, Lehmann G, Putz N, Rudel S, Kremmer E et al. Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. RNA 2011; 17: 737–749.

    Article  CAS  Google Scholar 

  51. Aleman LM, Doench J, Sharp PA . Comparison of siRNA-induced off-target RNA and protein effects. RNA 2007; 13: 385–395.

    Article  CAS  Google Scholar 

  52. Wu L, Fan J, Belasco JG . Importance of translation and nonnucleolytic ago proteins for on-target RNA interference. Curr Biol 2008; 18: 1327–1332.

    Article  CAS  Google Scholar 

  53. Vickers TA, Lima WF, Wu H, Nichols JG, Linsley PS, Crooke ST . Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1. Nucleic Acids Res 2009; 37: 6927–6941.

    Article  CAS  Google Scholar 

  54. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC . Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806–811.

    Article  CAS  Google Scholar 

  55. Ruijter JM, Thygesen HH, Schoneveld OJ, Das AT, Berkhout B, Lamers WH . Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology. Retrovirology 2006; 3: 2.

    Article  Google Scholar 

  56. Herrera-Carrillo E, Gao ZL, Harwig A, Heemskerk MT, Berkhout B . The influence of the 5-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site. Nucleic Acids Res 2017; 45: 4036–4050.

    Article  CAS  Google Scholar 

  57. Seppen J, Rijnberg M, Cooreman MP, Oude Elferink RP . Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes. J Hepatol 2002; 36: 459–465.

    Article  CAS  Google Scholar 

  58. Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA . A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 2000; 74: 4839–4852.

    Article  CAS  Google Scholar 

  59. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Konstantinova P, de Haan P, Das AT, Berkhout B . Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1. Nucleic acids Res 2006; 34: 2206–2218.

    Article  CAS  Google Scholar 

  61. Eekels JJ, Pasternak AO, Schut AM, Geerts D, Jeeninga RE, Berkhout B . A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation. Gene Ther 2012; 19: 1058–1064.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek—Chemische Wetenschappen (NWO-CW, Top Grant) and Zorg Onderzoek Nederland—Medische Wetenschappen (ZonMw, Translational Gene Therapy Grant). We thank Berend Hooibrink for expertise in cell sorting and maintenance of the flow cytometry facility. We thank Cosimo Cristella for valuable help with the statistical analyses.

Author information

Authors and Affiliations

  1. Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

    E Herrera-Carrillo, A Harwig & B Berkhout

Authors
  1. E Herrera-Carrillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Harwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B Berkhout
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B Berkhout.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Herrera-Carrillo, E., Harwig, A. & Berkhout, B. Silencing of HIV-1 by AgoshRNA molecules. Gene Ther 24, 453–461 (2017). https://doi.org/10.1038/gt.2017.44

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2017.44

Search

Advanced search

Quick links