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Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Nature Immunology volume 16, pages 1025–1033 (2015)Cite this article

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Abstract

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

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Figure 1: Immunostimulation by stem-loop-forming structures in HIV-1 sstDNA depends on unpaired guanosines flanking the stem.
Figure 2: Guanosine extensions render short DNA duplexes highly immunostimulatory.
Figure 3: Induction of IFN-α/β by Y-form DNA is independent of the G quadruplex.
Figure 4: G-YSD activates the cGAS-STING axis.
Figure 5: The interferon response induced by HIV-1 infection correlates with the presence of (−)-strand DNA but not with the presence of (+)-strand DNA.
Figure 6: Long ssDNA comprising the 5′-terminal HIV-1 (−)-strand sequence is highly immunostimulatory, and the recognition of endogenous retroelement-derived ssDNA depends on unpaired guanosines.

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References

  1. Krieg, A.M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Hartmann, G. et al. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells. Eur. J. Immunol. 33, 1633–1641 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fernandes-Alnemri, T., Yu, J.W., Datta, P., Wu, J. & Alnemri, E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bürckstümmer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat. Immunol. 10, 266–272 (2009).

    Article  PubMed  CAS  Google Scholar 

  7. Ablasser, A. et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat. Immunol. 10, 1065–1072 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Chiu, Y.H., Macmillan, J.B. & Chen, Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576–591 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schlee, M. et al. Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 31, 25–34 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cavlar, T., Ablasser, A. & Hornung, V. Induction of type I IFNs by intracellular DNA-sensing pathways. Immunol. Cell Biol. 90, 474–482 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Stetson, D.B. & Medzhitov, R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24, 93–103 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Yang, P. et al. The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway. Nat. Immunol. 11, 487–494 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Bagashev, A. et al. Leucine-rich repeat (in Flightless I) interacting protein-1 regulates a rapid type I interferon response. J. Interferon Cytokine Res. 30, 843–852 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhang, X. et al. Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN. J. Immunol. 186, 4541–4545 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Kim, T. et al. Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells. Proc. Natl. Acad. Sci. USA 107, 15181–15186 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ishikawa, H., Ma, Z. & Barber, G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhong, B. et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 29, 538–550 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Unterholzner, L. et al. IFI16 is an innate immune sensor for intracellular DNA. Nat. Immunol. 11, 997–1004 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Jakobsen, M.R. & Paludan, S.R. IFI16: At the interphase between innate DNA sensing and genome regulation. Cytokine Growth Factor Rev. 25, 649–655 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Civril, F. et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 498, 332–337 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kato, K. et al. Structural and functional analyses of DNA-sensing and immune activation by human cGAS. PLoS ONE 8, e76983 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kranzusch, P.J., Lee, A.S., Berger, J.M. & Doudna, J.A. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Rep. 3, 1362–1368 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gao, D. et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 341, 903–906 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Zhang, X. et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Rep. 6, 421–430 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li, X. et al. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity 39, 1019–1031 (2013).

    Article  CAS  PubMed  Google Scholar 

  30. Li, X.D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Lam, E., Stein, S. & Falck-Pedersen, E. Adenovirus detection by the cGAS/STING/TBK1 DNA sensing cascade. J. Virol. 88, 974–981 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Lahaye, X. et al. The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells. Immunity 39, 1132–1142 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Rasaiyaah, J. et al. HIV-1 evades innate immune recognition through specific cofactor recruitment. Nature 503, 402–405 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Suzuki, K. et al. Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. Proc. Natl. Acad. Sci. USA 96, 2285–2290 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ishii, K.J. et al. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat. Immunol. 7, 40–48 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Jakobsen, M.R. et al. From the Cover: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication. Proc. Natl. Acad. Sci. USA 110, E4571–E4580 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Galvis, A.E., Fisher, H.E., Nitta, T., Fan, H. & Camerini, D. Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown. J. Virol. 88, 7054–7069 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Gehrke, N. et al. Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing. Immunity 39, 482–495 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Huppert, J.L. Four-stranded nucleic acids: structure, function and targeting of G-quadruplexes. Chem. Soc. Rev. 37, 1375–1384 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Murchie, A.I. & Lilley, D.M. Retinoblastoma susceptibility genes contain 5′ sequences with a high propensity to form guanine-tetrad structures. Nucleic Acids Res. 20, 49–53 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hagmann, C.A. et al. RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells. PLoS ONE 8, e62872 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Takaoka, A. et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448, 501–505 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, Z. et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat. Immunol. 12, 959–965 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fisher, T.S., Darden, T. & Prasad, V.R. Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template. J. Virol. 77, 5837–5845 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Mankan, A.K. et al. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis. EMBO J. 33, 2937–2946 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Stetson, D.B., Ko, J.S., Heidmann, T. & Medzhitov, R. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 134, 587–598 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ablasser, A. et al. TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner. J. Immunol. 192, 5993–5997 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Yoh, S.M. et al. PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1. Cell 161, 1293–1305 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Watts, J.M. et al. Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460, 711–716 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. van der Kuyl, A.C. & Berkhout, B. The biased nucleotide composition of the HIV genome: a constant factor in a highly variable virus. Retrovirology 9, 92 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. van Hemert, F.J., van der Kuyl, A.C. & Berkhout, B. The A-nucleotide preference of HIV-1 in the context of its structured RNA genome. RNA Biol. 10, 211–215 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Martin-Gayo, E. et al. Potent cell-intrinsic immune responses in dendritic cells facilitate HIV-1-specific T cell immunity in HIV-1 elite controllers. PLoS Pathog. 11, e1004930 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Gall, A. et al. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 36, 120–131 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bauernfeind, F. et al. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J. Immunol. 187, 613–617 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Jin, T. et al. Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 36, 561–571 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Siering for help with circular dichroism spectroscopy, and S. Schmitt for discussions. Supported by Deutsche Forschungsgemeinschaft (SFB670 to M.S., W.B., V.H. and G.H.; DFG SCHL1930/1–1 to M.S.; SFB704 to G.H., V.H. and W.B.; and SFB832 and KFO177 to C.C. and G.H.), the Deutsche Forschungsgemeinschaft Excellence Cluster ImmunoSensation (G.H., M.S., V.H., E.B. and W.B.), BONFOR of the University of Bonn (E.B.) and the German Center of Infectious Disease (G.H., V.H. and W.B.).

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Author notes

  1. Cristina Amparo Hagmann & Damian Ackermann

    Present address: Present addresses: Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA (C.A.H.), and Microsynth, Balgach, Switzerland (D.A.).,

  2. Anna-Maria Herzner, Cristina Amparo Hagmann, Gunther Hartmann and Martin Schlee: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany

    Anna-Maria Herzner, Cristina Amparo Hagmann, Marion Goldeck, Steven Wolter, Christina Mertens, Thomas Zillinger, Eva Bartok, Christoph Coch, Janos Ludwig, Winfried Barchet, Gunther Hartmann & Martin Schlee

  2. Department of Obstetrics and Gynecology, Center for Integrated Oncology, University of Bonn, Bonn, Germany

    Kirsten Kübler

  3. Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg,, Erlangen, Germany

    Sabine Wittmann & Thomas Gramberg

  4. Department Biochemistry, Gene Center, Ludwig-Maximilians University, Munich, Germany

    Liudmila Andreeva & Karl-Peter Hopfner

  5. German Center of Infectious Disease, Cologne-Bonn, Germany.,

    Thomas Zillinger & Winfried Barchet

  6. School of Life Sciences, University of Science and Technology of China,, Hefei, China

    Tengchuan Jin

  7. Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

    Tsan Sam Xiao

  8. LIMES Institute, Chemical Biology, University of Bonn, Bonn, Germany

    Damian Ackermann

  9. Institute of Molecular Medicine, University Hospital, University of Bonn, Bonn, Germany

    Veit Hornung

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Contributions

M.S., A.-M.H., J.L., C.A.H. and G.H., conceptualization; M.S., A.-M.H., C.A.H., M.G., S. Wolter, K.K., T.G., L.A., K.-P.H., T.Z., C.M., T.J., T.S.X. and D.A., methodology; A.-M.H., C.A.H., M.G., D.A., T.J., T.S.X. and M.S., formal analysis; A.-M.H., M.S., C.A.H., M.G., S. Wolter, T.G., L.A., T.Z., C.M. and T.J., investigation; M.S., A.-M.H., E.B., C.A.H., V.H., W.B. and G.H., writing of the original draft; A.-M.H., M.S., E.B., W.B., C.C. and G.H., writing (review and editing); M.S., G.H., V.H., W.B., C.C., E.B., K.-P.H. and T.G., funding acquisition; T.J., T.S.X., S. Wittmann, T.G., L.A. and K.-P.H., resources; and M.S., T.G., K.-P.H., C.C., W.B. and G.H., supervision.

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Correspondence to Anna-Maria Herzner or Martin Schlee.

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Integrated supplementary information

Supplementary Figure 1 Stimulation of various human blood cells and structural analysis of SL2 derivatives.

(a– c) ELISA of IFN-α in the supernatant of human primary cells, treated for 20 h. (a) Chloroquine-treated PBMCs, transfected with blunt-ended non-palindromic DNAs of different lengths as indicated by models below plot. Numbers adjacent to models, stem length. IFN-α results are presented as relative to those of cells transfected with 84-nucleotide DNA, set as 100%. (b) Cells were transfected with the TLR9-ligand CpG ODN 2216 (CpG), ISD-pathway activating human genomic DNA (gDNA), TLR9- and ISD-pathway-activating plasmid DNA (pDNA), ISD-pathway activating poly(dAdT) or treated with medium alone (Med). Top: IFN-α secretion by CD14-depleted human PBMCs (white bars), untreated PBMCs (gray) or monocytes (black). Bottom: IFN-α secretion by human untreated PBMCs (white bars), chloroquine-treated PBMCs (light gray) or pDC-depleted PBMCs (dark gray). (c) Choroquine-treated PBMCs transfected with SL2 variants derived from HIV-1 strain HXB2 (SL2 HIV-1 HXB2; as used in Fig. 1), isolate 45_cpx.CD.97.97CD_MBFE185.FN392874 (SL2 HIV-1 #45) as well as a Simian Immunodeficiency Virus strain (SL2 SIV; SIV isolate CPZ.US.85.US_Marilyn.AF103818). Structures calculated by mFOLD server, below plots. (d) Left: Native PAGE analysis of HIV-1-derived ssDNA species (15%), staining with GelGreen. 1: SL2+3 wt, 2: SL2+3 ∆G, 3: SL2 wt, 4: SL2 ∆G, as in Fig.1. Right: Melting curve analysis of structured HIV-1-derived ssDNA species. Numbering as in left panel. (e,f) ELISA of IFN-α in the supernatant of monocytes (e) or monocyte-derived macrophages (f), 20 h (e) or 36 h (f) after transfection of DNA structures as in Fig.1, transfection with human genomic DNA (gDNA) or treatment with medium alone (Med). (a–f) Data are pooled from two (a), four (c) or three (e,f) with two biological replicates in each ((a,c,e,f; mean and s.e.m. of n = 4 donors (a), n = 8 donors (c) or n = 6 donors (e,f)) or are one representative of two experiments (b,d); technical duplicates are displayed in b (mean and s.d.). Sequences of DNA-structures, Supplementary Table 1.

Supplementary Figure 2 G-YSD motif variation and stimulation of monocytes, monocyte-derived macrophages and mouse macrophages.

(a,b) ELISA of IFN-α in the supernatant of human chloroquine-treated PBMC, treated for 20 h. (a) Transfection with DNAs with variations of guanosine numbers and positions in 5′ or 3′ overhangs of YSDs, as indicated by models below plots, transfection with genomic DNA (gDNA) or treatment with medium alone (Med). (b) Transfection of ISD, or derivatives of ISD with addition of indicated G3- or C3-overhangs, as indicated by models below plots, transfection of G3-YSD or treatment with medium alone. Results are presented as relative to those of cells transfected with G3-YSD-transfection (Pos.6), set as 100%. Statistical analysis by one-way-ANOVA and Tukey’s post-hoc test. *, P ≤ 0,001; NS, not significant. (c,d): ELISA of IFN-α in the supernatant of monocytes (c) or monocyte-derived macrophages (d), 20 h (c) or 36 h (d) after transfection. Med, treatment with medium alone. (e) Ifnb mRNA expression by murine immortalized macrophages, 6 h after transfection with the indicated DNA stimuli. Results are displayed relative to those of cells transfected with G3-YSD (Pos.1), set as 100%. Below plots, models of secondary structures (sequences, Supplementary Table 1). Numbers close to stems, stem length. (a–e) Data are pooled from two (a,b) or three (c,d,e) experiments with one or two biological replicates in each (mean and s.e.m.; a,b, n = 4 donors; c,d, n = 6 donors; e, n = 3 independent experiments)..

Supplementary Figure 3 G3 overhangs do not increase cytosolic availability or confer stability against DNases.

(a) Denaturing polyacrylamide gel electrophoresis (PAGE) of 5′-IRD700-labeled DNA, recovered from cytosolic lysates of transfected THP-1 cells, 4 h or 8 h after transfection. The labeled strands were visualized by IRD700 fluorescence detection. Arrow tips mark free IRD700 dye (species can neither be digested with DNaseI nor ethanol-precipitated). Slowly migrating species blunt-ended DNAs (2+3) represent non-denatured duplexes (determined by gel staining with gel dyes differentially or equally-staining dsDNA vs ssDNA, i.e. GelRed and GelGreen). Loading controls: Hybridized, untransfected DNA, treated with Proteinase K. Data shown is representative of two experiments. (b) TREX-1 digestion of the indicated DNA species. DNA integrity was measured by SYBR Green Fluorescence intensity over time. (c) Calculated half-lives on the representative DNA species in the presence of the indicated DNases. Half-lives, their 95% confidence interval (95% Conf. int.) and r2 were calculated by one-phase decay analysis using Prism 6 software.

Supplementary Figure 4 RNAi controls and IFI16 interaction in solution.

(a) Expression of MAVS mRNA (left) or TMEM173 mRNA (STING encoding, right) in cells treated with siRNAs for 72 h as in Fig. 4a. (b) Determination of Kd values of IFI16 binding to YSD or short blunt DNA by fluorescence polarization. (c) Controls for Fig. 4e: Left: Expression of MB21D1 mRNA (cGAS encoding) in cells treated with the indicated siRNAs for 48 h. Right: IFN-α/β activity in the supernatant of cells treated with the indicated siRNAs, and afterwards stimulated for 20 h with 3P-dsRNA. Results are presented relative to those of cells treated with siRNA control. (d) RNAi targeting IFI16 or STING in THP-1 cells. Left: Expression of IFI16 mRNA in siRNA-treated cells, 72 h after electroporation. Right: THP-1 cells were treated with the indicated siRNAs for 72 h and stimulated afterwards for 20 h. IFN-α/β activities in the supernatant displayed relative to those of cells treated with siRNA control. (a–d) Data are representative of three independent experiments (b) or pooled from three (a), four (c) or six (d) experiments with biological replicates (mean and s.e.m.). Statistical analysis by one-way-ANOVA and Tukey’s post-hoc test. *, P ≤ 0.01; **, P ≤ 0.001. P-value is indicated only if results of siRNA-treated cells significantly differ from those of control siRNA treated cells. Sequences of DNA-structures, Supplementary Table 1.

Supplementary Figure 5 Illustration of strand-specific qPCR and HIV-1 RT(N265D).

(a) Illustration of strand-specific detection of HIV-1 RT products. Left: (–)-strand detection, right: (+)-strand detection. For the specific detection of (–) or (+)-strand DNA, DNA was denatured, then linker-primers were annealed to the single strand (at the 5′ end for (–)-strand detection (5′Linker-LTR.2) for (–)-strand detection and 3′-end (3′Linker-LTR.1) for (+)-strand detection). The sequence complementary to the HIV-1 RT-derived DNA (LTR.1 or LTR.2) has a low annealing temperature (34–36 °C) to avoid priming at later stages. First-strand synthesis is started by the addition of dNTPs and Klenow fragment and stopped by heat inactivation. The first strand comprising a linker-sequence at the 5′ end, followed by HIV-1 sequence complementary to the detected strand serves as the template for qPCR with primers (3′- or 5′Linker and LTR.3 or 4) with high annealing temperatures (~ 70°C; 5′Linker and 3′LTR-Primer for (–)-strand detection and 3′Linker and 5′LTR-Primer for (+)-strand detection). (b) Illustration of HIV-1 RT(N265D) mutation. Top: The DNA polymerase activity as well as RNaseH activity of both, WT and N265D HIV-1 RT, first generate single-stranded DNA by RNA-templated DNA polymerization followed by RNA degradation. Primed by the polypurine-tracts (ppt) that are resistant to RNaseH degradation, second strand synthesis depends on the DNA-templated DNA polymerization activity of the HIV-1 RT, which is impaired by the N265D mutation, leading to decreased (+)-strand synthesis and therefore reduced dsDNA levels.

Supplementary Figure 6 Generation of single-stranded long DNA.

(a) Schematic illustration of ssDNA generation from PCR products. PCR-Products are generated, comprising a 5′ biotin and 5′ phosphorothioate(pto)-linkages at the 5′ end of the DNA strand corresponding to the (–)-strand of HIV-1, followed by a short spacer and an ApaI restriction site as well as a phosphate group at the 5′ end of the strand corresponding to the (–)-strand. PCR products are subjected to lambda exonuclease digestion, primed by the (+)-strand 5′phosphate and inhibited by the 5′phosphothioate linkages of the (–)-strand. To remove residual partially double-stranded products, the DNA is immobilized on NeutrAvidin beads by the 5′ biotin of the (–)-strand. DNA species that are still double-stranded at the ApaI restriction site are dissociated from the beads by ApaI digestion and removed by washing. To elute the single-stranded DNA with concomitant removal of the non-HIV-1 sequences and modifications, a short DNA oligomer, complementary to the ApaI restriction site and several flanking bases is annealed and the ssDNA eluted by ApaI-mediated cleavage. Residual DNA oligomers are removed by silica-column-mediated DNA purification. (b) PAGE analysis (6%) of generated ssDNA species used in Fig. 6a. ss-116 is identical to SL2+3 as in Fig. 1, ss-180 and ss-381 were generated from PCR products. 50 ng were loaded per lane and the gel stained with GelRed. Data is representative of two independent experiments.

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Supplementary Figures 1–6 (PDF 1519 kb)

Supplementary Table 1

Sequences of indicated DNA-structures (XLSX 57 kb)

Supplementary Table 2

Primers sequences (XLSX 45 kb)

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Herzner, AM., Hagmann, C., Goldeck, M. et al. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA. Nat Immunol 16, 1025–1033 (2015). https://doi.org/10.1038/ni.3267

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