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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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.).
The authors declare no competing financial interests.
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.
Supplementary Text and Figures
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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