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

Unpaired guanosines in Y-form DNA are a robust yet flexible PAMP

Next we investigated whether a closed loop of SL2 was essential for immunostimulation or if Y-form DNA junctions (the transition from dsDNA to ssDNA) were sufficient for immunoactivation. Opening the loop led to a slight yet insignificant reduction in the immunoactivity of the SL2 variants without mismatches (Fig. 2a), which showed that the unpaired stretches rather than the closed loop were necessary for recognition of the short stem-loop structures. Finally, simple, short DNA duplexes (20–21 bp) with various stem sequences (HIV derived, random non-palindromic, and palindromic) flanked by unpaired guanosine trimer (G₃) ends exhibited the same IFN-α/β-inducing activity as did the guanosine-enriched SL2 hairpin or genomic DNA with removal of mismatches (Fig. 2b). That finding prompted us to further study the flexibility of this recognition motif, which we called ‘G_n-ended Y-form short DNA’ (G_n-YSD, where ‘n’ indicates the number of unpaired guanosines at each 3′, and 5′, end).

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Figure 2

Guanosine extensions render short DNA duplexes highly immunostimulatory. (a,b) ELISA of IFN-α in the supernatant of chloroquine-treated human PBMCs 20 h after treatment with medium alone or transfection of genomic DNA (as in Fig. 1) or the SL2 variant with mismatches and bulges removed from the stem (SL2 ds) and with a closed loop (first and third bars) or open loop (second and fourth bars) comprising the unmodified unpaired regions (first and second bars) or with conversion of various nucleotides to guanosine (as in Fig. 1j; third and fourth bars) (a) or with short duplexes comprising variants of single-stranded regions (first bar as in the third bar in a; second bar as in the fourth bar in a; third bar with G₃ at each end) as well as different stem elements (third, fourth and fifth bars), randomized stem sequence (R) or palindromic stem sequence (P) (b). (c) ELISA of IFN-α in chloroquine-treated human PBMCs as in a,b, transfected with palindromic G₃-YSD, A₃-YSD, T₃-YSD or C₃-YSD, or with G₃-YSD hybridized to C₃-YSD or T₃-YSD, the latter two at a twofold excess (‡); results are presented relative to those of cells transfected with G₃-YSD, set as 100%. (d) ELISA of IFN-α in chloroquine-treated human PBMCs as in a,b, transfected with G₃-YSD or YSD with various numbers and positions of unpaired G trimers flanking 20-nucleotide dsDNA (presented as in c). (e) ELISA of IFN-α in chloroquine-treated human PBMCs as in a,b, transfected with G₃-YSD of various duplex lengths (12–28 bp) or with poly(dAdT) (AT); results are presented relative to those of cells transfected with G₃-YSD with a 20-bp duplex, set as 100%. (f) ELISA of IFN-α in chloroquine-treated human PBMCs as in a,b, transfected with G₃-YSD of various bulge-loop sizes in the base-paired region. Below plots, models of secondary structures (sequences, Supplementary Table 1); numbers adjacent to stems indicate stem length. ND, not detectable. NS, not significant (one-way-ANOVA followed by Fisher’s least-significant difference test). Data are pooled from two (a–c,e,f) or three (d) experiments with one or two biological replicates in each (mean and s.e.m. of n = 4 donors (a–c,f), n = 5 donors (d) or n = 3 donors (e)).

In line with those observations, substitution of the terminal G₃ with adenosine (A₃-YSD), thymidine (T₃-YSD) or cytidine (C₃-YSD) almost completely abrogated the induction of IFN-α (Fig. 2c). Furthermore, the stimulatory effect of the G₃ overhangs was neutralized by hybridization of G₃-YSD with the fully complementary C₃-YSD, but not by hybridization with the T₃-YSD control, since the latter left free G₃ ends available for recognition (Fig. 2c). Further experiments revealed that two G₃ overhangs at opposite ends of the DNA duplex (regardless of whether it was at the 3′ end or 5′ end) were sufficient for robust induction of IFN-α (Fig. 2d). Nevertheless, the sequence requirements for recognition of the unpaired overhangs demonstrated considerable flexibility; one guanosine within one of five unpaired bases at each 5′ and 3′ end of a DNA duplex was sufficient to initiate an IFN-α/β response (Supplementary Fig. 2a). While a decrease in the length of the core duplexes of G₃-YSD to 12 bp elicited a diminished but still substantial IFN-α response, an increase in its length did not enhance IFN-α induction (Fig. 2e). Notably, G₃ ends also further enhanced the already potent immunostimulatory activity of the prototypic blunt 45-nucleotide ISD¹¹ by approximately fourfold (Supplementary Fig. 2b). Mismatches or bulges of up to 4 bp in the dsDNA core sequence of G₃-YSD were tolerated, although this correlated with diminished immunoactivity (Fig. 2f).

We also observed specific recognition of G₃-YSD in purified human monocytes as well as in monocyte-derived macrophages, even though both cell types responded weakly to ISD (Supplementary Fig. 2c,d). Finally, G end–dependent recognition of Y-form DNA was not restricted to human cells, as immortalized mouse macrophages also showed a similar response to YSD (Supplementary Fig. 2d), although these cells also responded to 30-nucleotide blunt-ended dsDNA, as reported¹¹. Together, these results demonstrated that unpaired guanosine extensions potently enhanced the immunoactivity of short DNA duplexes.

G-ended Y-form DNA is active as a monomeric duplex

To address whether variations in DNA stability and transfection efficiency might be responsible for the differences observed in the stimulatory activity of the short DNA duplexes³⁸, we transfected THP-1 cells with 5′-labeled stimulatory DNA (G₃-YSD; ISD) or non-stimulatory DNA (C₃-YSD; blunt-ended 26-nucleotide DNA). At 4 h and 8 h after transfection, we assessed their presence in cytosolic lysates by denaturing polyacrylamide-gel electrophoresis (PAGE) of equal lysate proportions and subsequent detection of label fluorescence. Here, G₃-YSD exhibited fluorescence intensity in THP-1 cytosolic cell lysates similar to or somewhat less than that of the non-stimulatory C₃-YSD or blunt-ended 26-nucleotide DNA (Supplementary Fig. 3a). Furthermore, in vitro digestion of the same unlabeled DNA stimuli with recombinant DNase TREX1, DNase I or DNase II³⁸ did not substantially reduce degradation (Supplementary Fig. 3b,c). Thus, we concluded that direct ligand-receptor interaction, not stability or translocation effects, was relevant for the induction of IFN-α/β by guanosine-ended Y-form DNA.

One characteristic feature of guanosine-rich DNA sequences is their ability to assemble into guanosine quadruplex (‘G quadruplex’) complexes (‘G tetrad’) (Fig. 3a). One possible explanation for the enhanced recognition of guanosine-ended YSD was the potential formation of long chains by such intermolecular G-quadruplex interactions. By native PAGE, high-molecular-weight bands were visible for G₄-YSD or G₅-YSD (Fig. 3c), which indicated G quadruplex–mediated oligomerization (sequences and structures, Fig. 3b), while G₂-YSD or G₃-YSD migrated as a single, low-molecular-weight band (Fig. 3c). Since strong electric fields may interrupt weak interactions during electrophoresis, we investigated the molecular interactions in solution. Anti-parallel G quadruplexes are arranged chirally and are optically active at 295 nm (ref. 39). Their formation can be detected by circular dichroism spectroscopy, which measures the difference in the absorption of left-circularly polarized light and that of right-circularly polarized light (ellipticity) of the analyte at 30–70 °C. G₄-YSD and G₅-YSD exhibited a substantial positive ellipticity at 295 nm that was lost at high temperatures (≥70 °C) (Fig. 3d), which indicated G-quadruplex formation at physiological temperatures. In contrast, G₂-YSD or G₃-YSD, like the negative controls A₃-YSD and a blunt-ended 30-nucleotide DNA, did not show considerable optical activity at 295 nm (Fig. 3d), which indicated that G₂-YSD and G₃-YSD were present as monomeric duplexes. Notably, the quadruplex-forming YSDs did not induce larger amounts of IFN-α than the prototype G₃-YSDs did (Fig. 3e). To exclude the intracellular formation of G quadruplexes, we made use of 26-nucleotide G₂-YSD, which was as active as 20-nucleotide G₃-YSD (Fig. 3e). Here, we replaced the terminal guanosines with 7-deaza-guanosine or inosine. Substitution of nitrogen N7 of the guanine by a C-H group (7-deaza-guanosine) and the missing amine group at carbon C3 of inosine disables formation of the hydrogen bonds essential for the G-quadruplex arrangement⁴⁰. Substitution with 7-deaza-guanosine as well as substitution with inosine did not substantially alter the immunostimulatory activity (Fig. 3f). We concluded that intermolecular G-quadruplex interactions were not involved in the recognition of G-ended YSD but that the monomeric duplex was detected and induced IFN-α/β.

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Figure 3

Induction of IFN-α/β by Y-form DNA is independent of the G quadruplex. (a) G-quadruplex assembly: four guanosines form a planar structure, mediated by hydrogen bonds and stabilized by a central cation (gray). R, deoxyribose backbone. (b) Structures and sequences of YSD or short blunt DNA used in c–e. (c) Native PAGE of G_n-YSD variants (numbers above lanes correspond to models in b). (d) Circular-dichroism melting curve at 295 nm, with the ellipticity of YSD variants or blunt-end DNA (numbers in parentheses and diagrams at left match those in b) measured at 30–70 °C. (e) ELISA of IFN-α in supernatants of chloroquine-treated human PBMCs 20 h after transfection of G_n-YSD variants (numbers and diagrams below plots match those in b); results are presented relative to those of cells transfected with G₃-YSD variant 3 (left) or variant 5 (right), set as 100%. (f) ELISA of IFN-α (bottom) as in e, after transfection of 26-nucleotide G₂-YSD with stepwise substitution of terminal guanosines (G) with deazaguanosine (Z; left) or inosine (i; right); top, chemical structure of 2′-deoxy-7-deazaguanosine and 2′-deoxy-inosine. Numbers adjacent to stems (b,d–f) indicate stem length (sequences of DNA structures, Supplementary Table 1). Data are representative of two experiments (c,d) or are pooled from two (e, f, right) or three (f, right) experiments with one or two biological replicates in each (e,f; mean and s.e.m. of n = 3 donors (e), n = 6 donors (f, left) or n = 4 donors (f, right)).

G-ended Y-form DNA activates cGAS via direct interaction

Of all human cell lines we tested, only the human monocytic cell line THP-1 was responsive to G₃-YSD (data not shown), in line with what has been described for dsDNA with a random base composition⁴¹. The RNA helicase RIG-I has been linked to the detection of long AT-rich DNA^7,8. To exclude the possibility of involvement of signaling via RIG-I and the signaling adaptor MAVS in the recognition of YSD in this cell line, we performed RNA-mediated interference (RNAi) with small interfering (siRNA) targeting MAVS or STING. Knockdown of STING substantially repressed the induction of IFN-α/β by G₃-YSD and plasmid DNA, but knockdown of MAVS did not; instead, knockdown of MAVS repressed the response to the RIG-I ligand 3P-dsRNA (triphosphorylated double-stranded RNA) but not to any of the DNA ligands (Fig. 4a and Supplementary Fig. 4a). These results demonstrated that G₃-YSD indeed induced secretion of IFN-α/β via a STING-dependent pathway.

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Figure 4

G-YSD activates the cGAS-STING axis. (a) IFN-α/β activity in supernatants of THP-1 cells treated with control siRNA or siRNA targeting MAVS or STING (either of two target sequences (STING.1 or STING.2)) (key) and then, 72 h later, stimulated for 20 h with the RIG-I ligand 3P-dsRNA, plasmid DNA (pDNA), G₃-YSD or C₃-YSD; results are presented relative to those of THP-1 cells treated with control siRNA and stimulated with G₃-YSD, set as 100%. (b) Secondary structures of biotinylated DNA duplexes used in interaction assays in c,d: G₃-YSD or C₃-YSD, or short, 26-nucleotide (26-mer) or long, 79-nucleotide (79-mer) blunt dsDNA. (c) Immunoblot analysis of Flag-tagged receptor candidates (left margin) with lysate alone (0.1 volume of the lysate used in the precipitation assays; Input) at the beginning (0 h) of the assay (far left lane) or after precipitation (Ppt) with empty beads (middle left lane) or with G₃-YSD or C₃-YSD (above lanes; as in b). (d) Immunoblot analysis of Flag-tagged IFI16 or cGAS or of the nonspecific DNA-binding control Ku80 with lysate alone (Input) at the beginning (0 h) or end (2.5 h) of the experiment, or after precipitation with empty beads, G₃-YSD, C₃-YSD (as in c), or with short (26-nucleotide) or long (79-nucleotide) blunt dsDNA (as in b) (above lanes). ‡, lysate and precipitate detection are from the same blot with one empty lane removed. (e) IFN-α/β in supernatants of THP-1 cells treated with control siRNA or siRNA targeting cGAS (one of three target sequences (cGAS.2, cGAS.3 or cGAS.4)), MAVS or STING (horizontal axes) and then, 48 h later, stimulated for 20 h with G₃-YSD (top) or plasmid DNA (bottom); results are presented relative to those of THP-1 cells treated with control siRNA, set as 100%. (f) HPLC detecting the conversion of ATP and GTP to cG2′5′AMP (cyclic [G(2′,5′)pA(3′,5′)p]) by cGAS in the presence of ISD, G₃-YSD, C₃-YSD or the 26-nucleotide blunt DNA (without biotin) in b, presented (in milli-absorption units (mAU)) as absorption at 254 nm (A₂₅₄). Sequences of DNA structures, Supplementary Table 1. *P ≤ 0.01 and **P ≤ 0.001, presented only for results significantly different from control (two-way ANOVA followed by Bonferroni’s post-hoc test (a) or one-way ANOVA followed by Tukey’s post-hoc test (e)). Data are pooled from three (a,e, bottom) or four (e, top) independent experiments with biological replicates (mean and s.e.m.) or are representative of three experiments (c,d,f).

To identify the receptor responsible for the recognition of G₃-YSD, we expressed Flag-tagged candidate receptors in HEK293T human embryonic kidney cells and performed bead-coupled co-precipitation experiments with immobilized DNA ligands (Fig. 4b–d). IFI16, ZBP1, DDX41 and cGAS have been described as IFN-α/β-inducing cytosolic receptors for dsDNA in fibroblasts, monocytes, macrophages or conventional dendritic cells^{18,19,29,42,43}. Among those candidates, IFI16 and cGAS bound to both G₃-YSD and C₃-YSD, while ZBP1, DDX41 and the negative control RIG-I did not detectably precipitate together with YSD (Fig. 4c,d). Both IFI16 and cGAS bound to long (79-nucleotide) blunt-ended DNA, whereas short (26-nucleotide) blunt-ended DNA did not precipitate these candidates (Fig. 4d). Only Ku80, a nonspecific DNA-binding control, did not show substantial ‘preference’ for any DNA structure or length (Fig. 4d). Of note, IFI16 showed a slight (threefold) ‘preference’ for the immunostimulatory YSD (G₃-YSD over C₃-YSD; Fig. 4c,d and Supplementary Fig. 4b).

We performed RNAi experiments with siRNA in THP-1 cells to determine the contribution of IFI16 and cGAS to the secretion of IFN-α/β after stimulation with dsDNA. RNAi of cGAS almost completely abrogated the induction of IFN-α/β by dsDNA species, including G₃-YSD (Fig. 4e and Supplementary Fig. 4c). In contrast, RNAi of IFI16 did not substantially suppress the induction of IFN-α/β by dsDNA (Supplementary Fig. 4d). Moreover, RNAi of MAVS again inhibited only RIG-I-dependent, 3P-dsRNA-induced secretion of IFN-α/β, not the G₃-YSD-dependent response (Fig. 4e and Supplementary Fig. 4c). These results demonstrated that G₃-YSD-induced secretion of IFN-α/β depended on the cGAS-STING-axis.

To investigate whether guanosines were directly detected by cGAS, we performed in vitro–activation assays of truncated recombinant cGAS (amino acids 155–522). In the presence of a ligand, cGAS catalyzes the conversion of ATP and GTP to the non-canonical cyclic dinucleotide cGAMP. We used untagged G₃-YSD, C₃-YSD and 26-nucleotide blunt DNA, as well as 45-nucleotide ISD as a positive control¹¹: As expected, in the presence of ISD, ATP and GTP were converted to cGAMP, as visualized by the corresponding ultraviolet irradiation–absorption maxima in the HPLC chromatogram (Fig. 4f). While G₃-YSD activated cGAS to the same extent as ISD did, C₃-YSD and the blunt-ended 26-nucleotide DNA resulted in less conversion (Fig. 4f). Thus, we concluded that G-rich overhangs of short duplexes specifically and directly enhanced the activation of cGAS, which led to STING-dependent secretion of IFN-α/β.

The interferon response correlates with viral ssDNA content

During the life cycle of lentiviruses, reverse transcription of (−) ssDNA is followed by replication of second strand ((+)-strand) DNA, which leads to the generation of dsDNA³⁷. A published study has shown detection of HIV-1 ssDNA predominantly in the cytosol, with dsDNA in the nuclear and perinuclear fraction³⁷. Thus, we hypothesized that recognition of ssDNA by cGAS might be especially important during early infection. To investigate the kinetics of ssDNA formation, we developed a strand-specific quantitative PCR protocol to quantitatively detect (−)-strand DNA and (+)-strand DNA in infected cells (Supplementary Fig. 5a). To determine if dsDNA or ssDNA is crucial for cGAS stimulation, we generated replication-deficient HIV-1-derived lentiviral particles containing a mutant reverse transcriptase, RT(N265D) (Supplementary Fig. 5b), reported to be impaired in its use of DNA as template during replication⁴⁴. Thus, we expected this mutant to produce little or no dsDNA, represented by the presence of (+)-strand DNA. We infected THP-1 cells for 4 h with these HIV-1 particles during inhibition of SAMHD1 (which controls reverse transcription) by the nonstructural protein Vpx, delivered by virus-like-particles, and analyzed cytosol-enriched fractions of these cells. To confirm that the induction of interferon-stimulated genes by either wild-type particles or RT(N265D) particles was cGAS dependent, we infected wild-type THP-1 cells as well as cGAS-deficient THP-1 cells⁴⁵. As reported³⁷, in both wild-type and cGAS-deficient cells, we identified (−)-strand DNA as the predominant species in the cytosol (Fig. 5a). Compared with strand synthesis induced by wild-type RT particles, for the RT(N265D) particles, (+)strand synthesis was impaired but (−)-strand synthesis was not (Fig. 5a). Despite the diminished induction of (+)-strand synthesis by RT(N265D), induction of the interferon-stimulated gene IFIT2 by RT(N265D) particles was not diminished compared with its induction by wild-type particles but instead was slightly increased, in the responsive wild-type THP-1 cells (Fig. 5b). In addition, the induction of IFIT2 by both strains, as well as by the transfection of genomic DNA, but not by the transfection of 3P-dsRNA, was lost in cGAS-deficient cells (Fig. 5b,c), which demonstrated cGAS-mediated induction of an interferon-stimulated gene.

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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. (a) Strand-specific quantitative detection of the HIV-1 (+) strand and (−) strand (key) in cytosol-enriched nucleic acid preparations of wild-type (WT) or cGAS-deficient (cGAS-KO) THP-1 cells 4 h after infection with HIV-1 particles containing wild-type (HIV: WT) or mutant (HIV: N265D) reverse transcriptase or without infection (HIV: −); results are presented as ssDNA copies per mitochondrial genome (mit gen). (b,c) IFIT2 mRNA in THP-1 cells 4 h after treatment as in a (b) or after transfection of genomic DNA or 3P-dsRNA or treatment with medium alone (Med) (c); results are presented as copies of IFIT2 mRNA per copy of control GAPDH mRNA (c), or that value relative to the results of wild-type THP-1 cells infected with wild-type particles (b). (d) Strand-specific quantitative detection of the HIV-1 (+) and (−) strand (key) in cytosol-enriched nucleic acid preparations of monocyte-derived macrophages 4 h after no infection (Med) or infection with HIV-1 particles as in a (presented as in a). (e) Ratio of (+) strand to (−) strand in d. (f,g) IFNB1 mRNA (f) and IFIT2 mRNA (g) in monocyte-derived macrophages 4 h after stimulation with HIV-1 particles as in a or transfection of genomic DNA or 3P-dsRNA; results are presented as copies of IFNB1 mRNA (f) or IFIT2 mRNA (g) per copy of control GAPDH mRNA. *P ≤ 0.01 (ratio-paired t-test). Data are from three (a,b) i or two (c) independent experiments (mean and s.e.m.) or are pooled from two experiments with two biological replicates in each (d–g; mean and s.e.m. of n = 4 donors).

To verify that effect in primary cells, we also infected monocyte-derived macrophages, since these are particularly physiologically relevant for HIV infection. Again, we identified (−)ssDNA as the predominant DNA species in the cytosol-enriched fraction, with an even greater portion of (−)ssDNA than of (+)DNA (Fig. 5d); this confirmed both the presence and excess of ssDNA. While macrophages infected with RT(N265D) particles had slightly more (−)ssDNA than did cells infected with wild-type particles (Fig. 5d), the abundance of (+)-strand DNA was reduced in the former cells, which led to a significantly lower ratio of (+) strand to (−) strand (Fig. 5e). Notably, the induction of both IFNB1 and IFIT2 was greater in cells infected with the mutant RT(N265D) particles than in cells infected with wild-type particles (Fig. 5f,g), which demonstrated a correlation with the presence of the (−) strand rather than the (+) strand for the induction of IFN-β and interferon-stimulated genes and linked ssDNA to the recognition of early HIV-1 infection. Together these data offered evidence that (−)-strand ssDNA was the predominant DNA species detected by cGAS, at least during early stages of HIV-1 infection, and that the impairment of (+)-strand synthesis did not hinder the activation of cGAS. Furthermore, these data emphasized the importance of the detection of ssDNA by cGAS in vivo.

Long ssDNA is highly immunostimulatory

Since the probability of forming cGAS-stimulating YSD structures increases with ssDNA length, we hypothesized that the induction of IFN-α/β might correlate with length of the HIV (−)ssDNA strand. Thus, we established a protocol to enrich very pure ssDNA from PCR products comprising the first 180 or 381 nucleotides of the (−) strand emerging during infection with HIV-1 strain HXB2. Here, we combined digestion of the (+) strand with 5′ phosphate–dependent λ-exonuclease with purification via biotin-mediated immobilization and digestion with a dsDNA-specific restriction enzyme (Supplementary Fig. 6a). Using this protocol, we produced very pure ssDNA (≥100-fold excess of (−) strand over (+) strand), with each product appearing as one prominent band by PAGE (Supplementary Fig. 6b). Stimulation of monocyte-derived macrophages with these ssDNA species revealed a correlation between length and immunoactivity that culminated in the induction of IFN-α by the 381-nucleotide species that was comparable to that induced by G₃-YSD and was even greater than that induced by human genomic DNA (Fig. 6a). This indicated that long ssDNAs emerging during HIV-1 infection could be as immunoactive as comparable dsDNA sequences. In conclusion, these data supported the hypothesis that the detection of HIV-1 depends on the recognition of ssDNA.

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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. (a) ELISA of IFN-α in supernatants of monocyte-derived macrophages 36 h after treatment with medium alone (Med) or transfection of genomic DNA, G₃-YSD or ssDNA species of various lengths (116 nucleotides (ss-116; this is SL2 plus SL3 as in Fig. 1c), 180 nucleotides (ss-180) or 381 nucleotides (ss-381)); results are presented relative to those of cells transfected with G₃-YSD, set as 100%. (b) IFN-α in supernatants of chloroquine-treated PBMCs 24 h after treatment with medium alone (Med) or transfection of wild-type (WT) or mutant (ΔG) HERV-E or ERV3.1 (right), and mFOLD-derived models of the secondary structures of HERV-E or ERV3.1 (left); circles indicate mutation of guanosine (black, G to C; gray: G to A). *P ≤ 0.05 (one-way ANOVA followed by Tukey’s post-hoc test). Data are pooled from two experiments with one or two biological replicates in each (mean and s.e.m. of n = 3 donors (a) or n = 4 donors (b)).

Stimulatory nucleic acids

Synthetic DNA was obtained from Metabion, IDT, Invitrogen or EUROGENTEC (deazaguanosine-DNA). pDNA (pBluescript) was isolated from transformed Escherichia coli K12 with a PureLink HiPure Plasmid Filter Maxiprep Kit (Life Technologies). Poly(dAdT) was obtained from Sigma-Aldrich. 3P-dsRNA (in vitro transcript 4 (IVT4)) was prepared as described⁹. DNA was hybridized in 25 mM Tris-HCl, pH 7.4 and 25 mM NaCl. DNA was heated to 72 °C for 5 min and was then cooled to 18 °C at a cooling rate of 1 °C/min. DNA with a melting temperature of >70 °C was heated to 95 °C and then was cooled to 18 °C at a cooling rate of 1 °C/min.

Cell culture and stimulation

Cell lines were cultured in RPMI-1640 medium (THP-1 cells) or DMEM (HEK293T cells (immortalized mouse macrophages⁵⁴); Gibco), supplemented with 10% FCS and penicillin/streptomycin (standard medium; Gibco). Human PBMCs were isolated from buffy coats derived from human blood from healthy, voluntary donors by Ficoll-Hypaque density-gradient centrifugation (Biochrom). Monocytes were generated from PBMCs by magnetic cell sorting of CD14⁺ cells (Miltenyi Biotec). Human macrophages were differentiated in vitro from CD14⁺ monocytes in the presence of recombinant cytokine GM-CSF (50 U/ml) in standard RPMI-1640 medium. The generation of CD14^hiCD80^hiCD163⁻ M1-polarized macrophages was confirmed by flow cytometry after 5 d (BD Biosciences).

For stimulation experiments, 4 × 10⁵ PBMCs, 2 × 10⁵ monocytes 1.5 × 10⁵ macrophages or 6 × 10⁴ THP-1 cells per well were seeded into 96-well plates in standard RPMI-1640 medium. For prevention of TLR9 activation, PBMCs were incubated in 2.5 μg/ml chloroqine 30 min before stimulation. Cells were stimulated for 20 h (PBMCs, monocytes or THP-1 cells) or 36 h (monocyte-derived macrophages). Nucleic acid stimuli were used at a final concentration of 0.8 μg/ml (PBMCs, monocytes or monocyte-derived macrophages) or 1.3 μg/ml (THP-1 cells; immortalized mouse macrophages). RNA and DNA stimuli were used in complex with Lipofectamine 2000 (Invitrogen). Secretion of IFN-α was measured with ELISA kits supplied by eBioscience. Human IFN-α/β activity was quantified by incubation of the IFN-α/β-sensing reporter cell line HEK-Blue IFN-α/β (Invivogen) with supernatants of stimulated cells. Upregulation of Ifnb1 mRNA in immortalized mouse macrophages was detected by quantitative PCR 6 h after stimulation. The induction of IFNB1 or IFIT2 mRNA in infection experiments was measured 4 h after infection or transfection (described below in the subsection entitled “Infection of monocyte-derived macrophages and THP-1 cells”).

PAGE of nucleic acids

Samples were separated by electrophoresis through native polyacrylamide gels (15%) in KCl-supplemented TBE buffer (45 mM Tris-borate, pH 8.0, 1 mM EDTA and 20 mM KCl) at 12.5 V/cm and 4 °C. DNA was annealed in a buffer of 100 mM KCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA; 1 μg DNA was loaded per lane and, after electrophoresis, DNA was stained with methylene blue. For denaturing PAGE, nucleic acids or proteinase K–digested lysate (described below in the subsection entitled “Detection of cytosolic 5′ IRD700–labeled DNA”) were denatured for 10 min at 65 °C (long ssDNA) or for 5 min at 90 °C (short DNA duplexes) in an excess of formamide supplied with Orange G and then were ‘snap-cooled’ on ice. Samples were loaded onto a TBE-buffered 6% polyacrylamide gel (long ssDNA) or 15% polyacrylamide gel (short DNA duplexes) containing 48% (wt/vol) urea and the gel was run at 12.5 V/cm and 18–20 °C. DNA was detected by infrared fluorescence of the 5′-linked dye IRD-700 (described below) or by staining with GelRed or GelGreen (Biotium).

Circular dichroism spectroscopy

Circular-dichroism spectroscopy was recorded on a Jobin–Yvon Dichrograph model CD6 in a 1-cm quartz cell. For measurement of circular-dichroism, DNA was resolved in NaCl buffer (150 mM NaCl and 10 mM TRIS-HCl, pH 8) and was subsequently heated to 70 °C.

Detection of cytosolic 5′ IRD-700–labeled DNA

THP-1 cells seeded into 24-well plates (3 × 10⁵ cells per well) were transfected with 1 μg hybridized DNA labeled at the 5′ end with IRD-700 (Metabion), followed by incubation for 4 h or 8 h. The cells were washed with PBS and then were lysed for 5 min on ice with 40 μl NP40 buffer (25 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 5 mM KCl, 0.5% NP-40 and 10 mM EDTA). Nuclei were precipitated by centrifugation at 300g (5 min at 4 °C), then supernatants were cleared by another centrifugation step at 5,000g (5 min at 4 °C). Lysates (5 μl each) were mixed with 5 μl proteinase K mix (1% (wt/vol) SDS, 20 mM EDTA, 60 mM Tris-HCl, pH 6.8, and 0.2 mg/ml proteinase K), followed by digestion for 20 min at 50 °C, then by heat inactivation for 10 min at 90 °C. Samples were separated by electrophoresis through denaturing gels, and IRD-700 fluorescence was detected with an Odyssey Imaging System (Li-COR). Loading controls were prepared as follows: hybridized DNA (2.5 ng) was mixed with 5 μl proteinase K mix and then was incubated, denatured with formamide and separated by electrophoresis as described above.

DNA interaction assays

FLAG-tagged proteins were expressed by transient transfection into HEK293T cells (seeded in six-well plates) via CaPO₄ complexes. After 24 h, the cells were mechanically detached and then were resuspended in ice-cold PBS, precipitated and washed. Cells were lysed in DNA lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% (vol/vol) glycerol, 0.3% (vol/vol) Triton X-100, 1× Phosphatase-Inhibitor-Cocktail and 1× complete protease inhibitor (Roche); 100 μl per six wells), then nuclei were precipitated for 30 min at 20,000g and 4 °C. Hybridized DNA tagged with biotin at the 3′ end (5 μg ssDNA, or 10 μg dsDNA) was incubated for 30 min at 18–20 °C with 30 μl supernatant. Neutravidin beads (10 μl per reaction; Thermo Scientific) were washed twice with DNA lysis buffer and were loaded onto polyethylene filter columns (0.8 ml; Thermo Scientific). DNA-lysate mixes were added, followed by incubation for further 2 h. After that, the columns were opened at the bottom and the supernatant was collected in a 1.5 ml reaction tube. The beads were washed twice with DNA lysis buffer and once with DNA lysis buffer with a higher NaCl concentration (300 mM). Bound proteins were eluted for 30 min at 40 °C with Lämmli buffer (40 μl per reaction). A volume of 15 μl eluate or 3 μl cell lysate was assessed by standard immunoblot analysis with the monoclonal antibody ANTI-FLAG M2-Peroxidase (Sigma). The interaction assay analyzing the binding of the HIN domain of IFI16 to DNA in solution was performed as described⁵⁵.

cGAS in vitro activity assay

For in vitro reactions of cGAS in the presence of varying dsDNA stimuli, 2 μM recombinant human cGAS (truncated version: residues 155–522) was mixed with 88 ng/μl hybridized DNA, 0.1 mM ATP and 0.1 mM GTP in buffer A (100 mM NaCl, 40 mM Tris, pH 7.5, and 10 mM MgCl₂). After 90 min of incubation at 37 °C, the reaction mixture was analyzed by reverse-phase HPLC. Samples were prepared in 300 mM triethylammonium acetate, then were applied to a 4.1- × 250-mm PRP-1 column (Hamilton) and separated for 18 min at a flow rate of 1 ml/min in an isocratic gradient of 100 mM ammonium acetate.

RNAi

THP-1 cells were transfected by electroporation with siRNA at 72 h (Fig. 3a and Supplementary Fig. 2b) or 48 h (Fig. 3e) before stimulation. The siRNAs with 3′ dTdT modification (Biomers) targeted the following sequences: STING.1, 5′-GGCAUCAAGGAUCGGGUUU-3′; STING.2, 5′-GGUCAUAUUACAUCGGAUA-3′; MAVS, 5′-UUAAAGGAGUUUAUCG AUGUA-3′; cGAS.2, 5′-GGAAGAAAUUAACGACAUU-3′; cGAS.3, 5′-GAAGA AACAUGGCGGCUAU-3′; cGAS.4, 5′-AGAGAAAUGUUGCAGGAAA-3′; IFI16.2, 5′-UCAGAAGACCACAAUCUAC-3′; IFI16.3, 5′-GGUGCUGAACGCA ACAGAAUCAUUU-3′; and controls, 5′-CAUAAGGCAAUGAAGAGAUAG-3 ′ (luciferase; Fig. 3a) or 5′-GCACCCCAAUAACGAGCUU-3′ (no target; Fig. 3e and Supplementary Fig. 2b). mRNA expression at the time point of stimulation was analyzed by quantitative PCR.

Quantitative PCR (RNA detection)

Cells were lysed in 350 μl RLT buffer (Qiagen), then 350 μl 70% ethanol was added and the mixture was loaded onto a Zymo-SpinTM IIIC column. The column was washed sequentially with 350 μl buffer RW1 (Qiagen) and 350 μl Zymo RNA Wash buffer (Zymo). The columns were dried by centrifugation and RNA was eluted with desalted RNase-free water. For the detection of mouse Ifnb1 mRNA, 1 h of on-column digestion (at 18–20 °C) with DNase I (Zymo) and an additional wash step (500 μl Zymo RNA Wash buffer) were included after the application of Zymo RNA Wash buffer. cDNA was synthesized with a RevertAid RT Kit according to the manufacturer’s protocol (Thermo Scientific), with random hexamers. Quantitative PCR was performed with EvaGreen QPCR-Mix II (ROX) (Biobudget) in an ABI7900HT cycler (primer sequences, Supplementary Table 2).

Lentiviral particles

Lentiviral particles containing wild-type reverse transcriptase or the mutant RT(N265D) reverse transcriptase were produced by cotransfection of HEK293T cells (by the calcium phosphate method) with expression plasmids encoding vesicular stomatitis virus glycoprotein (pVSV-G), HIV-1 transactivator of transcription (Tat) (pcTat), or regulator of the expression of virion proteins (Rev) protein of Rous sarcoma virus (pRev). HIV-1 group-associated antigen and polymerase (Gag-Pol) sequence encoding wild-type reverse transcriptase (pMDL) or the mutant RT(N265D) reverse transcriptase (pMDL-N256D), and a lentiviral reporter construct expressing green fluorescent protein under the control of a cytomegalovirus promoter (pHR-CMV-GFP W Sin18) at a ratio of 1.5:1:1:3.5:3.5. For all transfection, the culture medium was replaced after 6 h. The supernatants were harvested 48 h after transfection, then were filtered (0.4 μm) and then purified by ultracentrifugation (90 min at 175,000g) with a sucrose cushion (20% sucrose, 10 mM Tris, pH 7.5, 1 mM EDTA and 100 mM NaCl), separated into aliquots and frozen at −80 °C. Reporter viruses were ‘titrated’ on HEK293T cells, and green fluorescent protein–positive cells were quantified at 72 h after infection by flow cytometry. In addition, particles containing wild-type or RT(N265D) reverse transcriptase were also normalized for the amount of p24 capsid protein, measured by ELISA (Innogenetics). The mutation encoding the N265D reverse transcriptase mutant⁴⁴ was introduced by cloning of a fusion PCR product into the AgeI and MfeI sites of the Gag-Pol gene of pMDL.

Infection of monocyte-derived macrophages and THP-1 cells

For infection, THP-1 cells (1 × 10⁵ cells per well) or monocyte-derived macrophages (1.5 × 10⁵ cells per well) were seeded into 96-well plates. Cells were preincubated for 2 h with Vpx-delivering virus-like particles and then were infected with lentiviral particles containing wild-type reverse transcriptase at a multiplicity of infection of 1 (THP-1 cells) or 0.67 (monocyte-derived macrophages; 1 × 10⁵ infectious units per well). Infection with lentiviral particles generated with the N265D mutant reverse transcriptase was adjusted according to the concentration of p24 (details in figure legends and Results). At 4 h after infection, cells were washed twice with PBS and then were gently lysed for 5 min on ice in Nonidet-P40 buffer (0.1 M NaCl, 10 mM Tris, pH 8, 2 mM EDTA, 1% β-mercaptoethanol and 0.5% Nonidet-P40). Nuclei were precipitated by centrifugation at 300g for 5 min at 4 °C, and the supernatant cleared by further centrifugation at 5,000g for 5 min at 4 °C. Supernatants were diluted in RLT buffer (Qiagen) and nucleic acids were isolated by a standard RNA-purification protocol (described above in the subsection entitled “Quantitative PCR (RNA detection)”). This protocol did not markedly affect DNA yield compared with results obtained by other protocols (data not shown).

Strand-specific detection of HIV reverse transcripts

Specific detection of HIV (+) and (−) strands included two steps: first, introduction of a strand-specific linker by reaction with DNA polymerase I without exonuclease activity (Klenow exo ), and second, linker-specific quantitative PCR. For the strand-specific quantitative PCR (first step), linker-introducing primers (3 ′ Linker-LTR.1 for (+)-strand detection; 5′ Linker-LTR.2 for (−)-strand detection; Supplementary Table 2) were used for first-strand synthesis. Here, cytosolic nucleic acid extracts, mixed with the respective linker primer (4 μM), in 1× FastDigest Buffer (Thermo Scientific), were heated to 95 °C for 5 min, then were cooled to 37 °C. Next, dNTPs (final concentration, 0.1 mM each) and Klenow exo–(1 U/10 μl; Thermo Scientific) were added in 1× FastDigest Buffer for the initiation of first-strand synthesis. After incubation for 15 min at 37 °C, the reaction was stopped by incubation for 10 min at 95 °C.

For the second step, the reaction mix was diluted 1:2 in water and used for quantitative PCR as described for classic quantitative PCR, except that a two-step PCR-protocol was used (50 s at 72 °C and 15 s at 95 °C for each cycle). Primers were 3′-Linker+5′LTR.3 for (+)-strand detection and 5′Linker+3′LTR.4 for (−)-strand detection (Supplementary Table 2). In each experiment, serial dilution of a plasmid (Sigma SHC003) was used as a standard in the Klenow reaction and quantitative PCR. Copy numbers were normalized to the number of mitochondrial genome copies, determined by classic three-step quantitative PCR.

Preparation of ssDNA from PCR products

PCR products were amplified from gBlocks long synthetic DNA (IDT) by Dreamtaq polymerase (Thermo Scientific), with (−)-strand primers comprising a 5′ biotin tag, 5 phosphothioate linkages at the 5′ end and a ApaI restriction site, followed by a sequence complementary to the HIV-1 RNA genomic sequence immediately 5′ of the original primer-binding site (identical to the 3′ end of the genomic RNA). The (+)-strand primers had a 5′ phosphate and were derived from the (+)strand sequence 180 bp or 381 bp from the 3′ end of the RNA genome. Primer sequences are in Supplementary Table 2. PCR products were purified with an AnalytikJena PCRpure kit and were digested for 30 min with Lambda Exonuclease (Thermo Scientific), followed by heat inactivation for 10 min at 95 °C. DNA was diluted in PBS and then was coupled to NeutrAvidin beads overnight at 4 °C. Beads were spun down (3800g and 4 °C for 30 s), then were resuspended in 1× FastDigest buffer containing FastDigest ApaI, followed by incubation for 15 min at 37 °C. PBS was added in excess, and the beads were incubated again for 2 h at 4 °C. The beads were washed with PBS twice, then were resuspended in 1× FastDigest Buffer containing ApaI anti-sense DNA oligonucleotide and were incubated for 30 min at 18–20 °C. Then, FastDigest ApaI was added again and the beads were incubated for 30 min at 37 °C. The ssDNA was again purified with an AnalytikJena PCRpure kit. Single-strandedness was controlled by strand-specific quantitative PCR (a ratio of (−) strand to (+) strand >100) and quality was assessed by denaturing gel electrophoresis (6%).

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.).