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

Anna-Maria Herzner; Cristina Amparo Hagmann; Marion Goldeck; Steven Wolter; Kirsten Kübler; Sabine Wittmann; Thomas Gramberg; Liudmila Andreeva; Karl-Peter Hopfner; Christina Mertens; Thomas Zillinger; Tengchuan Jin; Tsan Sam Xiao; Eva Bartok; Christoph Coch; Damian Ackermann; Veit Hornung; Janos Ludwig; Winfried Barchet; Gunther Hartmann; Martin Schlee

doi:10.1038/ni.3267

Figure 1

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Immunostimulation by stem-loop-forming structures in HIV-1 sstDNA depends on unpaired guanosines flanking the stem. (a) mFOLD model of HIV-1 sstDNA, the primary lysyl-tRNA (tRNA^Lys3)-primed reverse transcript of the 5′ untranslated region (R-U5). Gag-Pol and Env, regions encoding HIV-1 group-associated antigen (Gag), polymerase (Pol) and envelope (Env) proteins; U3-R, 3′ untranslated region and repeat. (b–j) Enzyme-linked immunosorbent assay (ELISA) of IFN-α in supernatants of chloroquine-treated human PBMCs 20 h after treatment with medium alone (Med) or transfection of genomic DNA (gDNA) or wild-type stem-loop structures SL1, SL2 or SL3 (b), or of wild-type SL2 (c–j) or a combination of SL2 and SL3 (SL2+3) (c), SL2 with introduced C:G base pairs (SL2 +G:C) or A:T base pairs (SL2 +A:T) (d), SL2 with mismatches and bulges removed from the stem (SL2 ds) (e), SL2 (SL2 blunt) or the SL2 mutant in e (SL2 ds blunt) with single-stranded parts removed (f), SL2 with substitution of guanosines (SL2 ΔG) or conversion of various nucleotides (color loops) to guanosine (SL2+G) in the loop as well as the 3′ and 5′ ends (g), the combined SL2 plus SL3 in c with (SL2+3 ΔG) or without (SL2+3) substitution of unpaired guanosines (h), the SL2 mutant in e with (SL2 ds ΔG) or without (SL2 ds) substitution of unpaired guanosines (i), or SL2 with conversion of various nucleotides (color loops) to guanosine (SL2+G), removal of mismatches and bases (SL2 ds) or a combination of those changes (SL2 ds+G) (j). Below plots (b–j), mFOLD models of secondary structures (sequences, Supplementary Table 1): colors indicate mutations (key; ‘N’ is any base.); letters adjacent to stems indicate bases removed. NS, not significant (P > 0.05); *P ≤ 0.05; **P ≤ 0.01 and ***P ≤ 0.001 (one-way analysis of variance (ANOVA) followed by Fisher’s least-significant difference LSD test.). Data are pooled from two (b,d,e,g–j) or four (c,f) experiments with two biological replicates in each (mean and s.e.m. of n = 4 donors (b,d,e,g–j; same experiments) or n = 8 donors (c,f; same experiments)).