The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING

Abstract

The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

Figure 1. Variable responsiveness of human STING variants to cyclic-di-nucleotides maps to arginine 232

(A) THP-1 cells were transduced with vectors encoding an shRNA targeting STING or a control shRNA. Cells were then stimulated with cyclic-di-GMP (cdG), dsDNA, cyclic-di-AMP (cdA), poly-inosine:cytosine (pI:C), or Sendai Virus, and induction of human interferon-β mRNA was assessed by quantitative reverse transcriptase PCR. (B) Western blotting confirmed that knockdown of STING was effective. (C) HEK293T cells were transfected with the indicated amounts of various mouse (m) or human (h) STING expression plasmid and then stimulated 6h later by transfection with synthetic cdG (5µM). GT denotes the null I199N allele of STING from Goldenticket (Gt) mice. STING activation was assessed by use of a cotransfected IFN-luciferase reporter construct. (D) Gt (STING-null) macrophages were transduced with retroviral vectors encoding the indicated STING alleles and were then stimulated 48h later by transfection with cdG (5µM) or dsDNA 70-mer oligonucleotide (0.5µg/mL). IFN induction was measured by qRT-PCR. ND, not detected. (E) Binding assay of STING to ³²P-c-di-GMP. STING proteins were expressed in HEK293T cells and cell lysates were subjected to UVcrosslinking with ³²P-cdG, and resolved by SDS-PAGE. Binding was quantified by autoradiography. Western blots of cell lysates with an anti-STING polyclonal antibody confirmed similar expression of the various STING proteins. (F) Responsiveness of mSTING to cGAMP is affected by mutations of R231. The indicated mutants were tested as in C. Data are representative of at least three independent experiments and are presented as mean. Error is represented as standard error of the mean. ***, P < 0.0001; **. P < 0.005. See also Figure S1.

Figure 2. STING variants are responsive to cGAS

(A) HEK293T cells were transfected with the indicated STING alleles and with human and mouse cGAS (wt and GS>AA mutants) (Sun et al., 2013) as indicated. STING activation was assessed by a co-transfected IFN-luciferase reporter construct. (B) HEK293T cells were transfected with the indicated STING alleles and with a mammalian expression vector encoding a cGAMP synthase (DncV) from V. cholerae. STING activation was assessed as in A. (C) In vitro enzymatically generated products of rWspR, rDncV and rcGAS were transfected into digitonin permeabilized HEK293T cells expressing the indicated mouse and human STING proteins. Chemically synthesized cyclic-di-GMP (cdG) and cGAMP were included as controls. STING activation was assessed as in A and B. Data are representative of at least three independent experiments and are presented as mean. Error is represented as standard error of the mean. See also Figure S2.

Figure 3. cGAS produces a non-canonical cyclic dinucleotide

(A) Purified recombinant WspR, DncV and cGAS were mixed with α³²P-GTP or α³²P-ATP and the indicated unlabeled nucleotides. Reactions were mixed with TLC running buffer and nucleic acid species were resolved on a PEI-Cellulose TLC plate. (B) WspR, DncV and cGAS products labeled with α³²PGTP were digested with nuclease P1 or Snake Venom Phosphodiesterase (SVPD) and resolved on a PEI-Cellulose TLC plate. See also Figure S3.

Figure 4. cGAS produces a cyclic dinucleotide containing a 2′ -5′ phosphodiester linkage

(A) ¹H-³¹P HMBC of HPLC-purified cGAS product acquired at 600 MHz and 50° C. Critical through-bond correlations for the phosphodiester bonds are indicated. NMR elucidated structure of cGAS product is also shown. (B) Chemical structures of canonical c[G(3′ -5′)pA(3′ -5′)p] cGAMP (left) and non-canonical c[G(2′ -5′)pA(3′ -5′)p] cGAMP (right). See also Figure S4.