www.sciencemag.org/cgi/content/full/science.aab3632/DC1

Supplementary Materials for

Viruses transfer the antiviral second messenger cGAMP between cells

A. Bridgeman, J. Maelfait, T. Davenne, T. Partridge, Y. Peng, A. Mayer, T. Dong, V. Kaever, P. Borrow, J. Rehwinkel*

*Corresponding author. E-mail: jan.rehwinkel@imm.ox.ac.uk

Published 30 July 2015 on Science Express

DOI: 10.1126/science.aab3632

This PDF file includes:

Materials and Methods Figs. S1 to S13 References

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Materials and Methods Plasmids

FLAG-m-cGAS and FLAG-m-cGAS-G198A/S199A pcDNA constructs were from Z.J. Chen and have been described elsewhere (4). pNL4-3-deltaE-EGFP was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs. Haili Zhang, Yan Zhou, and Robert Siliciano (28). pCAAGS-THOV-G was from G. Kochs. pNL4.3 and pCR3.1/HA-based tetherin plasmids were from S. Neil (29). pHIV-2 ROD9 ΔEnvΔNef GFP was from N. Manel (30). pGreenFire-ISRE was purchased from System Biosciences. pSIV4+, pVSV-G, pCSGW, p8.91, p125-F-Luc and pRL-TK have been described before (31, 32). Cells

Murine BMDMs and BM-DCs were obtained from fresh bone marrow using 20% L929 supernatant and GM-CSF, respectively, as described before (31). Human MDDCs were derived from CD14+ monocytes with 40 ng/ml GM-CSF and 40 ng/ml IL-4 (Peprotech) for 5 days. The purity of dendritic cells was >95% according to DC-SIGN staining. CD14+ monocytes were isolated from PBMCs using MACS separation columns and CD14 microbeads (Miltenyi). PBMCs were harvested from CD leukocyte cones (NHS Blood & Transplant, Bristol, UK) using lymphoprep (Alere, UK). MEFs, HEK293 cells and 293T cells were grown in DMEM medium. Jurkat cells, THP1 cells, BMDMs, BM-DCs and hMDDCs were grown in RPMI 1640 medium. All media contained 10% FCS and 2 mM glutamine. 100 units/ml penicillin, 100 mg/ml streptomycin and 50 µM 2-mercaptoethanol were additionally added to the RPMI medium used for BMDMs, BM-DCs and hMDDCs. All cells were grown at 37°C and 5% CO2. MEFs were cultivated at 3% O2. Western blot

α-STING and α-Caspase 3 antibodies were from Cell Signaling (cat. nb. 3337s & 9662; both 1:1,000). α-Syntenin antibody was from Tebu-bio (cat. nb. GTX108470; 1:1,000). α-p24 antibody was from Advance Bioscience (cat. nb. 4313; 1:5,000). α-VSV-G antibody was from Sigma (cat. nb. V4888; 1:10,000). These antibodies were used for Western blot with corresponding secondary HRP-coupled antibodies. α-FLAG and α-actin directly HRP conjugated antibodies were from Sigma (cat. nb. A8592, 1:5,000 and A3854, 1:10,000). FACS

FACS data were acquired on Beckmann Coulter CyAn or BD Biosciences LSRFortessa cell analysers. For HIV-1-GFP infection of HEK293 and 293T cells, 1 µg/ml DAPI (Sigma) was used to exclude dead cells. For hMDDCs, Fc receptors were blocked (Human TruStain FcX™, Biolegend, cat. nb. 422302) and dead cells were stained with a violet fluorescent reactive dye (Life Technologies, cat. nb. L34955). The following antibodies were then used for cell surface staining: α-hCD86 APC (clone 2331, BD Pharmingen or clone IT2.2, Biolegend); α-hHLA ABC: RPE (clone W6/32, AbD Serotec); α-hHLA-DR APC (clone G46-6, BD Pharmingen); α-hCD209 (DC-SIGN) APC (clone eB-h209, eBioscience). After fixation and permeabilization with perm/fix buffer (BD Biosciences), some cells were stained with α-p24 antibody (clone KC57, Beckman Coulter). For BM-DCs, Fc receptors were blocked (Fc block, BD Pharmingen, cat. nb. 553142) and dead cells were stained as above. The following antibodies were then used

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for cell surface staining: α-mCD80 PE (clone 16-10A1, eBioscience) and α-mCD40 PE-Cy7 (clone 3/23, Biolegend). Mice

STING-deficient animals (also known as Mpys-/-) have been described before (33) and are on a C57BL/6 background. cGAS-deficient mice were re-derived from frozen sperm (34), which was from EMMA (Request ID: 5217), strain ID: EM:04599, strain name: C57BL/6NTac-Mb21d1/lcs (cGAS knockout-first) (E101JM8N4-28). Cryopreserved spermatozoa were thawed, treated with FERTIUP® Mouse Preincubation Medium and CARD MEDIUM and then used to fertilize oocytes from super-ovulated C57BL6/J donor mice. Zygotes were cultured overnight at 37°C and 5% CO2 in HTF medium. The fertilization rate was approximately 85%. 18 to 20 two-cell embryos were surgically transferred via the infundibulum to each of two female recipients (one CD1 and one C57BL/6J x CBA F1) rendered pseudo-pregnant by mating with sterile males. Five live born pups were genotyped as described before (15) and one cGas+/- male was identified. This animal was crossed to its cGas+/+ littermates, and the resulting cGas+/- offspring were then intercrossed to generate cGas-/- mice. Lung fibroblasts obtained from both STING- and cGAS-deficient mice failed to secrete IFNα in response to transfected E. coli DNA but responded normally to RIG-I activation triggered by in-vitro transcribed RNA (Fig. S13).

Femurs, tibias and lungs were obtained from humanely killed animals aged 2-3 months and from age and gender matched C57BL/6 wild-type control animals. Lung fibroblasts were isolated as previously described (15, 35) and MEFs were generated by standard methods. Detailed laboratory protocols are available upon request. This work was performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines for animal care. This work was approved by a project licence granted by the UK Home Office (PPL No. 40/3583) and was also approved by the Institutional Animal Ethics Committee Review Board at the University of Oxford. IFN bioassay

Human IFN reporter cells were generated by transducing HEK293 cells with pGreenFire-ISRE derived lentivirus. Single clones were established by limiting dilution and clone 3C11 was selected based on its responsiveness to IFN. For the bioassay, cells were overlaid with cell culture supernatant and after 24 hours luciferase expression was quantified using One-Glo Luciferase Assay System (Promega) according to manufacturer’s instructions. The detection limit of the bioassay is 1.6 U/ml hIFNα2 (R&D systems) as shown in Fig. S1E. RT Q-PCR

RNA extraction from cells, reverse transcription and quantitative PCR have been described before (31). Pre-developed Taqman assay reagents containing primers and fluorescent probe for human or mouse GAPDH, IFNβ, CXCL11, IFI44, IFIT1, CXCL12, or HLA-DPA1 were from Applied Biosystems. Data were analyzed by the comparative Ct method. Lentivirus production

HIV-1-GFP was produced in 293T cells by plasmid transfection using FuGene HD (Promega, cat. nb. E2311):

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• VSV-G pseudotyped HIV-1-GFP was made with pNL4-3-deltaE-EGFP, pVSV-G and m-cGAS (or m-cGAS-AA) at a ratio of 2:1:2.

• THOV-G pseudotyped virus was produced with pCAAGS-THOV-G instead of VSV-G and using a plasmid ratio of 1:1:1.

• HIV-1-GFP containing biotin-cGAMP was produced as above in 293T cells transfected with pNL4-3-deltaE-EGFP, pVSV-G and biotin-cGAMP (Biolog, cat. nb. 176-001) at a ratio of 2:1:1.6.

• VLPs were produced by transfecting 293T cells with pSIV4+ (Vpx deficient), pVSV-G and m-cGAS (or m-cGAS-AA) at a ratio of 2:1:2.

• Vpu-deficient lentivectors were made with pCSGW, p8.91, pVSV-G, m-cGAS and pCR3.1/HA-tetherin (or pCR3.1/HA-tetherin-ΔGPI) at a ratio of 2:2:1:2:2.

Medium was replaced 24 hours after transfection. At this point, DEVD (60 µM; A0835, Sigma) or GW4869 (20 µM; D1692, Sigma) were added in some experiments. After an additional 48 hours, supernatants were filtered (0.22 µm) and, if required, concentrated by centrifugation over a 20% sucrose cushion (64,000 g, 2.5 hours, 4°C). Viral titres were determined as infectious units/ml by infection of 293T cells with a dilution series of virus stocks, followed by FACS analysis of GFP expression.

Fully infectious HIV-1 was produced in a BSL3 laboratory following the same protocol. pNL4.3 and m-cGAS (or m-cGAS-AA) were transfected at a ratio of 1:1. Titers were determined by infection of Jurkat cells and FACS analysis of p24+ cells.

HIV-2-GFP was produced in 293T cells by plasmid transfection using FuGene HD using pHIV-2 ROD9 ΔEnvΔNef GFP, pVSV-G and m-cGAS (or m-cGAS-AA) at a ratio of 2:1:2. Lentivirus infection

105 HEK293 were seeded in 24-wells. After 24 hours, HIV-GFP stocks were added in the presence of polybrene (8 µg/ml). 18 hours later the medium was exchanged. After additional 48 to 72 hours supernatants and cells were harvested for IFN bioassay and FACS analysis or RT Q-PCR analysis, respectively.

For the IFNβ promoter reporter assay, cells were additionally transfected with 125 ng p125-F-Luc and with 25 ng pRL-TK using lipofectamine 2000. This was done 6-8 hours prior to infection. Luciferase activity was analysed 24 hours after infection and F-Luc activity was normalized to R-Luc.

In some experiments, cells were treated for 1 hour prior to infection with nevirapine (5 µM; cat. nb. 4666; obtained through the NIH AIDS reagent program, Division of AIDS, NIAID, NIH from Division of AIDS, NIAID) or raltegravir (5 µM; cat. nb. 11680; obtained through the NIH AIDS reagent program, Division of AIDS, NIAID, NIH from Merck & Company, Inc.). Alternatively, virus stocks or cGAS plasmid were pre-treated with DNase I (40 µg/ml; Roche, 11284932001) for 1 hour at 37°C prior to infection.

3x105 hMDDCs were seeded in 96-wells and infected with HIV-1-GFP or HIV-1 in the presence of polybrene (8 µg/ml). Cells were harvested for FACS analysis after 16 hours. In some experiments, hMDDCs were seeded in 24-wells, infected and washed after 2 hours to remove the inoculum. Cells were then incubated in fresh medium for 48 hours before the supernatant was tested by IFN bioassay.

4-8x105 BMDMs were seeded in 12-wells and, after O/N incubation, were infected with HIV-1-GFP in the presence of polybrene (8 µg/ml) by spin-infection (1100 g; 90 min; room

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temperature). The inoculum was then removed and fresh medium was added. Supernatant and cells were harvested 24 hours later. mIFNα was detected by ELISA as described in (31). 4x105 BM-DCs were seeded into 24-wells and were infected in the presence of polybrene (8 µg/ml) for 2 hours after which the viral inoculum was washed away. Cells were collected after 48 hours for FACS analysis. Iodixanol gradients

HIV-1-GFP stocks were pelleted [centrifugation over a 20% sucrose cushion; 64,000 g, 2.5 hours, 4°C], re-suspended in PBS and loaded on a pre-formed 6-18% iodixanol (Sigma, cat. nb. D1556) sedimentation velocity gradient made of 1 ml increments of 1.2% iodixanol. The gradient was centrifuged at 200,000 g for 1.5 hours at 4oC in a SW41 Ti rotor in a Beckman XL-80K ultracentrifuge. Fractions were collected by taking 1 ml at a time from the meniscus using tips with the end cut off.

HIV-1 containing supernatants from virus producer cells were pre-cleared (20,000 g for 30 minutes at 4oC) and then spun at 100,000 g in a SW28 rotor for 2 hours. The pellet was resuspended in 1 ml PBS and loaded on iodixanol gradients as above. Adenovirus

Adenovirus was produced in HEK293 cells transfected with m-cGAS or m-cGAS-AA using FuGene HD or in untransfected cells. 2 hours after transfection, cells were infected with AdenoCreGfp virus (cat. nb. 1700, Vector Biolabs) at an MOI of 1. Virus was harvested 48 hours later from cells by three cycles of freeze, thaw and sonication.

Fresh HEK293 cells were infected for 18 hours. Cells were then washed and provided with new medium. After 48 additional hours, supernatants were harvested for analysis with the IFN bioassay and cells were collected for FACS analysis. Sendai virus

Sendai virus was from LGC standards (cat. nb. VR-907). Cells were infected by addition of Sendai virus to the culture medium. MCMV

MCMV-GFP virus (referred to as MCMV herein) was obtained from Paul Klenerman (University of Oxford, UK) and was originally from John Hamilton. MCMV was propagated in primary MEFs by infection using an MOI of 0.01. Virus spread was monitored with a fluorescence microscope and progeny virus was harvested after 5-7 days when >90% of cells expressed GFP. Virus containing cell culture supernatants were pre-cleared by centrifugation (1,500 rpm, 5 min), filtered (0.22 µm), and concentrated by centrifugation over a 20% sucrose cushion (64,000 g, 2.5 hours, 4°C). Viral titres were determined as infectious units/ml by over night infection of primary Sting-/- MEFs with a dilution series of virus stocks, followed by FACS analysis of GFP expression. Cells were infected by addition of MCMV to the culture medium. Small molecule extractions and THP1 stimulation

The method for small molecule extraction from virions was adapted from (24). Pelleted virions were resuspended in lysis buffer (1% Triton X-100, 10 mM Tris.Hcl pH 7.4, 1 mM NaCl, 1 mM EDTA and 3 mM MgCl2) and left on ice for 20 min. Lysates were clarified by centrifugation for 10 min at 1000 g at 4°C. To remove nucleic acids, samples were treated for 45

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min with 50 U/ml benzonase (Sigma) on ice. Next, proteins were eliminated by two sequential phenol-chloroform extractions followed by a chloroform wash to remove traces of phenol. The extract was filtered using Amicon Ultra 3 kDa centrifugal filters (Millipore, cat. nb. UFC500396) and the filtrate was concentrated by centrifugation under vacuum. Samples were resuspended in 20 µl water and stored at -80°C until further use.

cGAMP activity in these extracts was measured using a protocol adapted from (36). 100,000 THP-1 cells treated with 30 ng/ml PMA were seeded in 96-well plates and left overnight. Cells were then washed with medium and overlaid with 25 µl permeabilisation buffer (10 µg/ml digitonin, 50 mM Hepes pH 7.4, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP and 0.1 mM GTP) containing virion extracts or 2’3’-cGAMP standard (cat. nb. C161-005, Biolog) for 30 min at 37°C. Next, cells were washed with medium and 75 µl fresh medium was added. After 24 hours, supernatant was tested in the IFN bioassay.

In some experiments, 5 µl extract or 1 µg cGAMP were incubated in 50 mM Tris pH8.8, 10 mM MgCl2 with or without 0.002 units SVPDE (cat. nb. P3243, Sigma) at 37°C for 1 hour. Treated extracts and cGAMP were then serially diluted in permeabilisation buffer and added to THP1 cells as above. Mass spectrometry

Ammonium acetate was obtained from Sigma-Aldrich. Acetic acid and solvents used (methanol, acetonitrile and water) were of HPLC-grade and purchased from J.T. Baker. Stock solution of 2’3’-cGAMP (obtained from Biolog) was prepared in HPLC-grade water. Calibration curves were constructed using seven calibrators ranging from 0.64 to 10,000 nM. Tenofovir (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Division of AIDS, NIAID) was applied as internal standard.

Reversed Phase (RP) chromatographic separation of calibrators or sample extracts was performed using an HPLC-system (Shimadzu), consisting of two HPLC-Pumps, a temperature controlled autosampler, a degasser, an oven, and a control unit. A Zorbax eclipse XDB-C18 1.8 µm column (50 x 4.6 mm) from Agilent, connected to a C18-Security guard (Phenomenex) and a 2 µm column saver (Supelco) kept at 25°C was used. The mobile phases were 3/97 methanol/water [v/v] (A) and 97/3 methanol/water [v/v] (B), each containing 50 mM ammonium acetate and 0.1% acetic acid. The following gradient was applied: 0 to 5 min, 0 to 50% B and 5 to 8 min, 0% B. The flow rate was 400 µl/min. Detection and quantification of cGAMP was carried out by a tandem mass spectrometer, 5500QTRAP (AB Sciex), equipped with an electrospray ionization source, operating in positive ionization mode. For SRM detection, the following mass transitions [M+H]+ were identified for 2’3’-cGAMP: m/z 675.0 è 136.2 (quantifier), m/z 675.0 è 152.1 (identifier) and for tenofovir: m/z 288.0 è 176.0 (quantifier), m/z 288.0 è 159.1 (identifier). Dot blot

Virus preparations were resuspended in lysis buffer (1% Triton X-100, 10 mM Tris.HCl pH 7.4, 1 mM NaCl, 1 mM EDTA and 3 mM MgCl2) and blotted onto a nylon membrane (Zeta-probe GT membrane, cat. nb. 162-0197, Biorad), which was left to dry and UV cross-linked (UV Stratalinker 2400; 2x Autocross link, 120,000 µJ/cm2). Biotin-cGAMP was detected with streptavidin-HRP (cat. nb. 3310-9, Mabtech, 1:1,000). HRP was inactivated with 0.2% sodium azide and the absence of residual signal was validated by exposing the membrane for one hour. The membrane was then reprobed with mouse α-p24 (cat. nb. 4313, Advance Bioscience

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Laboratory, 1:5,000) followed by α-mouse HRP (cat. nb. NA931VS, GE Healthcare Life Sciences, 1:3,000). Lung fibroblast infections

20,000 primary lung fibroblasts were seeded in 96-well clear bottom plates in MEM containing 20% FCS. After 24 hours, cells were treated with medium alone, with IFN-A/D (cat. nb. I4401, Sigma) or with HIV-1-GFP (all in the presence of 8 µg/ml polybrene). After an additional 24 hours, cells were infected with EMCV or HSV-1 (kind gifts from Caetano Reis e Sousa). After further 24 hours, cells were fixed in 10% formalin and stained with crystal violet (cat. nb. HT90132-1L, Sigma). Data presentation and statistical analysis

Bar graphs show the mean of the indicated number of replicates, and individual values (n=2) or standard deviation (n≥3) are indicated. Data were analyzed with Graphpad Prism software by unpaired t-test or, in the case of multiple comparisons, by two-way analysis of variance (ANOVA). Differences with a P value of

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Fig. S1. Supplementary Fig. 1. HIV-1-GFP produced in cGAS-reconstituted 293T cells induces IFN in HEK293 cells. (A) Experimental setup. (B) Wild-type and mutant cGAS are expressed equally in virus producer cells. 293T virus producer cells were lysed 72 hours after transfection and cGAS protein levels were determined by Western blot using a monoclonal antibody recognizing the FLAG-tag. (C) HEK293 cells endogenously express STING. Extracts from THP1 and HEK293

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cells were tested by Western blot for STING expression. An irrelevant intervening lane was spliced out during preparation of the figure. (D) HEK293 cells induce IFN in response to cGAMP. 2x105 HEK293 cells were transiently transfected with the IFNβ promoter reporter (p125-F-Luc) and with pRL-TK for normalization. After 24 hours, cells were transfected with 2 µg 2’3’-cGAMP or with lipofectamine only (control). Luciferase activity was analyzed after 24 additional hours and F-Luc activity is shown normalized to R-Luc. N=2 biological replicates; average and individual values are indicated. (E) IFN bioassay. HEK293 cells were transduced with pGreenFire-ISRE using lentiviral delivery. Clones were obtained by serial dilution and, based on the responses to IFN, clone 3C11 was selected. 25,000 3C11 cells were seeded in 96-well plates and recombinant human IFNα2 was added. After 24 hours, cells were lysed and firefly luciferase activity was measured. Background bioluminescence in untreated cells was set to 1. N=2 biological replicates; average and individual values are indicated. (F) HIV-1-GFP infected cells (wedges: MOI=10, 5, 2, 1, 0.5, 0.1) were washed after 24 hours. After additional 48 hours, secreted IFN was measured by bioassay (left, N=4 biological replicates) and cells were analyzed by FACS (right). (G) The indicated mRNAs from cells infected as in (F) (MOI=0.5) were quantified by RT Q-PCR relative to GAPDH mRNA. N=6 replicates. In F and G error bars are ±SD and significance was determined by ANOVA (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). Data are representative of three or more independent experiments.

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Fig. S2 HIV-1-GFP from cGAS-expressing virus producer cells induces IFN and ISG expression in THP1 cells. 100,000 THP1 cells were spin-infected with HIV-1-GFP (MOI=1) collected from cells expressing cGAS as indicated, SeV (MOI=0.1), or were left uninfected. Cells were washed after spin-infection. After over-night incubation, secreted IFN was measured by bioassay (A) and the indicated mRNAs were quantified by RT Q-PCR relative to GAPDH mRNA (B). N=6 replicates. Error bars indicate SD and significance was determined by unpaired t-test (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). Data are representative of two independent experiments.

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Fig. S3 IFN induction triggered by HIV-1-GFP from cGAS expressing cells is independent of viral nucleic acids. HEK293 cells were infected for 24 hours, washed and after additional 48 hours, secreted IFN was analyzed by bioassay (A-E) and the fraction of infected cells was determined by FACS (B,C,E). (A) Virus stocks were treated or not with DNase I and then used at MOI=1. Medium and m-cGAS plasmid were incubated with DNase I and then added to cells or transfected, respectively. N=4 biological replicates. (B) HIV-1-GFP directly collected from producer cells (sup 1), supernatant collected after pelleting (sup 2) or pelleted virus (pel) were tested. Wedges: 10-fold dilutions; open circles: not determined. N=3 biological replicates. (C) The effects of nevirapine (Nev) and raltegravir (Ral) treatment of the infected cells were tested. N=2 biological replicates; average and individual values are indicated. (D) Cells were infected with supernatant (wedges: 100, 50, 5 µl) from cells producing HIV-1-GFP or virus like particles (VLPs). N=2 biological replicates; average and individual values are indicated. (E) HIV-1-GFP was pseudotyped with VSV-G or THOV-G and supernatants (VSV-G: 1 and 0.1 µl; THOV-G: 100 and 10 µl) from producer cells were used. N=2 biological replicates; average and individual values are indicated. In A and B error bars are ±SD and significance was determined by ANOVA (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). Data are representative of two or more independent experiments.

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Fig. S4 Setup of the experiment shown in Figure 3A. AG = 2’3’-cGAMP.

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Fig. S5 Detection of cGAMP by mass spectrometry. (A) Synthetic 2’3’-cGAMP at the indicated concentrations in water was analyzed by MS. The analyte peak area is shown in counts. (B) Synthetic 2’3’-cGAMP at the indicated concentration was subjected to the extraction protocol described in Materials and Methods and summarized in Fig. S4. The resulting extract was resuspended in an equal volume of water and analyzed by MS. 2’3’-cGAMP concentration was determined based on (A), showing that ~36% of 2’3’-cGAMP is retained during extraction. N=2 technical replicates; average and individual values are indicated. Data are representative of three or more independent experiments.

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Fig. S6 HIV-2-GFP produced in cGAS-reconstituted 293T cells induces IFN in HEK293 cells. HEK293 cells were infected as indicated with HIV-2-GFP or HIV-1-GFP (wedges: 1:10 dilution series starting with 30 µl supernatant from virus producer cells) and were washed after 24 hours. After additional 48 hours, secreted IFN was measured by bioassay (top) and cells were analyzed by FACS (bottom). N=2 biological replicates; average and individual values are indicated. Data are representative of two independent experiments.

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Fig. S7 The gating strategy for FACS data in Fig. 4A is shown.

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Fig. S8 Infection of human dendritic cells with HIV-1-GFP from cGAS-reconstituted producer cells induces expression co-stimulatory molecules and IFN secretion. (A-C) hMDDCs from two blood donors were infected with HIV-1-GFP at the indicated MOIs. After 48 hours, CD86 expression was analyzed by FACS. (A) The percentage of CD86+ cells is shown. (B) The CD86 median fluorescence intensity (MFI) is shown. (C) Supernatant was tested in the IFN bioassay. Note that these effects were observed in the absence of Vpx. N=2 infections per donor; average and individual values are indicated. Data are representative of two independent experiments.

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Fig. S9 cGAMP-loaded HIV-1-GFP triggers upregulation of MHC molecules on human dendritic cells. hMDDCs from three blood donors were infected with HIV-1-GFP and analyzed by FACS. The gating strategy is shown in panel (A) and MFIs are shown in panel (B) for the indicated markers. N=3 biological replicates. Error bars are ±SD and significance was determined by ANOVA (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). Data are representative of two independent experiments.

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Fig. S10 Setup of the experiments shown in Figure 4E-I.

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Fig. S11 Adenovirus-GFP (Ad-GFP) produced in cGAS-reconstituted cells does not trigger IFN in freshly infected target cells. (A) Ad-GFP was produced in HEK293 cells. Some virus producer cells were co-transfected with cGAS expression constructs. Virus stocks were then used to infect fresh HEK293 cells. Wedges represent 0.01 and 0.001 µl inoculum. IFN production by Ad-GFP infected cells was tested by bioassay. Recombinant IFNα at the indicated doses was used to demonstrate the responsiveness of the bioassay. N=2 biological replicates; average and individual values are indicated. (B) Infection was monitored by FACS. Data are representative of two independent experiments.

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Fig. S12 Infection of mouse dendritic cells with HIV-1-GFP from cGAS-reconstituted producer cells induces expression co-stimulatory molecules. BM-DCs from two mice were infected with HIV-1-GFP and analyzed by FACS. N=2 biological replicates; average and individual values are indicated. Data are representative of two independent experiments.

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Fig. S13 Validation of cGAS- and STING-deficient mice. 150,000 mouse lung fibroblasts of the indicated genotypes were seeded in 24-wells. After over night incubation, cells were transfected with 1 µg/well of E. coli DNA (USB, cat. nb. 14380) or Neo1-99 in-vitro transcribed RNA (32) (IVT-RNA) using lipofectamine 2000. Control cells were treated with transfection reagent alone. mIFNα was measured by ELISA in supernatants harvested 24 hours after transfection. N=3 mice for wt and cGas-/-; N=1 mouse for Sting-/-; n.d.: not detectable (lower limit of detection = 16 U/ml). Error bars indicate SD. Data are representative of two independent experiments.

SM.cover.page.blank.new.pdfViruses transfer the antiviral second messenger cGAMP between cells