RIG-I dependent sensing of poly(da-dt) via the induction of an RNA

Transcription

1 RI-I dependent sensing of via the induction of an RNA polymerase III transcribed RNA intermediate Andrea Ablasser 1*, Franz Bauernfeind 1*, unther Hartmann 1, Eicke Latz 2, Katherine A. Fitzgerald 2* and Veit Hornung 1* 1 Institute for linical hemistry and Pharmacology, niversity of Bonn, Bonn, ermany 2 Division of Infectious Diseases and Immunology, niversity of Massachusetts Medical School, Worcester, MA 165, SA *These authors contributed equally to this study orrespondence should be addressed to V.H. (veit.hornung@uni-bonn.de) Supplementary Figure 1-1 Supplementary Table 1-4 Supplementary References Nature Immunology: doi:1.138/ni.1779

2 a 4 pifn-β-luc (A) SEV EIF4A NS3-4A S139A NS3-4A WT MDA5 T789M RI-I T697M RI-I Δ ard EIF4A NS3-4A S139A NS3-4A WT MDA5 T789M RI-I T697M RI-I Δ ard EIF4A NS3-4A S139A NS3-4A WT MDA5 T789M RI-I T697M RI-I Δ ard b 3 3 NF-κB luc (A) pifn-β-luc (A) sirna control sirna RI-I TBK1 SEV 3 SEV 1 SEV 3 TBK1 SEV 3 SEV 1 SEV 3 Supplementary Figure 1: Dominant negative RI-I constructs or disruption of IPS-1 inhibits poly(dadt) triggered type I IFN induction and RI-I mediates NF-κB activation in response to. a, 293T cells were transfected with the indicated expression plasmids in conjunction with a pifn-β reporter plasmid. EIF4A was used as a control dead box helicase; NS3-4A S139A is the catalytically inactive point mutant of the hepatitis derived NS3-4A protease, the wild-type (WT) version of which cleaves and inactivates IPS-1; MDA5 T789M and RI-I T697M are helicase point mutants of MDA5 and RI-I respectively; RI-IΔard is RI-I devoid of its ard domain. After 24h cells were stimulated with SEV (3 HA/ml) or transfected with. After an additional 24h IFN-β promoter activation was assessed. b, 293T cells were transfected with sirna targeting RI-I or a control sirna. After 48h cells were transfected with an NF-κB reporter or a pifn-β reporter plasmid in conjunction with or TBK1 (5 ng) or were stimulated with SEV (3, 1 or 3 HA/ml). 24h later NF-κB activation or IFN-β promoter activation was assessed. Mean values ± s.e.m. of one representative experiment out of two (b) or three (a) experiments are depicted. Nature Immunology: doi:1.138/ni.1779

3 a IFN- α (ng/ml) p-a b IFN- α (ng/ml) ng 5ng 25ng 3pRNA RNA from transfected 293T 1 2 hloroquine (µg/ml) DNase I (/µl) Size (kb) c pifn-β-luc (A) pdna3 NS3-4A S139A NS3-4A WT RI-I Δ ard 3pRNA SEV RNA from untreated 293T RNA from transfected 293T RNA from SEV infected 293T Supplementary Figure 2: Stimulatory RNA from transfected cells is insensitive to DNase I and triggers type I IFN responses via RI-I in 293T cells. a, PBMs were chloroquine treated at the indicated concentrations and subsequently transfected with or p-a. 24h later IFN-α production was assessed by ELISA. b, RNA from transfected 293T cells was isolated and treated with DNase I at the indicated concentrations. 3pRNA and were treated the same way. hloroquine-treated PBMs were then transfected with the respective nucleic acids and IFN-α production was assessed 24h later. DNase I-treated RNA from transfected cells and was analyzed on an agarose gel. c, 293T cells were transfected with the indicated expression plasmids. 24h later pifn-β reporter plasmid was transfected in conjunction with 3pRNA, or RNA isolated from untreated, or SEVstimulated 293T cells. Direct infection with SEV (3 HA/ml) served as a positive control. After an additional period of 24h IFN-β promoter activity was assessed. Mean values ± s.e.m. of one representative experiment out of two (a, b, c) experiments are depicted. Nature Immunology: doi:1.138/ni.1779

4 12 IFN-α (% of control) oncentration (nm) DNA size (nt) Supplementary Figure 3: Dose response of stimulatory dsdnas in PBMs. dsdnas of different length generated from pcdna3 by PR were transfected into chloroquine-blocked PBMs at the indicated molar concentrations. IFN-α production was measured by ELISA and data were normalized to the 245nt dsdna transfected at 8.8 nm. Mean values ± s.e.m. of one representative experiment out of two independent experiments are depicted. Nature Immunology: doi:1.138/ni.1779

5 15 RNA from transfected cells 3pRNA IFN- α (ng/ml) 1 5 Alkaline phosphatase _ + _ + _ + Size (nt) Alkaline phosphatase 5 -pppnnnnnnnnnnnnn-3 5 -NNNNNNNNNNNNNNp-3 Supplementary Figure 4: Alkaline phosphatase treatment abolishes the activity of triggered RNA. RNA from transfected 293T cells was isolated and treated with alkaline phosphatase. Poly(dA-dT) or 3pRNA were treated the same way. hloroquine-blocked PBMs were then transfected with the respective nucleic acids and IFN-α production was assessed 24h later. In addition, the above nucleic acids were analyzed on an Agilent small RNA chip and the obtained data are depicted in a gel-like image. A schematic illustration of the enzymatic activity of alkaline phosphatase is depicted below the gellike image. Mean values ± s.e.m. of one representative experiment out of three experiments are depicted. Nature Immunology: doi:1.138/ni.1779

6 IFN- α (ng/ml) RNase T1 RNA from transfected cells 1(rA-r) 3pRNA _ + _ + _ + _ + 1(rA-r) 5 -pppaaaaaaaaaa-3 3 -AAAAAAAAAAppp-5 Size (nt) Supplementary Figure 5: A 2mer 5 triphosphate RNA consisting of alternating ra-r is insensitive to RNase T1 under denaturing conditions. RNA from transfected 293T cells, and 3pRNA were treated with RNase T1 under denaturing conditions. A 2mer 5 triphosphate RNA oligonucleotide consisting of alternating ra-r was treated the same way. hloroquine-treated PBMs were then transfected with the respective nucleic acids and IFN-α production was assessed 24h later. In addition, the above nucleic acids were analyzed on an Agilent small RNA chip and the obtained data are depicted in a gel-like image. Mean values ± s.e.m. of one representative experiment out of three experiments are depicted. Nature Immunology: doi:1.138/ni.1779

7 a 1 b 2 RNA from 293T cells IFN-α (pg/ml) c 3N (relative expression) AT+3N AT+6T+3N IFN-α (pg/ml) AT+3N: PS AT+3N PS: AT+3N AT+6T+3N Primer binding site for the 3N - 3 RAE PR 5 -ATATATATATA...AAAATAAAAA-3 5 -AAAATAAAAA-3 Supplementary Figure 6: Short synthetic triggers the formation of stimulatory RNA. a, Poly(dA-dT), AT+3N or AT+6T+3N was transfected into chloroquine-treated PBMs and IFN-α production was assessed 24h later. b, In addition, the above nucleic acids were transfected into 293T cells and RNA was isolated. The RNA was then transfected into chloroquine-treated PBMs and IFN-α production was assessed 24h later. c, Murine cds were transfected with the dsdna oligonucleotides AT+3N or PS, a dsdna template that only contains the 3N tag. 14h later, RNA was isolated and the expression of the 3N tag normalized to HPRT1 expression was assessed. Mean values ± s.e.m. of one representative experiment out of two (c) or three (a, b) experiments are depicted. Nature Immunology: doi:1.138/ni.1779

8 a DNA template: AT+3N: 35(dA-dT) 3N Primer 1 binding site b Transfection and RNA isolation: AT+3N 293T DNA RNA purified RNA c RNA transcript: Primer 1 binding site d Poly A tailing: Primer 1 binding site 5 - AAAAAAAAA-3 e Reverse transcription: Primer 1 binding site AAAAAAAAA-3 TTTTTTTTT -5 f PR: Primer AAAAAAAAA -3 TTTTTTTTT -5 RT primer Primer 2 binding site Primer 2 g PAE: Marker AT+3N Supplementary Figure 7: 3 RAE strategy to detect the transcription of a transcript. a, A dsdna template (AT+3N) containing a 7 nt homopolymeric stretch of 35 x da-dt was constructed to contain a non da-dt region at the 3 end (3N) that could be used as a specific primer binding site. b, This template was transfected into 293T cells and RNA (c) was isolated after 8h - 16h. d, Total RNA was polyadenylated using poly(a) polymerase and then (e) reverse transcribed using an oligo dt primer containing an additional primer binding site on its 5 end. f, The obtained cdna was then amplified in a PR reaction and analyzed on a polyacrylamide gel (schematic illustration) (g). Nature Immunology: doi:1.138/ni.1779

9 RP1 (relative expresison) RP3 (relative expresison) #1 #2 RP1 #1 #2 RP3 RP7 (relative expresison) RP2 (relative expresison) #1 #2 RP2 #1 #2 RP7 Supplementary Figure 8: sirna-mediated knock down of RP1, RP2, RP3 and RP7. 293T cells were transfected with two individual sirnas targeting the indicated genes or a control sirna. 48h after transfection expression of the respective target gene normalized to β2 microglobulin expression was analyzed by real time PR. Mean values ± s.e.m. of one representative experiment out of two experiments is depicted. Nature Immunology: doi:1.138/ni.1779

10 a EBER-1 RNA DNA template: NNNTTATTTTT-3 5 -NNNTTT-3 5 -NNNTTTTTTTT-3 3 -NNNAATAAAAA-5 3 -NNNAAA-5 3 -NNNAAAAAAAA-5 Putative RNA transcript: 5 3 A A A A A A A 3 b EBER-1 RNA EBER-1 RNA blunt end EBER-1 RNA blunt end + poly tail 3 25 pifn-β-luc (A) EBER-1+2 EBER-1 RNA EBER-1 RNA blunt end EBER-1 RNA blunt end + poly tail Supplementary Figure 9: Blunt end EBER-1 RNA is superior to natural EBER-1 RNA in IFN-β-activating capacity. The genomic locus that encodes EBER-1 and EBER-2, or the portion encoding only EBER-1, was amplified from EBV DNA by PR. In addition a modified EBER-1 locus was amplified lacking the naturally occurring 4 bases upstream of the poly(t) stretch termination signal and exchanging base No. 159 of the encoded EBER-1 RNA transcript from to T. This template thus encodes for a blunt end version of EBER-1 RNA (EBER-1 RNA blunt end). In addition, the latter template was modfied to contain an additional poly(t) stretch to obtain EBER-1 RNA blunt end + poly() tail. A schematic view of the 3 portions of the encoding dsdna templates and the expected terminal regions of the different EBER-1 RNA transcripts is depicted. Secondary structure prediction of the presumed RNA transcript was done using Mfold 5 (a). The respective DNA templates were transfected into IFN-primed 293T cells in conjunction with pifn-β reporter plasmid. 24h later IFN-β promoter activity was assessed (b). Mean values ± s.e.m. of one representative experiment out of three experiments is depicted. Nature Immunology: doi:1.138/ni.1779

11 a shrna KAT2B+ Transcription 6 promoter start site shrna gene 5 -TATTTTAAAAAAAAAAAAATTATTATAATTAAAATTATAATAATTTTTTTTTT-3 3 -ATAAAATTTTTTTTTTTTTAATAATATTAATTTTAATATTATTAAAAAAAAAA-5 shrna KAT2B- 6 promoter Transcription start site shrna gene 5 -TATTTTAAAAAAAAAAATTATTATAATTAAAATTATAATAATTTTTTTTTT-3 3 -ATAAAATTTTTTTTTTTAATAATATTAATTTTAATATTATTAAAAAAAAAA-5 b 16 c pifn-β-luc (A) pifn-β-luc (A) d 15 SEV IRF1 shrna KAT2B+ shrna KAT2B shrna KAT2B+ (ng) pifn-β-luc (% of control) shrna KAT2B+ IRF1 MDA5 RI-I IPS-1 Supplementary Figure 1: 6-driven shrna triggers potent type I IFN induction via RI-I. Two shrna constructs from the literature were studied. One of these constructs (shrna KAT2B+) has been shown to induce potent type I IFN responses, whereas a modified version of the latter (shrna KAT2B-) has been reported to be inert in terms of type I IFN induction 51. a, A schematic illustration of the two shrna genes is shown. b, 293T cells were transfected with plasmids encoding for shrna KAT2B+ or shrna KAT2b- in conjunction with a pifn-β reporter plasmid. IRF1, and SEV served as controls. After 24h IFN-β promoter activation was assessed. c, 293T cells were transfected as in (b) with the indicated amounts of shrna KAT2B+, whereas served as a control. d, 293T cells were transfected with sirnas targeting MDA5, RI-I, IPS1 or control sirnas. After 48h, cells were transfected with a pifn-β reporter plasmid in conjunction with IRF1 or shrna KAT2B+. 24h later IFN-β promoter activation was assessed. Two individual sirnas (#1 and #2) were used per gene and each sirna was tested in triplicate. Data are represented as mean values normalized to the set of control sirnas. Mean values ± s.e.m. of one representative experiment out of three (b, c, d) experiments are Nature Immunology: doi:1.138/ni.1779 depicted.

12 Species 1 ene Symbol (Alias) Forward Reverse Mm Hprt1 5 -TTAAAAATT-3 5 -TAATATATTATATAAA-3 Mm Ifnb1 5 -ATAAATATAA-3 5 -TTTTTATTA-3 Ppib (yclophilin B) 5 -AAAAATTATA TTTATTTATAA-3 Polr3a (RP1) 5 -ATAAATTAAA-3 5 -TATTTTTTAT-3 Polr3b (RP2) 5 -ATTAAAATT-3 5 -AAAAAAATTT-3 Polr3c (RP3) 5 -TTTTTTAAAT-3 5 -ATTATTTTT-3 Polr3g (RP7) 5 -AAAAAAAAAAAAA-3 5 -ATATTATTAT-3 B2m 5 -TTTTATTTTATT TTTTATTAA-3 n/a AT+3N or AT+6T+3N 5 -AAAATAA TATAATTTTT-3 2 5S rrna 5 -TTTTATATT TATAATTTTT-3 2 EBV EBER-1 RNA 5 -ATTTTTTTT TATAATTTTT-3 2 EBV EBER-2 RNA 5 -AAAAAATT TATAATTTTT-3 2 Supplementary Table 1: Primers used for real-time PR. 1 = homo sapiens, Mm = mus musculus, EBV = Epstein-Barr virus. 2 The transcripts that were polyadenylated using poly (A) polymerase for subsequent reverse transcription were amplified using a reverse primer that equals the 5 end of the 3-RAE-RT primer. Nature Immunology: doi:1.138/ni.1779

13 Name Sequence 1 AT+3N AT+6T+3N (AT)35 PS 5 - ATATATATATATATATATATATATATATATATATATATATATATATATATAT- ATATATATATATATATATAAAATAAAAA-3 5 -ATATATATATATATATATATATATATATATATATATATATATATATATATAT- ATATATATATATTTTTTTAAAATAAAAA-3 5 -TTATATATATATATATATATATATATATATATATATATATAT- ATATATATATATATATATATATATATATATAA-3 5 -AAAATAAAAA-3 3pRNA 2 5 -ATAATAATATATAAAAAAAAAAA-3 1(rA-r) ATAATAATATATTATATATATATATATATATAT-3 Supplementary Table 2: Sequences of the synthetic dsdnas and the in vitro transcription templates. 1 Only the sequence of the top strand is listed. 2 The sequence coding for the RNA transcript transcribed by T7 RNA polymerase is underlined. Nature Immunology: doi:1.138/ni.1779

14 Template Name Forward Reverse pdna3 dsdna 2 5 -AAATATTA TAATATTT-3 pdna3 dsdna 4 5 -AAATATTA-3 5 -TATTTTTATTATA-3 pdna3 dsdna AAATATTA-3 5 -TATTTTTATTATA-3 pdna3 dsdna AAATATTA-3 5 -TAATTTTTTT-3 pdna3 dsdna AAATATTA-3 5 -TAAAAAATAAA-3 pdna3- FP FP control 5 -ATTAAAA-3 5 -TTTAATTAT-3 EBV gdna EBER gene locus 5 - ATAAAATTAAAA TTAATTTTTA- AAA-3 EBV gdna EBER-1 gene locus 5 - ATAAAATTAAAA-3 5 -ATAAAAATAA-3 EBV gdna EBER-2 gene locus 5 - ATAAATTTATTTTAAT- TTAT TTAATTTTTA- AAA-3 EBV gdna EBV gdna EBER-1 gene locus blunt end EBER-1 gene locus blunt end + poly() tail 5 - ATAAAATTAAAA-3 5 -AAAATTATT ATAAAATTAAAA AAAAAAAAAT- TATT-3 Supplementary Table 3: Primers used for the generation of dsdna templates by PR. Nature Immunology: doi:1.138/ni.1779

15 Figure Species 1 ene symbol (Alias) Sequences 2 1a, 4b Tlr3 1: 5 -AAAdTdT-3 2: 5 -AAAAAAAdTdT-3 3: 5 -AAAAAAdTdT-3 1a, 4b, S1 Ifih1 (MDA5) 1a, 4b, S1 Ddx58 (RI-I) 1a, 4b, S1 Mavs (IPS-1) 1a, 4b, S1 n/a n/a () 1: 5 -AAAAAAdTdT-3 2: 5 -AAAAAdTdT-3 3: 5 -AAAAAAAdTdT-3 1: 5 -AAAAAAAAdTdT-3 2: 5 -AAAAAAAAAdTdT-3 3: 5 -AAAAAAAdTdT-3 1: 5 -AAdTdT-3 2: 5 -AAAAdTdT-3 3: 5 -AAAAAdTdT-3 1: 5 -AAAAAAA dtdt-3 2: 5 -AAAAA dtdt-3 Source Ambion Ambion Ambion Ambion Ambion 1b, 2e, S1b Ddx58 (RI-I) 5 - AAdTdT-3 Biomers 1b, 2e Mm Ddx58 (RI-I) 5 -AAAAAAAdTdT-3 Biomers 1b, 2e n/a n/a (; targets Luciferase) 5 -AAAAdTdT-3 Biomers 4 Oas1 1: 5 -AAAAAAdTdT-3 Ambion 2: 5 -AAAAAAAdTdT-3 3: 5 -AAAAdTdT-3 4 Oas2 1: 5 -AAAAdTdT-3 Ambion 2: 5 -AAAAAAAAdTdT-3 3: 5 -AAAAAAAAdTdT-3 4 Oas3 1: 5 -AAAAdTdT-3 Ambion 2: 5 -AAAAAdTdT-3 3: 5 -AAAAAAdTdT-3 4 Oasl 1: 5 -AAAAAdTdT-3 Ambion 2: 5 -AAAAdTdT-3 3: 5 -AAAAAdTdT-3 4 Rnasel 1: 5 -AAAAdTdT-3 Ambion 2: 5 -AAAAAAAAAAAdTdT-3 3: 5 -AAAAAAAdTdT-3 6e, 6f, 6g, Polr3a 1: 5 -TAATATTAATTAdTdT-3 Qiagen S8 (RP1) 2: 5 -AAAATATAAdTdT-3 3: 5 -AATATTATATAdTdT-3 4: 5 -TTTTATATTTAAdTdT-3 6e, 6f, 6g, S8 Polr3b (RP2) 1: 5 -AAAAAAAATTATAdTdT-3 2: 5 -AAAATTAATAATAdTdT-3 3: 5 -TAAAATTTTTTAAdTdT-3 4: 5 -ATTATATAATAAAdTdT-3 Qiagen Supplementary Table 4 (part 1): sirna sequences. 1 = homo sapiens, Mm = mus musculus. 2 Only the sense strand is depicted. Nature Immunology: doi:1.138/ni.1779

16 Figure Species 1 ene symbol (Alias) Sequences 2 Source 6e, 6f, 6g, S8 Polr3c (RP3) 1: 5 -ATTATAAAATAAdTdT-3 2: 5 -TAATTATATAAAdTdT-3 3: 5 -AAATAATTATTAdTdT-3 4: 5 -AATATATTAATAdTdT-3 Qiagen 6e, 6f, 6g, S8 Polr3g (RP7) 1: 5 -AAAAATATAATAdTdT-3 2: 5 -AATAATTTAAAdTdT-3 3: 5 -TTTTATAATTAAATAdTdT-3 4: 5 -TAATATAATTAAAdTdT-3 Qiagen 6e, 6f, 6g, 7f Ddx58 (RI-I) 1: 5 -TTTAAATTTTTATAdTdT-3 2: 5 -AAAAATTATATAdTdT-3 3: 5 -AATTTAAAAAATTTAdTdT-3 4: 5 -AAATTATAAATATdTdT-3 Qiagen 6e, 6f, 6g, 7f Mavs (IPS-1) 1: 5 -TTTAAAAAATATAdTdT-3 2: 5 -AAAAATATATAAdTdT-3 3: 5 -TATAATTTAAAdTdT-3 4: 5 -TTAAAATTTATATTAdTdT-3 Qiagen 6e, 6f, 6g, 7f, S1b, S8 n/a n/a () 1: AllStars Negative sirna (at. No. SI365318) 2: Negative sirna (at. No. SI365325) Qiagen Supplementary Table 4 (part 2): sirna sequences. 1 = homo sapiens, Mm = mus musculus. 2 Only the sense strand is depicted. Supplementary References: 5. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, (23). 51. Pebernard, S. & Iggo, R.D. Determinants of interferon-stimulated gene induction by RNAi vectors. Differentiation 72, (24). Nature Immunology: doi:1.138/ni.1779