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1 Supplementary Materials for Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness Gayathri Manokaran, Esteban Finol, Chunling Wang, Jayantha Gunaratne, Justin Bahl, Eugenia Z. Ong, Hwee Cheng Tan, October M. Sessions, Alex M. Ward, Duane J. Gubler, Eva Harris, Mariano A. Garcia-Blanco, Eng Eong Ooi* *Corresponding author. Published 2 July 2015 on Science Express DOI: /science.aab3369 This PDF file includes: Materials and Methods Figs. S1 to S13 Table S1 References

2 Materials and Methods Cells, viruses and infections HuH-7, C6/36, Vero and BHK-21 cells were purchased from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Primary monocytes were isolated from the principal investigator and cultured as described (28). The DENV-2 infected patient sera used in this study were gifts from the Dengue Branch of the Centers for Disease Control and Prevention, Puerto Rico. Low-passage stocks of virus isolates were generated by inoculating acute patient sera onto C6/36 cells as previously described (29). These virus stocks were propagated in the C6/36 cell line and harvested 7 days post-infection following filtration. All viral isolates included in this study were passaged less than 5 times. Viruses were confirmed to be DENV-2 by an immunofluorescence assay, aliquoted and stored at 80 C until use. Infectious titers were determined by plaque assay as previously reported (30). All infections were performed at a multiplicity of infection (MOI) of 1 for 24 h unless otherwise indicated. For primary monocytes, infection was performed at MOI 10 with an enhancing concentration of humanized 3H5 monoclonal antibody (0.391µg/ml) to permit efficient uptake of the viruses (30). Infectivity was measured using plaque assay and quantitative real-time PCR (qrt-pcr) analysis of viral genomes. For Nicaraguan clinical isolates, NI-1 clade consisted of DENV-2 Asian-American genotype viruses present in Managua, Nicaragua, through The NI-2B clade replaced NI-1, and was present from (5).

3 Sequence and structure analysis The complete coding and non-coding regions of the clinical isolates were sequenced previously (29). The maximum likelihood phylogenetic tree was constructed using MEGA6 (31) with the GTR+Γ4 model of nucleotide substitution from an alignment of 158 open reading frame DENV-2 sequences from Puerto Rico and neighboring countries. Neighbor-joining trees with a maximum likelihood optimized nucleotide model were generated using PAUP for full genomes of the 39 Puerto Rican isolates and 154 Nicaraguan isolates (32). Statistical support for the topology of all the trees was determined by 1000 bootstrap replications. For the amino acid and nucleotide substitutions listed in Fig. 1B, the total number of viruses in each clade was counted and 25% of this total number in each clade determined as x viruses. As long as a substitution was present in x or more than x viruses in each clade, it was included in the table. 25% was selected arbitrarily to rule out stochastic substitutions and due to the lack of a large sample size, the analysis was performed in this manner. The phylogenetic analysis of the prm and E protein amino acid sequences was performed using the Maximum Likelihood method based on the JTT matrix-based model (33). Individual gene trees were generated with the same method described above. Topological congruence of the gene trees with the genome tree was determined by the Shimodaira-Hasegawa and the Kashino- Hasegawa likelihood based tests (34, 35). To construct the secondary structures of the 3 UTR, the chemical probing model proposed by Chapman et al, 2014 was used (17). Since the substitutions in the Nicaraguan clade replacement event were found to be a covariation of a base pairing, the UNAFold webserver was used to estimate the impact of this covariation on the molecule s free energy (36).

4 Quantification of RNA Total RNA from infected cells was extracted using Trizol (Invitrogen) or the RNeasy kit (Qiagen). Superscript III reverse transcriptase and random hexamers (Invitrogen) were used in the reverse transcription reactions to generate cdna. qrt-pcr was performed using the iq SYBR Green Supermix Kit (Bio-Rad) according to the manufacturer's instructions. Quantification of grna and sfrna levels was performed as previously described (16). Primers used to detect the immune genes were obtained from OriGene. Quantification of DENV RNA after TRIM25 knockdown was performed as previously described (37). All PCR reactions were normalized against GAPDH. Reactions were run on a Roche Lightcycler 480 (Roche) and data analysis was carried out with the Lightcycler 480 software. qrt-pcr conditions to detect viral RNA: 95 C for 5 min, followed by 45 cycles of 95 C for 10 sec, 55 C for 5 sec and 72 C for 10 sec. Cycling conditions for the quantification of immune genes: 95 C for 5 min, followed by 45 cycles of 95 C for 5 sec, 53 C for 10 sec and 72 C for 10 sec. sirnas The sirnas used for IRF-3 silencing were obtained from Singapore Applied Biologics (SABio). They target three different sequences in IRF-3 RNA (IRF3-01: 5 GCAAAGAAGGGUUGCGUUU 3 ; IRF3-03: 5 AUGCACAGCAGGAGGAUUU 3 and IRF3-04: 5 GGGAAGAGUGGGAGUUCGA 3 ). AllStars negative control sirna (Qiagen) was used as the control sirna (sic). The sirna duplex used for TRIM25 knockdown including the negative control sirna was obtained from OriGene. Its sequence is 5 GGAAUUCUCAGACCGAUAGGAUGTC 3.

5 100 nm sirna was transfected using DharmaFect2 (Dharmacon) according to manufacturer s instructions 48 hours prior to infection. Knockdown efficiency was determined by Western blot using anti-irf3 (1:3000) (Cell Signaling) and anti-trim25 (1:5000) (BD Biosciences). Constructs The DENV-2 reporter replicon system (pdenrep-fh) has been described previously and was a gift from Eva Harris (20). This replicon has the backbone of the Thai DENV-2 strain (U ) and contains Renilla luciferase and a T7 promoter region. Point mutations were made to create the other replicons (pdenrep-fh/pr1, pdenrep-fh/pr2b/28nt, pdenrep-fh/pr2b/117nt and pdenrep-fh/pr2b) using the Quikchange site-directed mutagenesis kit (Stratagene) as per manufacturer s instructions. A Firefly luciferase containing construct ptnt-ffluc was used as the normalization control. In vitro transcription All DENV replicon DNA templates were generated by XbaI digestion of the corresponding plasmids, followed by phenol-chloroform extraction and ethanol precipitation. The T7 RiboMax large-scale RNA production system (Promega) and m 7 G(5')ppp(5')A RNA Cap Structure Analog (New England Biolabs) were used to generate capped RNA by in vitro transcription with the modifications to the manufacturer s protocol as previously described (38). Electroporation and dual-luciferase assay Briefly, HuH-7 cells were trypsinized and washed thrice with cold, sterile PBS after which they were counted and resuspended to cells/ml in cold Opti-MEM (Invitrogen).

6 800 µl of the cell suspension was aliquoted into each 4 mm electroporation cuvette (BioRad) followed by addition of 10 µg of purified replicon RNA and 1 µg of ptnt-ffluc. After one pulse at 950 μf and 250 V with a Gene Pulser II system (Bio-Rad), cells were incubated at room temperature for 10 min. Cells were gently transferred from each cuvette to 11.2 ml of prewarmed media and 500 µl of this final mixture was aliquoted into each well (sufficient for a 24- well plate). Cells were harvested at various time-points using the passive lysis buffer (Promega) and luciferase activity was measured using the Dual Luciferase Reporter Assay system (Promega) according to manufacturer s instructions. DENV-2 sfrna transfections DENV-2 3 UTR templates were PCR amplified from pdenrep-fh or viral cdna to add a T7 promoter immediately upstream of the DENV-2 stop codon and in vitro transcribed using the MEGAscript T7 transcription kit (Life Technologies). A size-matched control RNA was similarly prepared from the pgem-t vector (Life Technologies). The RNAs were decapped by tobacco acid pyrophosphatase treatment (Epicentre) and subjected to heat denaturation followed by cooling to form secondary structures. All RNAs were quantified spectrophotometrically and agarose gel electrophoresis was used to verify their integrity before use. 500 ng of the RNAs was either co-electroporated with pdenrep-fh or transfected into cells on a 24-well plate along with polyinosinic-polycytidylic acid (PolyIC) (1 g/ l per well) to stimulate the RIG-I pathway using Lipofectamine RNAiMax (Life Technologies) as per manufacturer s instructions. RNA-protein interactions DENV-2 3 UTR RNAs were made from viral cdna using AW005-PR-FOR (5 CGG GTA TGT GCG TCT GGA TCC TAT RGG AAG AGG AAG AGG CMG G 3 ) and AW135R

7 (5 TTG TTG CTG CGA TTT GTA AGG G 3 ). Size-matched control RNA was constructed from a plasmid as reported before (22). Tobramycin RNA affinity chromatography, mass spectrometry and data analysis was done as previously described (39). Briefly, a tobramycin RNA aptamer was incorporated into RNAs containing the partial 3 UTRs (the proximal ends of PR-1 or PR-2B 3 UTR, i.e. SL-II to SL-IV) or a size-matched control cloned from the DENV-2 NS2A open reading frame (22). RNA was bound to a tobramycin matrix, incubated with SILAClabeled HuH-7 cell lysates, washed and eluted using excess tobramycin. Eluates were analyzed by qms, allowing a direct comparison of proteins bound to the PR-1 3 UTR, PR-2B 3 UTR or control RNA. In order to identify the proteins involved in innate immunity, the list of proteins from the MS data was loaded into GO term finder. 32 out of 1202 proteins were represented in the GO term "innate response process" (40). RNA-immunoprecipitations were carried out using the MAGNA-RIP kit (Millipore) following manufacturer's recommendations. The levels of RNA in IP were determined by quantitative real-time RT-PCR and normalized to GAPDH mrna levels, control mouse IgG (TRIM25) and rabbit IgG (MAVS) IP following this formula: Fold enrichment = 2 -ddct where ddct= ((CT RNA,IP -CT GAPDH,IP )-(CT RNA,controlIP -CT GAPDH,controlIP )) Electrophoretic mobility shift assay (EMSA) Radiolabeled sfrna was in vitro transcribed from DENV-2 3 UTR templates using the MAXIscript T7 transcription kit (Life Technologies) and dutp α 32 P. Purified recombinant TRIM25 protein (OriGENE) was incubated at different concentrations with radiolabeled sfrna (30 nm) for 30 min at room temperature in a HEPES-based binding buffer. Lanes with no protein (i.e. 0 μm TRIM25) were used as a migration size control containing PR-1 and PR-2B

8 sfrna only. Complexes were resolved on a 4-20% acrylamide Tris borate EDTA nondenaturing gel (Bio-Rad) after a 45-min migration at room temperature, visualized and quantified by phosphorimaging. ImageJ software was used to calculate band intensity. Immunoprecipitations and Immunoblot analysis Immunoprecipitations were performed as previously detailed (30). Primary antibodies used were: anti-trim25 (1:200) (BD Biosciences), anti-mavs (1:1000) (Abcam), antiubiquitin (1:200) (P4D1, Santa Cruz) and anti-rig-i (1:300) (Alexis). Bands were visualized using ECL (Amersham) for chemiluminescence development. Statistical analysis All results are presented as mean ± SD of at least 3 independent experiments, unless otherwise indicated. Data were analyzed using unpaired, two-tailed Student's t-test or ANOVA and considered significant if p<0.05 (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

9 Fig. S1. Whole genome phylogenetic tree of DENV-2 clinical isolates from Puerto Rico and neighboring countries. Black dots indicate viruses used in this study.

10 Fig. S2. Phylogenetic analysis of the prm and E protein amino acid sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (33). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 56 amino acid sequences for the prm and E proteins of DENV-2 isolates from Puerto Rico and other Caribbean countries ( ). Isolates from the endemic lineage are labelled in blue and isolates from the epidemic lineage ( ) are highlighted in red.

11 Fig. S3. Amino acid and nucleotide substitutions in PR-1 and PR-2B viruses. Substitutions are present in the different genetic regions of at least 25% of the PR-1 and PR-2B viruses.

12 Fig. S4. Phylogenetic trees of the whole genome, coding and non-coding regions of Puerto Rican clinical isolates used in this study. Isolates belonging to the PR-1 lineage are marked in blue color while the isolates from the PR-2B lineage are highlighted in red. One exception is PR5828 (black colour) as it appears to be intermediate between the two clades. The p-values generated by the Shimodaira-Hasegawa test are shown below each tree in brackets. This test statistic is the score difference between the Maximum Likelihood tree (full genome phylogenetic tree) and every other tree compared. Comparison with NS1, NS3, NS5 regions and the 3 UTR yield p-values that are not significant, indicating that they are good explanations of the dataset, which separates the isolates into two clades.

13 Fig. S5. Phylogenetic trees of the coding and non-coding regions of Puerto Rican clinical isolates used in this study. Isolates belonging to the PR-1 lineage are marked in blue color while the isolates from the PR-2B lineage are highlighted in red. One exception is PR5828 (black colour) as it appears to be intermediate between the two clades. The p-values generated by the Shimodaira-Hasegawa test are shown below each tree in brackets. This test statistic is the score difference between the Maximum Likelihood tree (full genome phylogenetic tree) and every other tree compared. Comparison with 5 UTR, Capsid, prm (premembrane), Env (envelope), NS2A, NS2B, NS4A, and NS4B regions yield p-values that are significant, indicating that they are different from the full genome tree that separates the viruses into two clades.

14 Fig. S6. 3 UTR structure of Puerto Rican DENV-2 PR-1 constructed using the model proposed by E.G. Chapman et al., 2014 (17). The 3 substitutions present in the structure of PR-2B viruses are indicated in blue. Annotations are as follows: SL, stemloop; pk, pseudoknot; DB, dumbbell.

15 Fig. S7. Phylogenetic trees of the whole genome, coding and non-coding regions of Nicaraguan DENV-2 isolates. Major Nicaraguan clades are highlighted (black: Nicaraguan isolates from ; purple: NI-1; green: NI-2A; blue: NI-2B). The p-values generated by the Shimodaira-Hasegawa test are shown below each tree in brackets. This test statistic is the score difference between the Maximum Likelihood tree (full genome phylogenetic tree) and every other tree compared. Comparison with NS1, NS5 regions and the 3 UTR yield p-values that are not significant, indicating that they are good explanations of the dataset, which separates the viruses into NI-1 and NI-2B clades..

16 Fig. S8. Phylogenetic trees of the coding and non-coding regions of Nicaraguan DENV-2 isolates. Major Nicaraguan clades are highlighted (black: Nicaraguan isolates from ; purple: NI-1; green: NI-2A; blue: NI-2B) The p-values generated by the Shimodaira-Hasegawa test are shown below each tree in brackets. This test statistic is the score difference between the Maximum Likelihood tree (full genome phylogenetic tree) and every other tree compared. Comparison with 5 UTR, Capsid, prm (premembrane), Env (envelope), NS2A, NS2B, NS3, NS4A, and NS4B regions yield p-values that are significant, indicating that they are different from the full genome tree that separates the viruses into NI-1 and NI-2B clades.

17 Fig. S9. 3 UTR structure of Nicaraguan NI-1 DENV-2. Substitutions observed in the NI-2B clade are shown in red. Annotations are as follows: SL, stemloop; pk, pseudoknot; DB, dumbbell

18 Fig. S10. Validation of transfection efficiency. Quantification of PR-1 sfrna, PR-2B sfrna and size matched RNA control (Ctrl RNA) by qrt-pcr, 24 hours post-transfection with polyic. Results show that there is no significant difference in transfection efficiency between the three RNAs. Shown are representative examples of 3 experiments performed. All data are expressed as mean ± SD. Experiment was analyzed with T-test. n.s: non-significant.

19 Fig. S11. Detection of sfrna-binding proteins using SILAC-qMS. Volcano plots displaying proteins detected (n=1199) and enriched (n=198) with partial 3 UTRs (sequence ranging from the start of SL-II to the end of SL-IV) of PR-1 (A) or PR-2B (B) clades as compared to control RNA. Red dots represent significantly enriched proteins. The 4 labeled proteins (TRIM25, MAVS, Mitogen-Activated Protein Kinase 1 - MAPK1, Peroxiredoxin 3 - PRDX3) are known to be involved in signaling for IFN-β induction. Annotations as depicted: M/L (PR-1 3 UTR divided by control), H/L (PR-2B 3 UTR divided by control). Shown [(A) and (B)] are

20 representative examples of 3 experiments performed. Experiments were analyzed with T-test followed by multiple sampling correction using the Benjamini-Hochberg method (FDR). Corrected P values below 0.05 were considered as significant. (C) Dot plot highlighting the 32 proteins selected by the GO term finder to be involved in type-i interferon responses. Annotations as depicted: M/L (PR-1 3 UTR divided by control), H/L (PR-2B 3 UTR divided by control). Green dots show proteins with H/L and M/L ratios lower than 1.5 (i.e. not significantly enriched). Purple dots show proteins with H/L and M/L ratios higher than 1.5 (i.e. significantly enriched in presence of both 3 UTRs) and the blue dots show proteins with H/L ratio higher than 1.5 but M/L ratio lower than 1.5 (i.e. significantly enriched with PR-2B 3 UTR as compared to PR-1 3 UTR).

21 Fig. S12. RNA-immunoprecipitation of MAVS. RNA-immunoprecipitation (RIP) was performed on HuH-7 cells 24 hpi with 2 viruses, one from each clade (MOI 5). Infected cells were subjected to MAVS or anti-rabbit IgG control pull-down followed by immunoblotting with MAVS to confirm efficacy of pull-down. Input: Cell lysate before RIP; Sup: Supernatant following incubation of antibody-coated beads with cell lysate; Pellet: Final product after washing. The two bar charts show qrt-pcr results measuring fold enrichment of sfrna and grna upon pull-down with MAVS. Shown are representative examples of 3 experiments performed. All data are expressed as mean ± SD. Experiment was analyzed with T-test. n.s: non-significant.

22 Fig. S13. Proposed model depicting how sfrna inhibits RIG-I signaling and downregulates type I interferon response.

23 Table S1 Mass Spectrometry results. List of 1199 RNA binding proteins identified after pull-down with DENV-2 3 UTR (SL-II to SL-IV section) as compared to control RNA. Gene names are indicated. L2, L4 and L6 are normalized readings from three separate experiments. Annotations as depicted: M/L (PR-1 3 UTR divided by control), H/L (PR-2B 3 UTR divided by control). H/M (PR 2B 3 UTR divided by PR-1 3 UTR) Gene names Ratio M/L L2 Ratio M/L L4 Ratio M/L L6 Log (M/L) mean Pvalue (ttest) Ratio H/L L2 Ratio H/L L4 Ratio H/L L6 Log (H/L) mean Pvalue (ttest) Ratio H/M L2 Ratio H/M L4 Ratio H/M L6 Log (H/M) mean Pvalue (ttest) CAPRIN PRKRA SHMT RPS HARS PDLIM RARS AP3S RPL RBMS OTUD6B PRMT RPL RDX GLDC ESYT

24 ABCE EIF3E UFM DDX RPS HDLBP HSPA WARS RPL YTHDF MYO1E RPS4X HNRPDL RPL SNRPA DNM RBX HNRNPU S100P IST HMGB SSBP

25 ADRBK SND VASP NUDT ARSB LRRC PTMS GSDMD MAGEB DNAJB UBL SRSF KARS SERPINH MAP1A YTHDF PRMT FARSA SPTBN GSTK RBMS

26 EIF3K CCT6A TPT HNRNPR; HNRPR HSD17B YTHDF EEF1A1;E EF1A1P A1CF SNX GLT25D EPB41L NHP2L LDHA RPL IGF2BP SYNCRIP LMO AP2S MCM EEF1G EEF1E

27 RPL BRK AP3D STAT ARPC1A PPP1R14 A HDAC BCCIP RPL HIST1H3A PSMA GLUL RPL18A DHX EIF HNRNPM EIF4B MCM EPB41L TMEM189 ;UBE2V2; UBE2V

28 LSM SWAP TUFM PSMB TUBB4B RNASET GATM AKR1C1;A KR1C AHNAK POLR1C MSN NUMB;NU MBL EFTUD SPTAN KIAA PFKM ADAT PROSC EIF2AK EHHADH CORO1C

29 SUPT4H TUBB4A CLIC ASCC NCL NARS RPS15A TRA2B SRGAP1; SRGAP ACACA FOCAD RPS IGF2R TARS TPRKB FERMT RPS EIF RPS27A;U BC;UBB;U BA RPL

30 MAPK ACOX MCM RPL STRAP NXT2;NXT DARS;DK FZp781B MOV COMMD DYNC1LI PPA SNX EIF3I RPL RPS HIST1H1D ;HIST1H1 E;HIST1H 1C RPS PRDX S100A

31 HIST1H4A RPL TARDBP HNRNPA CLINT CORO1B PCBD PDCD GRWD HSPB TRMT TMA AP3B WASF UBE2O CALR FN KPNA TFAM RPS HNRNPK DYNLL

32 DRG CSDE SORD RPL CDC ERI3;PRN PIP SLC9A3R RPL36AL EIF4E SF3B RRBP EIF3A PABPC PAPOLA; PAPOLB MCM RPS CNOT AP3M PFKL PABPC

33 XRCC ATIC DHX COMMD RPS DDX SNX MAVS FAU ITIH SERF HMGB PSMB MAP EEF1D NAP1L LARS MCM HIST2H3A ;H3F3A;HI ST3H3;H3 F3C MSI

34 RPS3A EIF4G EPS8L MYH YBX FKBP CTPS PARG RPL ECH NCAPD DR TUBB TPP FTL RPS FGG TCP EEF1B C20orf RPL23A COMMD

35 XRCC HNRNPH GOLGA MTHFD AIMP RPS COMMD UPF ABHD NAP1L4b; NAP1L EZR GNB2L LAGE ARHGAP DDB PKM SPTBN PPHLN STXBP DHX RPP25L

36 COMMD EEA RPL PAIP PDAP ABI MAPRE STXBP RPS METAP FKBP PSMB ADSL S100A LRRC RPL13A VARS DIP2B HNRNPL CSNK2A SMG TJP

37 SET CUL4A ARRB MRPS VPS ACTN4;A CTN1;AC TN EIF3B AHSA LIN7C;LIN 7A RPS PLD TIMM DDX3X;D DX3Y CNOT RPL CAPN CBS APOB SPR KPNA

38 RPS EDF MATR AP2B POP CCDC RPS PCNA ABCF FAM129B HDGF HNRNPA NMT PRDX HSD17B DHX KIAA RPL7A BIN GCLM PSMA