Supplemental Material IRF3-dependent and NF- B-independent viral induction of the zinc-finger antiviral protein Nan Wang, Qingming Dong, Jingjing Li, Rohit K. Jangra, Meiyun Fan, Allan R. Brasier, Stanley M. Lemon, Lawrence M. Pfeffer, Kui Li Supplemental Tables Table S1. Genes differentially regulated > 2-fold by transient expression o f IRF3/5D in Hec1B cells Probe set a Signal b Fold change Change c Genbank ID Gene Gene Title 203153_at 3079.4 14.9 I NM_001548 IFIT1/ISG56 interferon-induced protein with tetratricopeptide repeats 1 205483_s_at 1662.9 9.2 I NM_005101 G1P2/ISG15 interferon, alpha-inducible protein, clone IFI-15K 217502_at 2105.4 5.3 I BE888744 IFIT2/ISG54 interferon-induced protein with tetratricopeptide repeats 2 lymphocyte cytosolic protein 2; SH2 domain containing leukocyte protein of 76kDa 205269_at 100.1 3.5 MI AI123251 LCP2/SLP-76 220104_at 475.9 3.5 I NM_020119 ZC3HAV1/ZAP zinc finger CCCH type, antiviral 1 204285_s_at 3414.1 3.5 I AI857639 PMAIP1/Noxa phorbol-12-myristate-13-acetate-induced protein 1 204286_s_at 366.7 3.2 I NM_021127 PMAIP1/Noxa phorbol-12-myristate-13-acetate-induced protein 1 204747_at 1324 3.0 I NM_001549 IFIT3/ISG60 interferon-induced protein with tetratricopeptide repeats 3 202621_at 3172.1 3.0 I NM_001571 IRF3 d interferon regulatory factor 3 210797_s_at 1803.4 2.8 I AF063612 OASL, p30 2'-5'-oligoadenylate synthetase-like, p30 205660_at 1516.2 2.5 I NM_003733 OASL, p59 2'-5'-oligoadenylate synthetase-like, p59 216453_at 412.5 2.3 I AL359578 zzn/a cdna DKFZp547N163, similar to ZNF287 Note: a Probe sets represent those of Affymetrix GeneChip U133A array b Signal represents the average difference (abundance) of cellular mrnas in the IRF3/5D expressing sample c I: increase; MI: marginal increase d The increase of IRF3 mrna abundance reflects signal from transfected IRF3/5D
Table S2. Primers for cloning of hzap and hoasl promoter constructs Oligo name Sequence (5-3 ) hzap -2881 Mluf agtcgacgcgttttagccaatatcctccagc hzap +340 XhoIr cttcactcgagggaaatcggaaatcgaaactt hzap -2486 Mluf cacgacgcgtcctttctctttcactttccctttctt hzap -2469 Mluf cacgacgcgtccctttcttttttcctttcac hzap -2339 Mluf cacgacgcgtcctttctttcctttccctc hzap -2302 Mluf cacgacgcgtcctttctctttcattttc hzap -2268 Mluf cacgacgcgttctctgtcactttccgtttccg hzap -2214 Mluf cacgacgcgtcaaaaggatcttcctctgttg hzap NheI -800f ctgacgctagcgccacagcacccagcccattg hoasl NheI -795f ctgacgctagccagcctgaccaaaatggtgag hoasl XhoI -1r cttcactcgagcccgagagtaccgctgctg Table S3. Primers for mutagenesis of ISREs in hzap and hoasl promoters Oligo name Sequence (5-3 ) hzap disre3-5f CTTCCTTTCTTTCCTTAAGGATCTTCCTCTG hzap disre3-5r CAGAGGAAGATCCTTAAGGAAAGAAAGGAAG hzap disre4-5f CCTTTCCCTTTCTCTTAAGGATCTTCCTCTG hzap disre4-5r CAGAGGAAGATCCTTAAGAGAAAGGGAAAGG hzap disre5f CTTTCTCTGTCACTTAAGGATCTTCCTCTG hzap disre5r CAGAGGAAGATCCTTAAGTGACAGAGAAAG hoasl mirse d f CAAACACTATCCAACTgCAGTTTCCCAACGTC hoasl misre d r GACGTTGGGAAACTGcAGTTGGATAGTGTTTG hoasl misre p f CTAAAAGTTGAGAATCGAtgCTGATGACAGATTGAC hoasl misre p r GTCAATCTGTCATCAGcaTCGATTCTCAACTTTTAG Table S4. Primers for mutagenesis of STAT-IV and -V sites in hzap promoter Oligo name Sequence (5-3 ) ZAPmST4f TCTGGGGCCTGGGGACGCGAcccGGGagtACTCGGTGGCATCTGGGCTCCGCTT ZAPmST4r AAGCGGAGCCCAGATGCCACCGAGTactCCCgggTCGCGTCCCCAGGCCCCAGA ZAPmST5f TCGCTAGGGTCTGCTCTcgagGGcccTGGGTGGGGTGCGCACTCCT ZAPmST5r AGGAGTGCGCACCCCACCCAgggCCctcgAGAGCAGACCCTAGCGA
Table S5. Q-PCR primers used in this study Oligo name Sequence (5-3 ) OAS1-F ACCTAACCCCCAAATCTATGTCAA OAS1-R TGGAGAACTCGCCCTCTTTC IFNβ-F CCAACAAGTGTCTCCTCCAAATT IFNβ-R GTAGGAATCCAAGCAAGTTGTAGCT MxA-F CAACCTGTGCAGCCAGTATGA MxA-R AGCCCGCAGGGAGTCAAT ZAP-F CCCGAGGGAACTGTCGTTTT ZAP-R GATGGCCAGCACCTTTCTGT 28S-F GACCCGCTGAATTTAAGCAT 28S-R GCCTCGATCAGAAGGACTTG Table S6. Primers for ChIP assay of hzap promoter Oligo name Sequence (5-3 ) Product size (bp) ISRE1-2-F1 ttttcccttcccagaactcc 134 ISRE1-2-R1 aggaaaggggaaaaggaagg ISRE3-5-F2 tccttccttttcccctttc 220 ISRE3-5-R2 acagaggaagatccttttgga STAT V-F3 cctcaagttcagggtgagga 161 STAT V-R3 gtcctgagagccctgttgag
Supplemental Figure Legends Fig.S1. Annotated sequence of the human ZAP promoter (-2881-+340). The putative ISRE/IRF-E (red letters) and STAT binding sites (blue letters) and TATA box (brown letters) are underlined. Fig.S2. The ISRE proximal to transcription start is important for activation of human OASL promoter by virus or IFN. (A) Positions (relative to transcription start) and sequences of the two putative ISREs (distal and proximal, underlined) in human OASL promoter. Letters in bold denote core ISRE motifs. (B) Activation of the wild-type (WT) and individual ISRE mutant hoasl promoter and the hoas2 p69 promoter by SeV in Hec1B cells. (C) Activation of the wild-type and individual ISRE mutant hoasl promoter and the hoas2 p69 promoter by SeV (solid bars) or IFN (hatched bars) in Huh7 cells.
Wang et al, Fig.S1-2881 -2533-2301 -1489-1257 -909-329 -97 TTTAGCCAATATCCTCCAGCTTTAGCTCTTTCAAGATCTGCCCCAGCATTCGAGCTGAAGTCATCCACTTCTCTGGTGGCCCCCACCGGTGACTGAGCAAGGCAGTACAAGGGCCT AGCCTTTTCTGTCCAAAGCTGGACTCCTACTCCAGAGCTCCTCATTGGGCTGGCAGAGACTTTGTTAGGTCCGCACAAAAGTCTGATAGGCCCCATGGATCAATCATGCTTTTTCC TTTGACTTTTTCTGGCATTATGCCCCAGAAAAAGTGCAGAGAAAGTTCTGCACTCTTAACTCCATGTCAGGATCTCCTTCCTGGAGAACCCAATCTCTGATGGCACATATTTCAAA ATTTTTCAAAATGTCTCCTTTTCCCTTCCCAGAACTCCTTAAATCACCTTTCTCTTTCACTTTCCCTTTCTTTTTTCCTTTCACTTTCCCTTCTTTTTTCCCCTTTTCCTTTTCCT STAT I ISRE1 ISRE2 TCCTTTTTTTCCTTTTCCTTCCTTTTCCCCTTTCCTTTCCCTTCCTTCCTTTCCTTTCTCTTCCTTTCCTTTCCCTTCCTTTCTTTCCTTTCCCTCCCTTTCCCTTTCCCTTTCCC ISRE3 TTTCTCTTTCATTTTCACTTTCCCTTTCCCTTTCTCTGTCACTTTCCGTTTCCGTTTCCCTTTCCCTTTCCCTTCTCTTCCCTTTCCAAAAGGATCTTCCTCTGTTGCCTTTGCTG ISRE4 ISRE5 GAGTGCAGTGGCACAATTGTAGCTCACTGCAACCTTGAACTCCCGGGCTCAAGAAATCCTCCCGCCGCAGCCTCCCAAGTAGTTGGGACTACAGGCACATACCACTATGTCTGGCT XmaI AATTTTTATTTTTAGTGGAGATGAGGTCTCGCTATGTTGCCCAGGTGGATCTCACAGTATGTTGCCCAGGATGGTCATGATCCTCCCGCCTTGGCCTCCCAAAGTGTTGGGATTAC AGGCACAAGCCACTGCACCTGGCCCTTAAATCATTTTTCATGGCTTCAAATTGGAGGAGATTCTATGTCTGTACTCCCAACCACTGTAGCCAAACTAGACTCTTCAGTCTCTCATC TTCTATATTCCCATTGTGGCATTTACCCATGAATTTGCTTATATTTCCATGCATTTTTTCATGTCTTCGCATACATGCTCCTTGAGTGCACGGACTGTTTTTTAAACTTCTGCATG TCCTTACATTACTAACTAATGTAAATACCACATAGAAGATAGTCAATAAACATCTAGAACTTAGTTGATTTGATAATAAAAGGTCCCTGGGAACCTGTCAGACCACAAATAAGGCC AAGTGGATTAAAAGACAACTATAGAAGCAGCAACTAGAGGGAACAAGATGTCCCTCTTAGACATCCTAAGATGATCCTATGTGGCCCCCATGTTTGGCTGGTTTTCATAACAAAGT ACTCTATTCCTGAAAGAGGGTTTATATACCATGAATGACAAAACTGAGGAGAATTCTCATTAATCATTTTGAAGAATTTCTAAGTTAGCAACCAGATTCTATTATTTTTTAAAAAG STAT II EcoRI ATGTATTTCTTATCTGATCAAATGACCCATCTATTTTATTTGAAGATGTTGGAAAAAAAAAACCTTTTTTCTCTTCAACTTGTGTCCTGTCTTTGGGGGCCTAAAAATTAACTGGC ACTAGACAGATAAATAGGAGAAAAGACAAAGTTTATTTACACATGCATGCAGGAGCTTCCAGTAACAGAAAACTCAAAAGTAAAGGTTTATATACCTCAACTTAACAAAAAGGAAA STAT III GTTTTAGGGCTTCAGTAGGAGAGTTTGGAAGTTTTGATTGGGTTGTTTTGTGTTTTTGAGACAAAGTCTCAGCGCTATCCCCCAGGCTGGAGTGCAGTGGCACAATCCCGGCTCGT TGCAGCCTCCTCCCGTGCTCAAGCAATCCTCCCACCTCAGGCTCCCGAGTAGCTGGGACTGCGGCTGCACACCACCATGCCCGGGTTGTTTTATTATTATTATTATTATTATTATT XmaI ATTATTATTGAAGAGAGCTCACTATGTTGCCCAGGCTAGTCTTGAACTCCTGGGTTCAAGCAATCCTCCCACTGCCTCGGCTTCCTAAAGCGCTGGGATTACGGGCTGAGCCACAG CACCCAGCCCATTGGGTTTTTTGGTGCTAATGGTAATGGGACGGTCTGTCTTCCTGGCAGCTGAATACTCCGAGAGGCTTTTAATGGCACCATGTTTTCGGGAGGTTTTGCTTTAG TCAGATAAGGGAAGTTTCATTAAGATTTCTTTCTACATCTTCTGTAGCTCAATGTTTTCACTTAAAAACAATCTTTATGTCAACTCTGGGGGTCAAAGTACGTCCCCACGGTGGAA AAGTTCTTGTCCCAATCTCTACGGGGAGGAACCGATCTGAAATCTTGGGATATGTCTTAGCATGTCCACCGTGATTGAGGTACAAGCAGTGGTTCAGGGCACCGAGTTAGCGATTC TAGGTAAGCAAGACCATGTCAAGAAGTGGTTCGTCCAAAAGTTCGTGAGAAGGCAGAAACGAGGGAGAGTGGCAATCGTTGTTCTTTAAGAGGGCACGGTGGTGGAGGAGAAACTT GCACATCGGGGCCTGGGGTACCACGATCTGGGGCCTGGGGACGCGATTTGGGGAAACTCGGTGGCATCTGGGCTCCGCTTGATGCCAGTGCGTGTTCGTGCAAGTTACTTCCTCAA STAT IV GTTCAGGGTGAGGATAATAATATCTACCATGTGGAGTTGCTGGGAAGATTCACTACCAACGGAGGCAGAGCGCACGGCTCGCAGGAAGCGCTCGCTAGGGTCTGCTCTTTCCGGGA STAT V ATGGGTGGGGTGCGCACTCCTCAACAGGGCTCTCAGGACTTGCGTCACACGCCTCAGTCGGCTGGCTTATAAAACACCGGCCAGAGCCCCAAGATCGCTTTTAGTTTCTCTTCTTT TATA box Transcription start CTAAAGAAGGCTCGCGGAGCCCGGCTGGAGAACCTCACCCTCGCCGAGCCTAGAACCGAGAGGGGGCCACCCCAGGCGGTCACCAGCAGATTTGCCCGCGCGTTCTCTTTCTTTCC ACCCAGTTGCCCTTGCGGCCGGCTGTAAACCTGCCACTAGGACCCGGTCGGTGAGATCTAGCCTCTTGACCTGAGAGCCGAGAGTGGATCGCTGGGCTGGGCTAACGGCGACGGAG AGCGCGCCCTCGCTGACTCCGGGCGCGCCCAGCAGTAGCACCGCCCGCGCCCGCCCCTGGACACTTGTAAGTTTCGATTTCCGATTTCC +340
Wang et al, Fig.S2 A ISRE d : -335 TATCCAACTTCAGTTTCCCAA -315 misre d : ---------g----------- ISRE p : -291 GTTGAGAATCGAAACTGATGA -271 misre p : ------------tg------- B Relative Luc Activity 70 60 50 40 30 20 10 Hec1B Mock SeV C Relative Luc Activity 8 6 4 2 Huh7 Mock IFN SeV 0 WT misre d hoasl misre p hoas2 p69 0 WT misre d hoasl misre p hoas2 p69