SUPPLEMENTARY INFORMATION. Divergent TLR7/9 signaling and type I interferon production distinguish
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1 SUPPLEMENTARY INFOATION Divergent TLR7/9 signaling and type I interferon production distinguish pathogenic and non-pathogenic AIDS-virus infections Judith N. Mandl, Ashley P. Barry, Thomas H. Vanderford, Natalia Kozyr, Rahul Chavan, Sara Klucking, Franck J. Barrat, Robert L. Coffman, Silvija I. Staprans, and Mark B. Feinberg
2 Percentage Ki67 + CD8 + T cells Time after infection (d) Percentage Ki67 + CD + T cells Time after infection (d) Supplementary Figure. Muted T cell activation during acute SIVsm infection of s compared to s. Percent proliferating (Ki67 + ) CD8 + T cells (left) and CD + T cells (right) in s (red) and s (blue). Mean ± s.e.m. are shown.
3 a Sooty mangabey Rhesus macaque CD.9.75 CD.7.76 HLA-DR HLA-DR C C R 5 C C R CD CD. b NS NS NS P <. Number pdc per µl 6 5 Percentage pdc of PBMCs Percentage CD + pdc Percentage CCR5 + CD + pdc Supplementary Figure. Phenotype of plasmacytoid dendritic cells in s and s. (a) Percent blood pdcs (gating as in Fig d) and the percent of pdcs expressing CCR5 and CD is shown in two representative animals from both s and s. Numbers on FACS plots indicate percentage of cells in the gates shown. (b) Number of pdcs per µl blood, frequency of pdcs as percentage of peripheral blood mononuclear cells, percent of pdcs expressing CD, and percent of pdcs expressing CCR5 and CD in s (black circles) and s (grey circles). Points represent individual animals, group means are denoted by a line. P values were calculated using a -sample t-test (NS = not significant). All s and s shown are SIV negative.
4 a Microvesicle control Live influenza A/aichi Fold change in IFN-α expression relative to h (log ) - isiv CpG C Time after stimulation (h) b Microvesicle control Live influenza A/aichi Fold change in IFN-β expression relative to h (log ) isiv CpG C Time after stimulation (h) c..5 Microvesicle control..5 Live influenza A/aichi Fold change in IRF-7 expression relative to h (log ) isiv CpG C Time after stimulation (h) Supplementary Figure. Time course of IFNα, IFNβ, and IRF-7 expression in s and s. PBMCs were stimulated from s or s with either microvesicle controls, live influenza A/Aichi, isiv or CpG C 95 and the expression of (a) IFNα, (b) IFNβ, or (c) IRF-7 assessed from the same samples using real-time quantitative RT-PCR calculated relative to a housekeeping gene for each individual animal and shown as log fold change in expression from prior to stimulation ( h). s are depicted by red lines and s by blue lines throughout. Mean ± s.e.m. are shown.
5 a IFN β promoter Human TGTAAATGACATAGGAAAACTGAAAGGGAGAAGTGAAAGTGGGAAATTCCTCTGAATAGAGAGAGGACCATCTCATATAAA..C...C...GG.....C...C...GG... PRD IV PRD III PRD I PRD II IFN α promoter TATA box Human GAGTGCATAAAGAAAGCAAAAAGAGAAGTAGAAAGTAACACAGGGGCATTTGGAAAATGTAAACGAGTATGTTCCCTATTTAA...G...T..A......G...T..A... IFN α promoter TATA box Human GAGTGCATGAAGGAAAGCAAAAACAGAAATGGAAAGTGGCCCAGAAGCATTAAGAAAGTGGAAATCAGTATGTTCCCTATTTAA...-..G G......G.A......G.A......G......G.A......G.A... TATA box b MyD Human MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLE...TEP......TEP Human IRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQ...H......H Human QQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIR Human QLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRVVVVSDDYLQSKECDFQTKFA Human LSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP Death Domain Toll/IL-R (TIR) Domain c Toll-like Receptor 7 (TLR7) Human MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIPGGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKPRSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLGQNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTEL.M..V...S...V...R...S...PR...T....M..V...S...V...R...S...PR...T Human YLYNNMIAKIQEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSNSLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRANLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLFKQFKRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVL...E...T...N...N......E...T...N...N Human EQLHYFRYDKYARSCRFKNKEASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQPLAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQKLMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKNSLSFLPSGVFDGMPPNLKNLSLAKNGLKSF...Y...T...I...R...D......Y...T...I...R...D Human SWKKLQCLKNLETLDLSHNQLTTVPERLSNCSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLLHHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNLILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTKDPAVTEWVLAELVAKLEDPR I.E..RY... I.E..RY...A Human EKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDKYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQAHPYFWQCLKNALATDNHVAYSQVFKETV Toll-like Receptor 9 (TLR9) Human MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNWLFLKSVPHFAAPRGNVTSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFLAVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYYKNPCRQALEVAPGALLGLGNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYN...C...MV..T...A...R...R...S.T...V...D...S..S...E......C...MV..T...A...R...S.T...V...D...S..S...E Human RIVKLAPEDLANLTALRVLDVGGNCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLNASWFRGLGNLRVLDLSENFLYKCITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSFGSLVALKELDMHGIFFRSLDETTLRPLARLPMLQTLRLQMNFINQAQLGIFRAFPGLRYVDLSDNRISGASELTATMGEADGGEKVWLQPGDLAPAPVDT..I...Q...H...H..V...S...Q.VV.....I...Q...H...V...S...Q.V Human PSSEDFRPNCSTLNFTLDLSRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLDLSHNKLDLYHEHSFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQQLCSTSLRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTLRNLPKSLQVLRLRDNYLAFFKWWSLHFLPKLEVLDLAGNQLK...R...S...R...H..DK...H...GN.IH...K......R...S...R...H..NK...GN.IH Human ALTNGSLPAGTRLRRLDVSCNSISFVAPGFFSKAKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQAAVPGLPSRVKCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLCGWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPGKTLF...D...P...I...T...S...QG...R......D...P...I...T...QG...R Human ENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE......C... Leucine-Rich Repeats (LRR) # N- and C-terminal LRR-like domains Undefined Transmembrane domain Toll/IL-R (TIR) Domain Supplementary Figure. The nucleotide sequences of the and IFN β, α and α promoters and the translated amino acid sequences of MyD88, TLR7 and TLR9 aligned with the homologous human sequences. (a) The positive regulatory domains (PRD) I-IV and TATA box are indicated under each alignment. The critical nucleotide sequences in the IFNα promoter recognized by the IRF-7 DNA-binding domain are positive regulatory domains (PRD) I and III for IFNβ, and PRD I and PRD III-like elements (PRD-LE) for the IFNα genes. The -specific polymorphism (an A to G mutation observed in of 6 s sequenced) in a PRD-LE in the IFN α promoter is highlighted in grey. (b) The and MyD88 translated amino acid sequences aligned with the human homolog with the death domain highlighted in blue and the TIR domain in red. Domains were identified using ART ( (c) The and TLR7 and TLR9 translated amino acid sequences aligned with their human homologs. Differences between the and sequences are indicated in either white or grey. The N- and C-terminal LRR-like domains, which cap the ends of the solenoid structure formed by the LRR domains, are highlighted in brown. A portion of TLR7 and 9 in the middle of the extracellular domain that does not correspond to any known structure is indicated in violet. The transmembrance domain is in green, and the TIR signaling domain in red. The LRR domains in the extracellular domain are underlined and numbered. LRRs were defined as in [Bell () Trends in Immunology]. All other domains were identified using ART.
6 Supplementary Table. Summary of Amino acid differences TLR / interferon signalling genes between humans, s and s. Gene Amino acid differences / homogous site Genbank Accession No. vs. Human vs Human vs (; ) Intracellular signalling molecules IKK 5 / 7 5 / 7 8 / 76 EU96; EU97 IRAK / 596 / 6 9 / 596 EU95; EU9 IRAK / 6 / 6 / 6 EU9; EU9 IRF / 9 / 9 / 9 EU9; EU9 IRF / 8 / 8 / EU98; EU99 IRF7 / 5 / 5 8 / 5 EU96; EU97 MyD88 / 96 / 96 / 96 EU95; EU9 TRAF6 / 5 9 / 5 / 5 EU98; EU99 Toll-like receptors TLR / / / 786 EU9; EU9 TLR / 78 8 / 78 / 78 EU9; EU9 TLR / 9 / 9 / 9 EU95; EU9 TLR 9 / 6 69 / 89 / 6 EU97; EU96 TLR5 / / 858 / 858 EU98; EU99 TLR6 / / 796 / 796 EU9; EU9 TLR7 / 9 / 9 / 9 EU9; EU9 TLR8 9 / 9 5 / / 9 EU95; EU9 TLR9 8 / 8 / / EU96; EU97
7 Supplementary Table. CD T cell counts and viral loads of SIV-infected s and s, and HIV-infected humans sampled cross-sectionally for gene expression analyses. Identifier CD T cell count/ml % CD Plasma viral load (Log RNA copies/ml) SU neg SU neg SU neg SU neg SU neg SU neg SU neg SU neg SU neg SU MEAN neg SI SI SI SI SI SI SI MEAN SU 65.. neg SU neg SU neg RU MEAN Neg RI RI RI RI <.9 RI RI RI RI RI MEAN HU neg HU neg HU 56.. neg HU neg HU neg HU MEAN Neg HI HI ND.85 HI HI. 9.. HI HI HI HI HI HI HI HI.6.8 HI MEAN 5.8. SU sooty mangabey uninfected; SI - sooty mangabey infected; RU rhesus macaque uninfected; RI rhesus macaque infected; HU - human uninfected; HI - human infected; ND - not determined; neg. - uninfected.
8 Supplementary Table. Primer and probe sets used for quantitative real-time RT-PCR assays. All IDs refer to assays from Applied Biosystems. See Materials and Methods for details. Gene Name Primer & probe set ID Human, Interferon, alpha (IFNA) Hs5688_s Hs5688_s Interferon, alpha (IFNA) Hs655_s Rh979_s Interferon beta (IFNAB) Hs5_g Rh97_s Myxovirus resistance (MX) Hs87_m Rh88_m Interferon regulatory factor 7 (IRF7) Hs8575_m Rh897_m Chemokine ligand (IP) Hs5_g Rh78858_m Interleukin 6 (IL6) Hs9856_m Rh679_u Interleukin B (ILB) Hs58_m Rh678_m Tumor necrosis factor alpha (TNF-alpha) Hs78_m Rh678_s House-keeping gene: Glucuronidase, beta (GUSB) 6E Rh78876_m
9 SUPPLEMENTARY METHODS Amplification and sequencing of Type I IFN signaling genes: Cellular RNA was extracted using RNEasy RNA Extraction kit (Qiagen) according to the manufacturer s instructions from HIV/SIV negative human,, or PBMCs. Cellular RNA was reverse transcribed with either Powerscript (Clontech) or Superscript II (Invitrogen Corporation) priming with an oligo-dt primer. PCR primers were designed using available primate sequences, or, when no lower primate sequences were available, human sequences. Short (~kb) fragments of each gene were PCR amplified using a : mixture of Takara s LA taq polymerase (Chemicon International) and Stratagene s pfuturbo in the Takara buffers. All PCRs were cycled using a touchdown PCR protocol modified from the protocol recommended by Takara. PCR products were gel purified and TOPOcloned into pcr for sequencing (Invitrogen). All genes were cloned in pieces except for MyD88, TLR7, 8 and 9 which were amplified and sequenced in a single fragment. Multiple colonies from each PCR reaction were sequenced in order to identify potential PCR-induced mutations. Sequencing was performed on automated sequencers at Lark Technologies. Contigs were assembled in Sequencher... The promoter regions and open reading frames of the IFN, IFN, and IFN genes were PCR amplified from genomic DNA extracted from and PBMCs using QIAamp DNA MiniKit (Qiagen). Gene specific PCR primers were designed from human genomic sequences and PCR products were cloned into vectors for sequencing, all as above. The identity of cloned IFN genes was confirmed by clustering on a neighbour-joining phylogenetic tree of an alignment of and IFN genes with the complete set of human IFN loci.
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