H5N1 Virus Activates Signaling Pathways in Human Endothelial Cells Resulting in a Specific Imbalanced Inflammatory Response

This information is current as Dorothee Viemann, Mirco Schmolke, Aloys Lueken, of September 25, 2021. Yvonne Boergeling, Judith Friesenhagen, Helmut Wittkowski, Stephan Ludwig and Johannes Roth J Immunol 2011; 186:164-173; Prepublished online 24 November 2010;

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Supplementary http://www.jimmunol.org/content/suppl/2010/11/24/jimmunol.090417 Material 0.DC1 http://www.jimmunol.org/ References This article cites 48 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/186/1/164.full#ref-list-1

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H5N1 Virus Activates Signaling Pathways in Human Endothelial Cells Resulting in a Specific Imbalanced Inflammatory Response

Dorothee Viemann,*,†,‡ Mirco Schmolke,x Aloys Lueken,*,‡ Yvonne Boergeling,x Judith Friesenhagen,* Helmut Wittkowski,*,† Stephan Ludwig,‡,x and Johannes Roth*,‡

H5N1 influenza virus infections in humans cause a characteristic systemic inflammatory response syndrome; however, the molec- ular mechanisms are largely unknown. Endothelial cells (ECs) play a pivotal role in hyperdynamic septic diseases. To unravel specific signaling networks activated by H5N1 we used a genome-wide comparative systems biology approach analyzing ex- pression in human ECs infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of

specific signaling pathways revealed that H5N1 induces an exceptionally NF-kB–dependent gene response in human endothelia. Downloaded from Additionally, the IFN-driven antiviral program in ECs is shown to be dependent on IFN regulatory factor 3 but significantly impaired upon H5N1 infection compared with low pathogenic influenza virus. As additional modulators of this H5N1-specific imbalanced gene response pattern, we identified HMGA1 as a novel specifically responsible for the over- whelming proinflammatory but not antiviral response, whereas NFATC4 was found to regulate transcription of specifically H5N1- induced . We describe for the first time, to our knowledge, defined signaling patterns specifically activated by H5N1, which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of an overwhelming proinflammatory and http://www.jimmunol.org/ impaired antiviral gene program. The Journal of Immunology, 2011, 186: 164–173.

nfluenza A viruses still pose a major threat due to their cell (EC) tropism (9–13). Recently, a-2,3-linked sialic acid re- pandemic potential. There is a huge natural virus reservoir in ceptors preferentially bound by avian influenza virus were shown to I birds, which provides a pool of viral genes that contribute to be present on human ECs, suggesting a crucial involvement of ECs the generation of novel pandemic virus strains. The highly path- in the characteristics of systemic H5N1 diseases (14). Isolated ogenic avian H5N1 virus is the first example of an avian virus re- suppression or knockdown of the response did not pro- ported to have infected and killed several hundreds of humans (1). tect against lethal outcomes (13, 15, 16). In human bronchial epi- by guest on September 25, 2021 Human-adapted influenza A viruses cause primarily infections of thelial cells, high pathogenic H5N1 variants replicate stronger than the upper respiratory tract with limited virus replication. Highly low pathogenic variants or the human H3N2 virus but elicit a much pathogenic avian influenza viruses (HPAIVs) rather cause systemic weaker and delayed IFN response (17). The overall pattern of infections with hemorrhagic sepsis in poultry. In humans, clinical proinflammatory and antiviral signaling pathways activated by manifestations of H5N1 infections are a systemic inflammatory H5N1 in specific tissues might be the clue to the pathogenicity of response syndrome (SIRS) leading to multiorgan failure (2–6). HPAIVs. There are two characteristics of H5N1 infections: an unusual The cardinal problem of treating H5N1 infections with neur- strong cytokine response (7, 8) and a neuro- as well as endothelial aminidase inhibitors is that it has to be initiated very early, oth- erwise virus replication and dissemination has already progressed too far (6). Evaluating the role of endothelial response programs x *Institute of Immunology, ‡Interdisciplinary Center of Clinical Research, and Institute with respect to proinflammatory and antiviral strategies and de- of Molecular Virology, University of Muenster; and †Department of Pediatrics, Uni- versity Hospital of Muenster, Muenster, Germany lineating the responsible signaling cascades will allow the de- velopment of novel therapeutic strategies. Received for publication December 28, 2009. Accepted for publication October 29, 2010. To approach to these objectives, we performed comparative This work was supported by the Bundesministerium fu¨r Bildung, Wissenschaft, For- global studies of ECs infected with HPAIV of the schung und Technologie Zoonosis Network FluResearchNet (Grant 01KI07130). H7N7 and H5N1 subtype and a low pathogenic human influenza D.V. and J.R. were supported by grants from the Interdisciplinary Center of Clinical virus of the H1N1 subtype to identify H5N1 specific gene response Research University of Muenster. subpatterns. In line with recent findings of our group, we confirm Address correspondence and reprint requests to Dr. Dorothee Viemann, Institute of k Immunology, University of Muenster, Roentgenstr. 21, D-48149 Muenster, Germany. a remarkable NF- B dependency of the H5N1-induced gene ex- E-mail address: [email protected] pression program (18), which we demonstrate to be much weaker The online version of this article contains supplemental material. or not relevant for infections with the other influenza strains. Abbreviations used in this paper: BS, binding site; EC, endothelial cell; FC, fold Concurrently, infections with H5N1 were characterized by a strik- change; FPV, A/FPV/Bratislava/79 (H7N7) fowl plague virus; HPAIV, highly patho- ing attenuation of the IFN-b–dependent antiviral response. Fur- genic avian influenza virus; IKK2kd, kinase-dead IkB kinase 2; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; MDCK, Madin-Darby canine kidney; thermore, we reveal HMGA1 as a novel transcription factor to be MOI, multiplicity of infection; PCA, principal component analysis; p.i., postinfection; specifically responsible for the overwhelming proinflammatory PR8, A/PR8/34 (H1N1); qRT-PCR, quantitative real-time RT-PCR; siRNA, short in- but not antiviral response in H5N1 infections, whereas NFATC4 terfering RNA; SIRS, systemic inflammatory response syndrome; wt, wild-type. was found to be a transcriptional regulator of specifically H5N1- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 induced genes. We describe for the first time, to our knowledge, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904170 The Journal of Immunology 165

MDCKII were cultured in MEM (PAA Laboratories, Pasching, Austria) containing 10% v/v FCS and 100 U/ml penicillin/0.1 mg/ml streptomycin (13 penicillin/streptomycin) (Life Technologies, Carlsbad, CA). A/PR8/ 34 (H1N1) (PR8) was cultivated in 11-d-old embryonic chicken as de- scribed previuosly (19). Allantoic liquid containing viral particles was harvested 50 h postinfection (p.i.). HUVECs were obtained from Cambrex (Charles City, IA) and cultured as described elsewhere (20, 21). HUVECs between passages 5 and 7 were infected with a multiplicity of infection (MOI) of 5 of each viral strain. For the plaque assay, MDCK cells grown 100% confluent in six-well dishes were washed with PBS and infected with serial dilutions of culture FIGURE 1. Infectibility of HUVECs with different strains of influenza supernatants in PBS containing 0.2% bovine serum albumin, 1 mM MgCl2, viruses. Lysates of uninfected HUVECs (ctrl) and PR8-, FPV-, and H5N1- 0.9 mM CaCl2, 100 U/ml penicillin, and 0.1 mg/ml streptomycin for 30 infected HUVECs were immunoblotted 3, 5, and 8 h p.i. for viral min at 37˚C. The inoculum was aspirated, and cells were incubated with PB1 (86 kDa), M1 (27 kDa), and NS1 (26 kDa). Immunostaining of ERK1/ 2 ml MEM medium containing 0.2% BSA and antibiotics supplemented with 0.6% agar (Oxoid, Cambridge, U.K.), 0.3% DEAE-dextran (GE 2 served as loading control. Healthcare Bio-Sciences AB, Uppsala, Sweden), and 1.5% NaHCO3 at 37˚C, 5% CO2 for 2 to 3 d. Virus plaques were visualized by staining with defined signaling patterns specifically activated by H5N1, which, neutral-red or Coomassie blue (0.1% Coomassie Brilliant blue G-250 in 40% methanol and 10% acetic acid). The FNX amphotropic retrovirus in contrast to low pathogenic influenza viruses, are responsible for producer cells were kindly provided by G. Nolan, Stanford, CA, and an imbalance of an overwhelming proinflammatory and impaired cultured as described elsewhere (21). Retroviral infection of HUVECs with antiviral gene program. the pCFG5-IEGZ vector containing an insert of kinase-dead IkB kinase 2 Downloaded from (IKK2kd) was performed as described (18, 21). Materials and Methods Immunofluorescence microscopy Viruses and cells To determine the infectious doses resulting in an 80% infection rate, The human H5N1 influenza A virus isolate A/Thailand/KAN-1/2004 was HUVECs were infected with different MOIs of each virus strain and fixed used with kind permission of Pilaipan Puthavathana, Bangkok, Thailand. with 3.7% formaldehyde.

The avian influenza virus A/FPV/79/Bratislava (H7N7, fowl plague virus They were stained against an influenza A group-specific nucleoprotein http://www.jimmunol.org/ [FPV]) and the human influenza virus strain A/Puerto-Rico/8/34 (Giessen (mouse mAB OBT0053, clone 1A52.9; Serotec, Dusseldorf, Germany) and variant) were obtained from the Institute of Virology in Giessen, Germany. counterstained with DAPI to determine the proportion of nucleoprotein- Viruses were propagated on Madin-Darby canine kidney (MDCK) II cells. positive HUVECs. Goat anti-mouse Cy5-labeled secondary Ab was pur- by guest on September 25, 2021

FIGURE 2. Differential gene profiles induced in HUVECs p.i. with different influenza strains. A, PCA comparing gene profiles of uninfected HUVECs (ctrl; black vector cloud) and PR8- (light gray), FPV- (medium gray), and H5N1-infected HUVECs (dark gray) 5 h p.i. Vector clouds representing a gene profile of one experiment are positioned in a three-dimensional vector space according to variance to each other. B, Venn diagram indicates the number and overlap of genes induced 5 h p.i. by H5N1, FPV, and PR8. Data are based on microarray analyses of three independent experiments. Plotted are functional gene groups according to annotations overrepresented in C, the intersection group of genes induced by PR8, FPV, and H5N1, or in D, the group of genes only upregulated by H5N1. Statistical significance (y-axis) was determined by applying the Fisher’s exact test. 166 H5N1 TRANSFORMS IMMUNE RESPONSES IN ENDOTHELIA chased from Dianova (Hamburg, Germany).The fluorescence was detected ERK2 (rabbit IgG, C-14; Santa Cruz Biotechnology, Heidelberg, Ger- using the Axioskop microscope from Zeiss (Go¨ttingen, Germany). many), anti-M1 (GA2B; AbD Serotec, Oxford, U.K.), anti-IFN regulatory factor (IRF) 3 (rabbit p-Ab; Zymed, San Francisco, CA), anti-IRF7 (F-1), Western blot anti-HMGA1 (FL-95), anti-NFATC4 (H-74), and anti-PB1 Ab (goat pAB, vK20) (all from Santa Cruz Biotechnology). A murine mAb against in- For immunoblotting, cells were lysed in RIPA buffer and separated by SDS- fluenza A virus NS1 was generated by V. Wixler (Institute of Molecular PAGE as described (22). Western blot staining was performed with anti- Virology, Muenster, Germany). Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Kinetic course of gene expression in HUVEC after HPAIV infection. A–H, HUVEC monolayers were infected with PR8 (rhombus), FPV (squares), and H5N1 (triangles) viruses. Gene expression changes of antiviral genes (A, IRF7; B, IFNB1; C, CXCL9; and D, IFIH1) and inflammatory genes (E, IL8; F, CCL2; G, CXCL2; and H, VCAM) were determined 5, 8, 10, and 16 h p.i. by qRT-PCR and plotted as mean of FCs compared with uninfected control HUVECs (n = 3). The Journal of Immunology 167

DNA microarray hybridization and statistical data analyses As an additional bioinformatics tool, we applied principal component analysis (PCA), which is a mathematical procedure that reduces the enor- Total cellular RNA was isolated from three independent experiments with mous number of possibly correlated gene expression values to a smaller wild-type (wt) HUVECs and three independent experiments with HUVECs number of uncorrelated variables called principal components (23). This that had been infected with empty retroviral expression vector or a vector method results in location of individual microarray data sets in a virtual containing the dominant-negative mutant of IKK2 (IKK2kd). Cells were three-dimensional vector space allowing comparisons of individual virus- infected for 5 h with the three influenza strains PR8, FPV, and H5N1 induced expression patterns regarding variations (different location) and (RNeasy , Qiagen, Hilden, Germany). Samples were processed for similarities (close location) in gene expression in an observer-independent microarray hybridization using Affymetrix U133 Plus 2.0 manner. For three-dimensional performance, we chose data transformation Gene Arrays according to the manufacturer’s instructions (Affymetrix, into three principal components. Santa Clara, CA). Fluorescent signals were detected by the GeneChip To identify overrepresented functional categories of genes, we compared Scanner 3000 and recorded and computed by GeneChip Operating Soft- the distribution of gene ontology annotations on the Affymetrix U133 Plus ware version 1.4 (Affymetrix). For a more sophisticated data analysis, we 2.0 Array (Affymetrix) with the gene group of interest applying Fisher’s used the Expressionist Suite software from GeneData (Basel, Switzerland) exact test. In case of genes that are represented by two or more probe sets, as described (20). Genes with a fold change (FC) of .2.0 and a p value # only one transcript was taken into account to avoid potential bias. 0.05 (paired t test) were considered. On/off-regulated genes were evaluated For the promoter analyses, we took advantage of a computational method as described (20) considering genes with on/off ratios of 0:3, 0:2, 1:3, 3:0, for transcriptional regulatory network inference, Computational Ascer- 2:0, and 3:1, respectively. From this group of on/off-regulated genes, tainment of Regulatory Relationships Inferred from Expression, described we only included regulations with a high fold-change of $5 and a p value by Haverty et al. (24). Briefly, microarray data and promoter sequence data of , 0.05 to exclude on/off phenomenons occurring around the background derived from TRANSFAC database 7.0 are merged and checked against threshold. Microarray data are Minimum Information About a Microarray each other. Thereby, promoter regions of a group of unregulated genes are Experiment compliant and deposited in Gene Expression Omnibus database compared with the promoters of a group of regulated genes regarding tran-

(http://www.ncbi.nlm.nih.gov/geo/; accession number GSE13637). Parts of scription factor binding sites (BSs) to compute the relative overabundance Downloaded from the data set concerning H5N1-infected HUVECs have also been used in of cis-elements in the group of regulated genes. So, overrepresented tran- a previous study of our group (18). scription factor BSs are detected and defined as potential regulators of http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Role of NF-kB for the gene expression profiles induced in HUVECs by different influenza strains. A, Lysates of wt HUVECs, empty vector- infected HUVECs, and HUVECs expressing IKK2kd mutant were immunoblotted against viral M1 protein (27 kDa) 5 h p.i. with PR8, FPV, and H5N1. Blot shows comparable expression of M1 in wt and empty vector-infected HUVECs. Blocking NF-kB did not significantly change M1 expression. PCA comparing gene profiles induced by PR8 (B), FPV (C), or H5N1 (D) virus in empty vector-transfected HUVECs (black clouds), in empty vector-transfected and virus-infected HUVECs (dark gray clouds), and in virus-infected HUVECs transfected with IKK2kd (light gray clouds). Data illustrate the strong NF- kB dependence of gene expression changes induced by H5N1. FCs of gene expression (y-axis) in monolayer of empty vector- (black bars) or IKK2kd- transfected (gray bars) HUVECs determined by qRT-PCR 5 h p.i. with PR8 (E, F), FPV (G, H), and H5N1 (I, J). Bars represent means of FCs 6 SD compared with uninfected control HUVECs (n = 5). Asterisks mark significant inhibition of gene inducibility by H5N1 resulting from blocking NF-kB. *p , 0.05; **p , 0.005; ***p , 0.001. 168 H5N1 TRANSFORMS IMMUNE RESPONSES IN ENDOTHELIA a gene group of interest according to the statistical significance of over- the highest number of genes (418 genes) due to a high proportion of representation. predominantly unspecific gene regulations with low FCs between Quantitative real-time RT-PCR 2.0 and 4.0 followed by the H5N1 strain (149 genes) and the FPV virus (80 genes) (Fig. 2B, Supplemental Tables I–III). Forty-seven m cDNA was synthesized from 4 g total RNA using RevertAid H Minus genes were induced by all influenza viruses (Supplemental Table M-MuLV Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany). Spe- cific primers for each gene (sequences shown in Supplemental Table XI) IV), whereas 33 genes were specifically induced by H5N1, 22 were designed using Primer Express software (Applied Biosystems, Foster genes specifically by FPV, and 291 genes specifically by PR8. City, CA) and obtained from MWG Biotech (Ebersberg, Germany). Functional clustering according to gene ontology annotations Quantitative real-time RT-PCR (qRT-PCR) was performed using the revealed that genes induced by all viruses primarily belonged to QuantiTect SYBR Green PCR kit (Qiagen) as described (20) and data acquired with the ABI PRISM 7900 (Applied Biosystems). Gene ex- inflammatory viral response genes and cell–cell signaling genes pression was normalized to the endogenous housekeeping control gene (Fig. 2C). A similar functional pattern was computed for PR8- RPL9, and relative expression of respective genes was calculated using specific genes, whereas FPV-specific genes had functions assigned the comparative threshold cycle method as described (25). to , cell proliferation, and cell cycle control (Supple- Short interfering RNA experiments mental Fig. 1). In contrast, in the group of H5N1-specific genes, an overwhelming overrepresentation of chemokines and chemotactic 3 5 A total of 1.2 10 cells were seeded on six-well plates. Twenty-four genes was observed (Fig. 2D). Chemokines specifically induced by hours later, each well was transfected with 200 pmol short interfering RNA (siRNA; Qiagen) and 6 ml Oligofectamine (Invitrogen) in 200 ml Opti- H5N1 included CCL7, CXCL1, CXCL5, CXCL6, and IL8 (Supple- MEM (Life Technologies). The siRNAs used in this study consisted of 21- mental Table V). nucleotide dsRNAs, each strand of which contained a 19-nucleotide target Downloaded from sequence and a two-uracil overhang at the 39 end and were predesigned for IRF3, IRF7, GATA6, and NFATC4 and validated for HMGA1 and negative Table I. Transcription factor profiles of H5N1-induced gene programs control siRNA (Supplemental Table XII). Efficiency of mRNA or protein in HUVECs knockdown was measured by qRT-PCR or Western blot, respectively. Forty-eight hours after siRNA transfection, HUVECs were infected with 5 Transcription Factor Binding Site p Valuea MOI of H5N1. In case of additional IFN-b pretreatment, HUVECs were incubated with 100 U/ml IFN-b1a (ImmunoTools, Friesoythe, Germany). Universe of H5N1-induced genes Then, 5 h p.i., RNA was collected and processed for qRT-PCR. Myogenic MADS factor MEF-2 1.52E-22 http://www.jimmunol.org/ Fetal Alz-50 clone 1 1.21E-21 Statistical analyses IRF8 9.25E-21 The results of all qRT-PCRs were assessed by Student t test and are ex- IRF1 9.25E-21 pressed as means 6 SD. IRF 3.34E-20 Retroviral TATA box 6.48E-17 ISRE 1.07E-15 IRF7 5.47E-14 Results E4BP4 3.75E-12 Infection of primary ECs with HPAIV GATA6 1.19E-10 We used three influenza virus isolates to infect monolayers of Hepatic factor 2.29E-10 by guest on September 25, 2021 Related to , C4 3.42E-10 HUVECs and analyzed the effects on gene expression changes: A/ TATA binding protein 1.31E-09 PR8/34 (H1N1) (PR8) (a low pathogenic human reference in- C/EBPd 1.74E-09 fluenza strain), FPV (highly pathogenic for birds) (26, 27), and an HMGA1 2.07E-09 H5N1 isolate A/Thailand/KAN-1/2004 (an HPAIV isolated from Meis-1b/HOXA9 heterodimeric binding 2.52E-09 Serum response factor 4.95E-09 a fatal human case) (28, 29). Infectious doses of all virus strains Forkhead box D3 1.02E-07 were adjusted to result in 80% of infected ECs as determined Meis-1a/HOXA9 heterodimeric binding 1.15E-07 by immunofluorescence staining of an influenza A group-specific Pax-6 2.34E-07 nucleoprotein (data not shown). Efficient infection was confirmed Cell division control protein 5 3.79E-07 k by immunoblotting of the viral proteins PB1, M1, and NS1 3, 5, NF- B 3.92E-07 Homeo domain factor Pbx-1 5.67E-07 and 8 h p.i., respectively (Fig. 1). Although at 5 h p.i. there were HNF-3 8.10E-07 slight differences in viral protein expression suggesting an NFATC4 8.59E-07 advantaged H5N1 propagation, the accumulation of viral proteins STAT5B 1.29E-06 at 8 h p.i did not greatly differ between the different strains, in- 58 KDA repressor protein 4.41E-06 Pit-1 4.41E-06 dicating that the replication efficiency of all three viruses was at Sex-determining region Y gene product 7.41E-06 least similar within the first replication cycle. To map primary Specifically H5N1-induced genes effects on gene expression, we therefore chose the time point of GATA6 9.39E-16 5 h p.i., well within the first replication cycle of all three viruses. STAT5A 1.87E-15 For 24 h p.i., we found a significant trend of 1 log level higher TTF-1, TITF1 3.67E-13 STAT5B 1.56E-12 viral titers in supernatants of H5N1-infected HUVECs compared Androgen 6.70E-11 with PR8 and FPV infections (data not shown). POU3F2 6.83E-08 TGIF 6.88E-08 Gene profiling of HPAIV-infected HUVECs Upstream stimulating factor 3.83E-07 After 5 h p.i., we processed total RNA of infected and uninfected NFATC4 1.14E-05 Serum response factor 2.16E-04 control HUVECs for microarray hybridization. We exclusively Transcriptional repressor CDP 4.48E-04 focused on upregulated genes because unspecific cap-snatching HMGA1 1.98E-03 mechanisms significantly contribute to the process of gene down- Boldface text indicates BSs that were not computed for any PR8- or FPV-related regulation by influenza virus (30, 31). PCA displaying all influenza- gene profile and additionally identified in the analysis of the entire H5N1-induced inducible genes on the particular gene profiles showed consistence gene profile as well as in the analysis of specifically H5N1-induced genes. ap values indicate the significance of overrepresentation of binding sites in pro- of the profiles within the same experimental group and clear sep- moters of H5N1-regulated genes compared with unregulated genes. aration of the different experimental groups (Fig. 2A). PR8 induced ISRE, IFN-stimulated response element. The Journal of Immunology 169

To screen whether the overrepresentation of inflammatory genes proinflammatory response in ECs (20), we blocked the NF-kB after H5N1 infections is temporal or sustained compared with PR8 signaling pathway by introducing retrovirally an IKK2kd mutant and FPV infections, we performed qRT-PCRs of representative into HUVECs (20, 21, 32). NF-kB blockade did not significantly genes of the antiviral and inflammatory gene program 5, 8, 10, and attenuate viral M1 expression of each viral strain, indicating ap- 16 h after HPAIV infection (Fig. 3). Expression differences propriate replication of all viruses in this EC system (Fig. 4A). detected 5 h p.i. also persisted 16 h p.i. Differences in genes PCAofmicroarraydataraisedintheseHUVECs5hp.i.dem- contributing to the antiviral response like IRF7, IFNB1, CXCL9, onstrated that blocking of NF-kB did not significantly alter the and IFIH1 were similar 5 h as well as 16 h p.i. They were strongest global gene profiles induced by PR8 (Fig. 4B). For FPV, we found induced by PR8 virus and significantly lower by FPV and H5N1 a slight shift toward the control profiles, suggesting a minor de- virus (Fig. 3A–D). We also confirmed microarray data to that ef- pendence on NF-kB (Fig. 4C) whereas H5N1-induced endothelial fect that H5N1 infections compared with PR8 and FPV viruses gene profiles significantly shifted back toward controls if NF-kB caused significantly stronger or even specific inductions of inflam- was blocked (Fig. 4D), confirming recent findings of our group in matory genes like IL8, CCL2, CXCL2, and VCAM in HUVECs terms of an essential impact of NF-kBsignalinginH5N1infec- beginning 5 h p.i. (first replication cycle) and becoming clearer 16 h tions (18). However, the NF-kB dependence of the H5N1 virus did p.i. So, the gene profiles identified within the first replication cycle not reach the same level that we observed in a previous study for of all three viruses represent reliable data of primary HPAIVeffects TNF-induced gene expression changes in human ECs, which are on endothelial gene expression. virtually to 100% dependent on NF-kB(20). To confirm microarray data concerning NF-kB dependence and

virus specificity, we performed qRT-PCRs (Supplemental Table Downloaded from k Role of NF- B for gene expression changes in influenza VI). Genes could be classified into three different groups: first, virus-infected HUVECs H5N1-strain specific genes; second, influenza virus inducible genes With chemokines being exceedingly overrepresented among with NF-kB dependence predominantly in the H5N1 strain; and H5N1-specific genes and knowing that NF-kBiscrucialfora third, genes induced in an NF-kB–dependent mode by all influenza http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Role of IRF3 and IRF7 in H5N1-mediated gene expression changes in HUVECs. A, Western blot shows the detection of IRF3 (50 kDa) in HUVECs transfected with IRF3 siRNA (24 h, 48 h, 72 h) and nontargeting control siRNA. Transfection of HUVECs with IRF3 siRNA knocks down IRF3 protein expression for 48 h, whereas after 72 h, IRF3 protein expression reappears. Detection of b-actin serves as protein loading control. B, Demonstration of lasting successful knockdown of IRF3 protein expression in HUVEC by siRNA pre- and p.i. with H5N1 virus. Detection of b-actin serves as protein loading control. C, Western blot shows induction of IRF7 (63 kDa) protein expression and knockdown by siRNA in uninfected and H5N1-infected HUVECs. D, Western blot shows the detection of IRF7 in control siRNA-transfected HUVECs, overexpression of IRF7 in IRF3 siRNA-transfected HUVECs, and successful knockdown of IRF7 in IRF7 siRNA-transfected HUVECs. Detection of b-actin serves as protein loading control. FCs of mRNA expression (y-axis) in H5N1-infected compared with uninfected HUVECs transfected with untargeted control siRNA (black bars), IRF3-directed siRNA (E, gray bars), and IRF7-directed siRNA (F, gray bars) determined by qRT-PCR. IRF7* in F shows the induction of IRF7 in HUVECs pretreated with IFN-b before siRNA transfections and H5N1 infection. Bars represent means 6 SD (n = 5). *p , 0.005; **p , 0.001. 170 H5N1 TRANSFORMS IMMUNE RESPONSES IN ENDOTHELIA virus. Primarily proinflammatory genes such as CCL5, CXCL10, IRF3 is essential for the initiation of an IFN response to H5N1 CXCL11, TNFSF10, CXCL2, TLR3, and VCAM belonged to the virus infections in HUVECs k third group of generally virus-inducible NF- B–dependent genes To identify additional transcriptional regulators crucial for H5N1- (Supplemental Table VI). IL8 and CCL2 proved true as H5N1- mediated gene expression, we performed promoter analyses (Table k specific NF- B–dependent genes 5 h p.i. (Fig. 4E,4G,4I). From I). This bioinformatic approach revealed that in ECs, next to NF- the second group of generally influenza-inducible genes with su- kB also transcription factor BSs of IRFs, namely IRF consensus k perior NF- B dependence in the H5N1 profile, we selected five BSs, IRF8, IRF1, IFN-stimulated response element, and IRF7, are genes for qRT-PCR (SELE, IFNB1, CXCL9, IFIH1, and IRF7). We significantly overrepresented in promoters of H5N1-induced genes k verified that the induction of none of these genes was NF- B de- (Table I) as well as in the group of PR8- and FPV-induced genes pendent in the case of PR8 infections (Fig. 4F). FPV only induced (Supplemental Tables VII, IX). In other cell systems than human SELE and IRF7 in an NF-kB–dependent manner (Fig. 4H). In ECs, IRF3 is important for the induction of the IFN–STAT1/2 contrast, virtually all of these genes were NF-kB dependently in- pathway, whereas IRF7 rather overtakes the tasks of IRF3 in the duced by H5N1 (Fig. 4J). Interestingly, in H5N1-infected ECs also, second stage (33, 34). To examine the relevance of IRF3 and IRF7 genes that launch an antiviral response, including IFNB1, CXCL9 in ECs, we knocked them down by targeted siRNA (Fig. 5A–D; and IFIH1, were NF-kB dependently induced, whereas this was not mean of 70% inhibition of IRF3 mRNA expression and 85% in- the case or tendentiously even contrary in PR8 or FPV infections. hibition of IRF7 mRNA expression according to qRT-PCR). IRF3 Besides the NF-kB dependence, it was very striking that the IFNB1 expression remained readily knocked down for 48 h after siRNA response induced by the H5N1 strain in ECs was significantly transfection (Fig. 5A) and also after H5N1 infection (Fig. 5B). weaker compared with PR8 and FPV (Fig. 4F,4H,4J). Because IRF7 is only hardly expressed in resting HUVECs, we Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. HMGA1 and NFATC4 regulate H5N1-mediated gene expression changes in HUVECs. A, Western blot shows knockdown of HMGA1 (18 kDa) protein expression by siRNA compared with control siRNA-transfected HUVECs pre- and p.i. with H5N1 virus. Detection of b-actin serves as protein loading control. B, FC of mRNA of NF-kB dependently induced genes in H5N1-infected compared with uninfected HUVEC transfected with control siRNA (black bars) or HMGA1-directed siRNA (gray bars) determined by qRT-PCR (n = 10). C, Western blot shows knockdown of NFATC4 (140 kDa) protein expression by siRNA in HUVEC pre- and p.i. with H5N1 virus. Detection of b-actin serves as protein loading control. D, FC of mRNA of NF-kB dependently induced genes in H5N1-infected compared with uninfected HUVECs transfected with control siRNA (black bars) or NFATC4-directed siRNA (gray bars) determined by qRT-PCR (n = 10). FC of mRNA of NF-kB independently induced genes in H5N1-infected compared with uninfected HUVECs transfected with control siRNA (black bars), HMGA1-directed siRNA (E, gray bars), or NFATC4-directed siRNA (F, gray bars) determined by qRT-PCR (n = 6). B and D–F, Bars represent means 6 SEM. *p , 0.05; **p , 0.005. The Journal of Immunology 171 tested its inducibility in uninfected HUVECs by IFN-b and veri- the H5N1-induced proinflammatory as well as antiviral gene pro- fied successful and lasting knockdown of IRF7 after H5N1 in- file independent of NF-kB. fection (Fig. 5C). Interestingly, IRF3 knockdown resulted in a The interaction of IRF3, NF- kB, HMGA1, and NFATC4 in considerable IRF7 overexpression (Fig. 5D). qRT-PCRs in H5N1- H5N1-infected HUVECs results in an imbalanced inflammatory infected control and knockdown cells confirmed that IRF3 is es- response with an overwhelming activation of common and specific sential for the induction of IFNB1 and CXCL9 in ECs by H5N1 proinflammatory gene programs and an impaired antiviral gene virus (Fig. 5E). Surprisingly, some proinflammatory genes, such as program compared with low pathogenic influenza strains (Fig. 7). CXCL11, VCAM, and SELE were strongly inducible in HUVECs with blocked IRF3. Knockdown of IRF7 in HUVECs neither Discussion changed the level of IRF3 expression nor did it significantly in- Diseases caused by HPAIVs are characterized by systemic dis- fluence H5N1-mediated gene expression changes 5 h p.i. (Fig. semination leading to an SIRS (2, 4, 6–8, 10). Given that ECs 5F). IRF7 mRNA itself showed no relevant changes after IRF7 significantly contribute to SIRS and considering the EC tropism of knockdown, maybe due to the very low basal expression level. We HPAIV, we examined the network of signaling pathways and therefore enhanced IRF7 expression by IFN-b treatment before transcriptional regulators specifically activated upon H5N1 virus IRF7 knockdown and H5N1 infection and found a clear inhibitory infection of HUVECs. For this purpose, we used a comparative effect on IRF7 mRNA induction by H5N1 virus (Fig. 5F, IRF7*). approach with three influenza strains of different pathogenicity for birds and humans. PR8 is a low pathogenic human influenza strain, HMGA1 and NFATC4 represent novel transcriptional FPV is a H7 virus highly pathogenic for birds, whereas H5N1

regulators in the response of H5N1 virus-infected HUVECs Downloaded from viruses are known to infect and kill humans. Next, we performed a promoter analysis of genes exclusively in- Within the first viral replication cycle, propagation efficiency of duced upon H5N1 virus infection (Table I) in ECs. The same dual all influenza virus strains was comparable. Each viral strain induced analysis was performed for PR8- and FPV-induced genes (Sup- a distinct characteristic gene profile. Functional clustering revealed plemental Tables VII–X). So, we established virtually virus strain- that genes induced by all strains were inflammatory viral response specific endothelial transcription factor profiles. We filtered out genes and cell–cell signaling genes. The functional pattern of PR8- those specifically H5N1-relevant transcription factors that are also specific genes was very similar to that of the intersection of gen- http://www.jimmunol.org/ overrepresented in the promoter regions of the entire H5N1- erally influenza-inducible genes. Among FPV-specific genes, no induced gene profile but not relevant for any PR8- or FPV-related relevant highly overrepresented gene groups were observed. On gene profile. In that way, three candidates of H5N1-specifc tran- the contrary, in the set of H5N1 specifically induced genes proin- scription factors were identified (gray shaded in Table I): GATA6, flammatory were enormously overrepresented, confirm- HMGA1, and NFATC4. ing the manifold described cytokine storm (7, 8). Our data suggest Using an siRNA approach, we could not confirm any regulatory that ECs may be a major source of cytokines in the pathogenesis of role of GATA6 for H5N1-induced genes (data not shown). H5N1-induced SIRS. In contrast, transfection of HMGA1-directed siRNA in HUVECs Mircroarray data from PR8-, FPV- and H5N1-infected HUVECs (Fig. 6A, mean of 80% inhibition of HMGA1 mRNA expression) for which NF-kB pathway had been blocked illustrated that NF- by guest on September 25, 2021 led to a pattern of inhibited or decoupled gene transcription upon kB is strictly required for the H5N1-induced gene response but H5N1 virus infection that differed significantly from the effects of less important for the FPV-induced profile and of minor relevance NF-kBorIRF3 blocking on gene transcription (Figs. 4I,4J,5E, for PR8-induced genes. Thereby, we confirm the pivotal role of 6C). The induction of several proinflammatory genes as SELE, NF-kB signaling for the endothelial response to H5N1 infections CXCL11, TNFSF10A, and CXCL2 was dependent on HMGA1 (Fig. worked out in a recent study of our group (18) and demonstrate 6B). Except for SELE, these genes belonged to the group of gen- additionally the specificity of NF-kB dependence for the induction erally NF-kB–dependent and virus-inducible genes not dependent of several inflammatory (IL8, CCL2, and SELE) and important on IRF3 (Fig. 5E, Supplemental Table VI). In contrast, genes such antiviral genes (IFNB1, CXCL9, IFIH1,andIRF7)inH5N1 as IL8, IFIH1,orIRF7 that were H5N1 specifically dependent on NF-kB (Fig. 4I,4J) and not IRF3 dependent (Fig. 5E) were not affected by HMGA1 knockdown (Fig. 6B). Interestingly, the in- duction of IFNB1 and CXCL9, both strictly IRF3 and H5N1 spe- cifically NF-kB dependent (Fig. 4C,4J), significantly increased upon knockdown of HMGA1 (Fig. 6B). The impact of NFATC4 knockdown in HUVECs (Fig. 6C, mean of 50% inhibition of NFATC4 mRNA expression) was differential compared with the knockdown of NF-kB, IRF3, or HMGA1 (Figs. 4, 5E,6B) and resulted in specific changes of the H5N1-induced gene profile in HUVECs. The induction of all selected H5N1- inducible genes was suppressed when NFATC4 was knocked down (Fig. 6D). According to microarray data, H5N1 infection does not induce HMGA1 mRNA and only slightly NFATC4 mRNA (Supplemental Tables I, V), whereas at the protein level, induction of none of could be observed (Fig. 6A,6C). To clarify the relation between k FIGURE 7. Signaling pathways activated by H5N1 virus in HUVECs. NF- B and HMGA1 and NFATC4, we focused on genes specifi- Model figure illustrates the signaling pathways activated by H5N1 virus in k cally induced by H5N1 in an NF- B–independent mode as CCL7, HUVECs. HMGA1 and NFATC4 are specifically involved by H5N1 (black CXCL5, and CXCL6 (Supplemental Table VI). All genes were arrows), whereas NF-kB and IRF3 are also activated by PR8 and FPV induced by H5N1 in an HMGA1- and NFATC4-dependent mode virus (gray arrows). Drawn arrows indicate positive and dotted lines (Fig. 6E,6F). Data demonstrate that HMGA1 and NFATC4 modify negative regulation. 172 H5N1 TRANSFORMS IMMUNE RESPONSES IN ENDOTHELIA infections compared with low-pathogenic influenza virus. Other by appropriate modulation of the acetylation status of HMGA1 data concerning the role of NF-kB in influenza infections are (46). conflicting most likely due to cell-specific responses to influenza NFATC4 was the third putative H5N1 virus-specific transcrip- infections (35) or due to the use of genetically modified cells or tion factor. In contrast to other NFAT family members (47), viruses (36). It has been argued that cells with low NF-kB activity NFATC4 has not been reported until now to regulate inflammatory were resistant to influenza virus infections (37) and that NF-kB genes. The factor was hitherto not known to be expressed in cells plays a supportive role for virus propagation itself (38). Lee et al. of the immune system but to regulate cell differentiation of car- (39) examined signaling pathways in H5N1-infected human diomyocytes and be expressed in perivascular tissue directing the macrophages and assigned a superior regulatory function for the development of ECs (47, 48). We verified that NFATC4 is also induction of the cytokine storm to p38 MAPK but negate an ex- expressed in HUVECs. After blocking NFATC4, the H5N1-induced ceptional role for NF-kB. However, their conclusion bases on the proinflammatory as well as antiviral gene program is inhibited. assumption of similar levels of NF-kB activation in H5N1 infec- No correlation to the mode of IRF3 or HMGA1 dependence was tions compared with a low pathogenic H1N1 virus without per- detectable. Moreover, both HMGA1 and NFATC4 clearly regulate forming blocking experiments. NF-kB independently H5N1-induced genes. All findings identify In our studies, we generally find an imperative necessity of NF- HMGA1 and NFATC4 for the first time, to our knowledge, to kB signaling to mediate strong proinflammatory gene responses in be transcription factors that independently modulate the proin- ECs as shown in this study in H5N1 infections and previously flammatory as well as antiviral endothelial gene program in an shown for TNF (20) and Candida albicans (32). NF-kB activation H5N1-specific manner. obviously hallmarks responses to inflammatory agents with ca- Taken together, we demonstrate that a systems biology approach Downloaded from pacity to cause SIRS or sepsis. is a very useful tool to evaluate proinflammatory and antiviral re- Besides a differential NF-kB dependence, representatives of sponse programs in ECs. With respect to H5N1 infections of antiviral genes as IFNB1 and CXCL9 are significantly weaker in- HUVECs, we observed an attenuation of the antiviral type I IFN duced 5 h p.i. by H5N1 than by PR8 or FPV. In a human bronchial response and identified HMGA1 and NFATC4 as two novel mo- epithelial cell model, Zeng et al. (17) similarly worked out that the lecular targets contributing to a strong overbalance of proinflam-

pathogenicity of different H5N1 strains correlated with the capa- matory mechanisms. Our data may help to design strategies for the http://www.jimmunol.org/ bility to attenuate the type I IFN response. Our data suggest that development of novel therapeutic approaches for the treatment of H5N1 causes an imbalance between a strong NF-kB–dependent H5N1 infections impeding a devastating proinflammatory response. proinflammatory response and an impaired induction of the anti- viral IFN program in ECs, which might be the key to the fatality of Acknowledgments H5N1 infections. Altogether, this specific gene program apparently We thank U. Nordhues for excellent technical assistance. ensures a survival and replication advantage for H5N1 virus in ECs as reflected by constantly higher H5N1 virus titers 24 h p.i. com- Disclosures pared with PR8 and FPV. The authors have no financial conflicts of interest. 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Supplemental Figures

Figure 1

A B 1.00E-11 1.00E-03 Main categories: Main categories: 1.00E-10 inflammatory response metabolism proliferation/cell cycle control 1.00E-09 signaling/transcriptional regulation 1.00E-08 proliferation/cell cycle control 1.00E-02 1.00E-07 1.00E-06 1.00E-05 p-value p-value

1.00E-04 1.00E-01 1.00E-03 activity space region 1.00E-02 activity factor activity response membrane acid modification transduction 1.00E-01 GO:0004597 peptide- proliferation GO:0006955 immune GO:0004871 signal transducer activity GO:0008283 cell GO:0005576 extracellular GO:0007165 signal GO:0005615 extracellular GO:0003700 transcription growth factor receptor factor growth GO:0030176 integral to GO:0018193 peptidyl-amino aspartate beta-dioxygenase GO:0005006 epidermal endoplasmic reticulum GO:0005488 binding 1.00E+00 1.00E+00

Legend Supplemental Figure 1: Overrepresentation of functional gene groups in the group of genes specifically upregulated by PR8 or FPV virus

Plotted are functional gene groups according to gene ontology (GO) annotations that are overrepresented in the group of genes only upregulated by PR8 (A) or FPV (B), respectively.

The statistical significance (y-axis) was determined by applying the Fisher’s exact test. Viemann et al. Supplemental data 2

Supplemental Tables

Table I H5N1-induced genes in HUVEC

Affymetrix Description Gene Symbol FCa p-valuea ID 204533_at chemokine (C-X-C motif) ligand 10 CXCL10 191.0 8.19E-05 208173_at interferon, beta 1, fibroblast IFNB1 165.7 1.10E-05 233092_s_at DKFZP434B061 protein DKFZP434B061 131.7 1.14E-05 203153_at interferon-induced protein with tetratricopeptide repeats 1 IFIT1 104.2 1.59E-06 235276_at Epithelial stromal interaction 1 (breast) EPSTI1 90.8 1.92E-05 226757_at interferon-induced protein with tetratricopeptide repeats 2 IFIT2 85.6 6.35E-06 211122_s_at chemokine (C-X-C motif) ligand 11 CXCL11 71.1 2.00E-04 219684_at 28kD interferon responsive protein IFRG28 67.8 5.04E-06 1405_i_at chemokine (C-C motif) ligand 5 CCL5 56.8 2.30E-04 242625_at radical S-adenosyl methionine domain containing 2 cig5 52.6 4.56E-05 229450_at interferon-induced protein with tetratricopeptide repeats 3 IFIT4 51.1 5.06E-07 241869_at apolipoprotein L, 6 APOL6 47.3 9.14E-04 226702_at hypothetical protein LOC129607 LOC129607 30.0 3.04E-05 204972_at 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 29.3 2.95E-04 202086_at myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 28.5 1.45E-04 204439_at interferon-induced protein 44-like C1orf29 27.9 2.98E-04 235574_at guanylate binding protein 4 GBP4 26.6 1.56E-04 210797_s_at 2'-5'-oligoadenylate synthetase-like OASL 23.0 7.68E-04 228439_at hypothetical protein BC012330 MGC20410 22.5 5.27E-04 232666_at 2'-5'-oligoadenylate synthetase 3, 100kDa OAS3 19.1 1.53E-04 219209_at interferon induced with C domain 1 IFIH1 18.4 3.41E-05 1557236_at Homo sapiens transcribed sequences 18.4 3.52E-02 209774_x_at chemokine (C-X-C motif) ligand 2 CXCL2 16.5 6.11E-05 219863_at hect domain and RLD 5 HERC5 16.2 3.18E-06 203915_at chemokine (C-X-C motif) ligand 9 CXCL9 15.7 2.42E-03 228230_at peroxisomal proliferator-activated receptor A interacting complex 285 PRIC285 15.3 9.60E-03 205552_s_at 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 14.3 5.79E-05 Transcribed , moderately similar to NP_055301.1 neuronal thread protein AD7c-NTP [Homo 238725_at 13.7 2.69E-03 sapiens] Viemann et al. Supplemental data 3

237084_at Sarcoma antigen NY-SAR-79 mRNA, partial cds 12.8 2.08E-06 207072_at interleukin 18 receptor accessory protein IL18RAP 12.3 1.96E-04 207850_at chemokine (C-X-C motif) ligand 3 CXCL3 11.6 4.71E-04 223502_s_at tumor necrosis factor (ligand) superfamily, member 13b TNFSF13B 11.3 3.69E-02 1556062_at hypothetical protein LOC283012 11.3 8.07E-03 236875_at Transcribed locus 9.9 1.90E-03 242234_at XIAP associated factor-1 HSXIAPAF1 9.6 1.08E-02 238850_at CDNA clone IMAGE:5260726, partial cds 9.3 2.39E-02 204698_at interferon stimulated gene 20kDa ISG20 8.8 1.70E-03 216598_s_at chemokine (C-C motif) ligand 2 CCL2 8.8 4.56E-04 206211_at selectin E (endothelial adhesion molecule 1) SELE 8.4 1.79E-04 223980_s_at SP110 nuclear body protein SP110 8.1 1.15E-03 203868_s_at vascular cell adhesion molecule 1 VCAM1 8.0 2.95E-06 238512_at Suppression of tumorigenicity 7 like 7.5 2.55E-02 239766_at HSPB (heat shock 27kDa) associated protein 1 7.0 2.62E-02 219691_at sterile alpha motif domain containing 9 C7orf5 6.8 3.59E-04 222139_at KIAA1466 gene 6.8 2.16E-03 209795_at CD69 antigen (p60, early T-cell activation antigen) CD69 6.7 3.45E-04 205483_s_at interferon, alpha-inducible protein (clone IFI-15K) (ISG15) G1P2 6.5 4.83E-06 227697_at suppressor of cytokine signaling 3 SOCS3 6.4 1.82E-04 222793_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 6.4 1.41E-03 214059_at Interferon-induced protein 44 IFI44 6.2 4.33E-02 1557116_at CDNA FLJ39947 fis, clone SPLEN2024232 6.1 1.02E-03 203596_s_at interferon-induced protein with tetratricopeptide repeats 5 IFIT5 6.0 1.50E-04 223220_s_at poly (ADP-ribose) polymerase family, member 9 BAL 5.9 1.23E-04 228325_at KIAA0146 protein KIAA0146 5.9 7.64E-03 225415_at deltex 3-like (Drosophila) BBAP 5.7 6.09E-04 823_at chemokine (C-X3-C motif) ligand 1 CX3CL1 5.6 1.79E-03 228278_at /X (CCAAT-binding transcription factor) NFIX 5.4 1.82E-03 202688_at tumor necrosis factor (ligand) superfamily, member 10 TNFSF10 5.4 1.05E-04 213006_at CCAAT/enhancer binding protein (C/EBP), delta KIAA0146 5.2 1.38E-02 243465_at Hypothetical protein FLJ11000 5.2 1.61E-02 202531_at interferon regulatory factor 1 IRF1 5.0 6.89E-03 1563075_s_at Homo sapiens clone IMAGE:110987 mRNA sequence 5.0 4.28E-02 204279_at proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional protease 2) PSMB9 4.7 1.38E-03 Viemann et al. Supplemental data 4

231577_s_at guanylate binding protein 1, interferon-inducible, 67kDa GBP1 4.7 3.67E-04 1555852_at proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional protease 7) PSMB8 4.5 5.13E-05 204470_at chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCL1 4.5 8.76E-04 235086_at Thrombospondin 1 THBS1 4.5 1.32E-04 214974_x_at chemokine (C-X-C motif) ligand 5 CXCL5 4.5 3.59E-03 202307_s_at transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) TAP1 4.3 2.49E-03 202672_s_at activating transcription factor 3 ATF3 4.2 4.45E-03 219352_at hect domain and RLD 6 HERC6 4.2 3.07E-04 211506_s_at interleukin 8 IL8 4.1 1.85E-02 214022_s_at interferon induced transmembrane protein 1 (9-27) IFITM1 4.0 8.23E-04 1569132_s_at Hypothetical protein DKFZp313G1735 DKFZp313G1735 4.0 2.49E-02 221766_s_at family with sequence similarity 46, member A C6orf37 4.0 2.63E-03 232150_at CDNA FLJ12885 fis, clone NT2RP2003988 3.9 2.74E-02 204415_at interferon, alpha-inducible protein (clone IFI-6-16) G1P3 3.9 8.18E-03 202638_s_at intercellular adhesion molecule 1 (CD54), human rhinovirus receptor ICAM1 3.8 1.76E-02 217446_x_at MRNA; cDNA DKFZp434M054 (from clone DKFZp434M054) 3.8 1.19E-02 221432_s_at solute carrier family 25, member 28 SLC25A28 3.7 1.35E-03 209417_s_at interferon-induced protein 35 IFI35 3.7 6.46E-05 219211_at specific protease 18 USP18 3.7 2.78E-03 224701_at poly (ADP-ribose) polymerase family, member 14 KIAA1268 3.7 2.24E-02 203148_s_at tripartite motif-containing 14 TRIM14 3.7 1.85E-03 208436_s_at interferon regulatory factor 7 IRF7 3.6 1.95E-02 228967_at Putative translation initiation factor SUI1 3.6 2.37E-03 230300_at CDNA FLJ42315 fis, clone TRACH2019661 3.5 1.90E-03 228395_at Glycosyltransferase AD-017 AD-017 3.5 6.28E-03 208075_s_at chemokine (C-C motif) ligand 7 CCL7 3.5 1.81E-03 Transcribed locus, weakly similar to NP_055301.1 neuronal thread protein AD7c-NTP [Homo 230657_at CLOCK 3.5 2.32E-03 sapiens] 230036_at 7 open reading frame 6 C7orf6 3.5 1.74E-03 225634_at CCCH type, antiviral 1 ZC3HAV1 3.4 8.02E-05 235061_at protein phosphatase 1K (PP2C domain containing) DKFZp761G058 3.2 1.63E-02 204804_at tripartite motif-containing 21 SSA1 3.2 7.96E-03 206271_at toll-like receptor 3 TLR3 3.2 6.00E-03 212427_at KIAA0368 KIAA0368 3.2 7.10E-03 211998_at H3 histone, family 3B (H3.3B) H3F3B 3.2 1.62E-02 Viemann et al. Supplemental data 5

204285_s_at phorbol-12-myristate-13-acetate-induced protein 1 PMAIP1 3.2 1.35E-03 1565830_at KIAA1731 protein KIAA1731 3.1 1.98E-02 212182_at nudix (nucleoside diphosphate linked moiety X)-type motif 4 NUDT4 3.1 2.04E-02 205207_at interleukin 6 (interferon, beta 2) IL6 3.1 5.92E-04 203882_at interferon-stimulated transcription factor 3, gamma 48kDa ISGF3G 3.0 1.41E-04 225929_s_at open reading frame 27 C17orf27 2.9 2.69E-02 228152_s_at hypothetical protein FLJ31033 FLJ31033 2.9 3.73E-02 1568807_a_at Nedd4 family interacting protein 2 2.9 2.38E-03 205932_s_at msh homeo box homolog 1 (Drosophila) MSX1 2.8 3.02E-03 204224_s_at GTP cyclohydrolase 1 (dopa-responsive dystonia) GCH1 2.8 2.04E-03 242389_at resistance-associated overexpressed protein LUC7A 2.8 2.20E-02 225886_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 DDX5 2.8 2.41E-02 226773_at MRNA (clone ICRFp507I1077) 2.8 8.73E-03 1558938_at Clone IMAGE:5222953, mRNA 2.7 4.14E-02 228987_at Family with sequence similarity 49, member B 2.7 4.54E-03 Transcribed sequence with weak similarity to protein sp:P39192 (H.sapiens) ALU5_HUMAN Alu 244350_at 2.7 5.42E-03 subfamily SC sequence contamination warning entry 1560071_a_at thioesterase superfamily member 2 THEM2 2.7 4.96E-02 206336_at chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) CXCL6 2.7 4.76E-03 211368_s_at caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase) CASP1 2.6 2.58E-02 242907_at guanylate binding protein 2, interferon-inducible GBP2 2.6 1.95E-02 221653_x_at apolipoprotein L, 2 APOL2 2.5 7.33E-03 203236_s_at lectin, galactoside-binding, soluble, 9 (galectin 9) LGALS9 2.5 3.49E-02 234987_at SAM domain and HD domain 1 2.5 1.33E-02 1566342_at superoxide dismutase 2, mitochondrial SOD2 2.5 2.02E-02 Transcribed locus, strongly similar to NP_001055.1 transketolase (Wernicke-Korsakoff syndrome) 230305_at 2.5 5.54E-03 [Homo sapiens] 201649_at ubiquitin-conjugating enzyme E2L 6 UBE2L6 2.4 8.18E-04 230405_at hypothetical gene supported by AL713721 2.4 1.74E-02 1557487_at Homo sapiens, clone IMAGE:4797099, mRNA 2.3 1.08E-02 237387_at Transcribed sequences 2.3 2.03E-02 229321_s_at BH3 interacting domain death agonist BID 2.3 2.38E-02 235964_x_at FLJ46365 protein FLJ46365 2.2 3.93E-03 204205_at apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G APOBEC3G 2.2 1.44E-02 203378_at pre-mRNA cleavage complex II protein Pcf11 PCF11 2.2 1.56E-02 Viemann et al. Supplemental data 6

204070_at responder (tazarotene induced) 3 RARRES3 2.2 2.86E-02 243299_at Vaccinia related kinase 2 2.2 5.47E-03 236270_at nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 NFATC4 2.2 3.90E-02 212783_at retinoblastoma binding protein 6 RBBP6 2.2 5.07E-03 239660_at KIAA1272 protein KIAA1272 2.2 2.34E-02 236978_at Thyroid associated protein 1 2.2 2.59E-02 221477_s_at FLJ43505 protein SOD2 2.2 5.78E-03 235084_x_at Tripartite motif-containing 38 TRIM38 2.1 1.86E-02 240466_at Mitochondrial tumor suppressor 1 2.1 3.32E-04 202411_at interferon, alpha-inducible protein 27 IFI27 2.1 1.21E-04 209969_s_at signal transducer and activator of transcription 1, 91kDa STAT1 2.1 1.78E-03 202644_s_at tumor necrosis factor, alpha-induced protein 3 TNFAIP3 2.1 8.94E-03 225076_s_at KIAA1404 protein KIAA1404 2.1 2.19E-02 221087_s_at apolipoprotein L, 3 APOL3 2.1 4.84E-02 226040_at MRNA; cDNA DKFZp762N156 (from clone DKFZp762N156) 2.1 4.57E-02 1558755_x_at hypothetical protein LOC284390 LOC284390 2.0 3.72E-02 226934_at Cleavage and polyadenylation specific factor 6, 68kDa CPSF6 2.0 3.01E-02 226713_at chromosome 3 open reading frame 6 C3orf6 2.0 4.86E-02 225842_at CDNA clone IMAGE:4514712, partial cds PHLDA1 2.0 4.04E-02 a FC and p-value were computed from 3 independent experiments determining gene expression changes in 5h H5N1-infected HUVEC by microarray analysis (U133Plus2 microarray, Affymetrix; see Material and Methods).

Viemann et al. Supplemental data 7

Table II FPV-induced genes in HUVEC

Name Description Gene Symbol FCa p-valuea 203153_at interferon-induced protein with tetratricopeptide repeats 1 IFIT1 1310.0 1.07E-04 204533_at chemokine (C-X-C motif) ligand 10 CXCL10 74.6 4.62E-03 202086_at myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 74.2 3.88E-04 242625_at radical S-adenosyl methionine domain containing 2 cig5 44.0 2.39E-04 208173_at interferon, beta 1, fibroblast IFNB1 40.5 8.13E-06 226757_at interferon-induced protein with tetratricopeptide repeats 2 IFIT2 37.9 3.20E-06 1405_i_at chemokine (C-C motif) ligand 5 CCL5 35.6 6.69E-03 229450_at interferon-induced protein with tetratricopeptide repeats 3 IFIT3 32.7 6.13E-06 239979_at Epithelial stromal interaction 1 (breast) 25.6 1.10E-02 205660_at 2'-5'-oligoadenylate synthetase-like OASL 23.9 7.56E-03 226702_at hypothetical protein LOC129607 LOC129607 21.4 6.40E-07 204972_at 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 20.2 1.88E-02 204994_at myxovirus (influenza virus) resistance 2 (mouse) MX2 19.9 1.22E-03 211122_s_at chemokine (C-X-C motif) ligand 11 CXCL11 17.3 3.93E-03 1558686_at Full length insert cDNA YP99D02 16.2 5.00E-02 1565913_at Zinc finger CCCH type, antiviral 1 16.2 4.19E-02 204439_at interferon-induced protein 44-like IFI44L 14.4 8.25E-04 232787_at hypothetical protein LOC200213 LOC200213 10.2 9.09E-03 228439_at hypothetical protein BC012330 MGC20410 10.0 4.42E-04 203915_at chemokine (C-X-C motif) ligand 9 CXCL9 9.8 3.40E-02 219863_at hect domain and RLD 5 HERC5 9.3 2.08E-04 1568915_at Homo sapiens, clone IMAGE:4822684, mRNA 9.0 2.24E-02 202437_s_at cytochrome P450, family 1, subfamily B, polypeptide 1 CYP1B1 9.0 3.13E-02 214993_at hypothetical protein LOC253982 LOC253982 8.7 1.35E-04 1569503_at KIAA1414 protein KIAA1414 7.9 4.36E-02 211149_at ubiquitously transcribed tetratricopeptide repeat gene, Y-linked UTY 7.4 1.54E-02 221766_s_at family with sequence similarity 46, member A C6orf37 7.3 8.00E-03 208392_x_at SP110 nuclear body protein SP110 6.9 3.09E-02 1555090_x_at hypothetical protein FLJ30294 FLJ30294 6.6 3.65E-02 244654_at IG MYO1G 6.6 2.45E-02 223137_at zinc finger, DHHC domain containing 4 ZDHHC4 6.4 3.44E-02 204415_at interferon, alpha-inducible protein (clone IFI-6-16) G1P3 6.4 3.13E-04 Viemann et al. Supplemental data 8

208851_s_at Thy-1 cell surface antigen THY1 6.1 6.82E-03 219209_at interferon induced with helicase C domain 1 IFIH1 6.1 2.92E-05 217210_at , cytoplasmic, light polypeptide 1 DNCL1 5.8 1.44E-03 1558392_at spectrin repeat containing, nuclear envelope 2 SYNE2 5.8 2.41E-02 202869_at 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 5.5 3.25E-04 202454_s_at v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) ERBB3 5.5 2.46E-02 228230_at peroxisomal proliferator-activated receptor A interacting complex 285 PRIC285 5.3 1.32E-04 230757_at FLJ44796 protein 5.3 4.75E-02 205483_s_at interferon, alpha-inducible protein (clone IFI-15K) G1P2 5.2 8.39E-04 214453_s_at interferon-induced protein 44 IFI44 4.7 1.16E-06 228531_at sterile alpha motif domain containing 9 SAMD9 4.4 3.80E-04 214022_s_at interferon induced transmembrane protein 1 (9-27) IFITM1 4.1 1.04E-04 239845_at Transcribed sequences 3.8 1.07E-02 244293_at Transcribed locus 3.8 5.37E-03 228617_at XIAP associated factor-1 HSXIAPAF1 3.7 2.40E-03 235298_at hypothetical protein MGC43690 MGC43690 3.5 3.67E-02 216263_s_at open reading frame 120 C14orf120 3.4 4.17E-02 230300_at CDNA FLJ42315 fis, clone TRACH2019661 3.3 3.16E-03 223220_s_at poly (ADP-ribose) polymerase family, member 9 PARP9 3.0 2.20E-03 235497_at Hypothetical protein LOC284591 LOC284591 2.9 1.89E-03 1568964_x_at sialophorin (gpL115, leukosialin, CD43) 2.8 1.99E-03 208436_s_at interferon regulatory factor 7 IRF7 2.7 3.10E-03 242003_at Hypothetical protein LOC157697 LOC157697 2.7 2.95E-02 1555852_at proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional protease 7) 2.7 2.66E-02 242389_at Cisplatin resistance-associated overexpressed protein CROP 2.6 3.56E-02 214329_x_at tumor necrosis factor (ligand) superfamily, member 10 2.6 3.59E-02 228967_at Putative translation initiation factor SUI1 2.6 3.63E-03 203595_s_at interferon-induced protein with tetratricopeptide repeats 5 IFIT5 2.5 4.07E-04 Transcribed locus, weakly similar to XP_519878.1 similar to ubiquitin-conjugating enzyme E2 variant 230449_x_at 2.5 2.09E-04 1 isoform c; DNA-binding protein [Pan troglodytes] 231577_s_at guanylate binding protein 1, interferon-inducible, 67kDa GBP1 2.4 1.48E-02 1553538_s_at 2.3 4.40E-02 220866_at A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 6 ADAMTS6 2.3 4.72E-02 228641_at Caspase recruitment domain family, member 8 CARD8 2.3 1.90E-03 222793_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 2.3 2.93E-03 Viemann et al. Supplemental data 9

209417_s_at interferon-induced protein 35 IFI35 2.2 2.53E-03 1563014_at Ribosomal protein S15 RPS15 2.2 2.87E-02 201649_at ubiquitin-conjugating enzyme E2L 6 UBE2L6 2.2 5.13E-04 1554672_at hypothetical protein FLJ12571 FLJ12571 2.1 3.72E-02 235061_at protein phosphatase 1K (PP2C domain containing) PPM1K 2.1 1.57E-02 201744_s_at lumican LUM 2.1 2.71E-02 228645_at Clone IMAGE:5221276, mRNA, partial cds 2.1 1.53E-04 230314_at Similar to hypothetical protein 628 2.1 1.91E-02 230036_at chromosome 7 open reading frame 6 C7orf6 2.1 3.60E-03 203236_s_at lectin, galactoside-binding, soluble, 9 (galectin 9) LGALS9 2.1 4.06E-02 211512_s_at opioid growth factor receptor OGFR 2.1 1.53E-02 202531_at interferon regulatory factor 1 IRF1 2.0 1.46E-02 225415_at deltex 3-like (Drosophila) DTX3L 2.0 2.63E-03 208190_s_at liver-specific bHLH-Zip transcription factor LISCH7 2.0 3.07E-02 a FC and p-value were computed from 3 independent experiments determining gene expression changes in 5h FPV-infected HUVEC by microarray analysis (U133Plus2 microarray, Affymetrix; see Material and Methods).

Viemann et al. Supplemental data 10

Table III PR8-induced genes in HUVEC Affymetrix Description Gene Symbol FCa p-valuea ID 203153_at interferon-induced protein with tetratricopeptide repeats 1 IFIT1 6607.3 4.51E-05 204533_at chemokine (C-X-C motif) ligand 10 CXCL10 3348.3 3.87E-04 1405_i_at chemokine (C-C motif) ligand 5 CCL5 2281.0 3.31E-04 208173_at interferon, beta 1, fibroblast IFNB1 1412.6 1.92E-07 205660_at 2'-5'-oligoadenylate synthetase-like OASL 873.9 4.10E-04 203915_at chemokine (C-X-C motif) ligand 9 CXCL9 705.2 3.07E-04 242625_at radical S-adenosyl methionine domain containing 2 cig5 582.8 2.76E-05 226757_at interferon-induced protein with tetratricopeptide repeats 2 IFIT2 477.1 3.77E-07 202086_at myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 412.3 1.09E-04 211122_s_at chemokine (C-X-C motif) ligand 11 CXCL11 314.8 1.11E-04 229450_at interferon-induced protein with tetratricopeptide repeats 3 IFIT4 264.4 7.00E-07 1557236_at Homo sapiens transcribed sequences 253.1 2.63E-04 219863_at hect domain and RLD 5 HERC5 227.8 1.38E-07 204994_at myxovirus (influenza virus) resistance 2 (mouse) MX2 203.3 1.11E-04 226702_at hypothetical protein LOC129607 LOC129607 141.3 7.09E-08 239979_at Epithelial stromal interaction 1 (breast) 136.9 1.45E-03 210029_at indoleamine-pyrrole 2,3 dioxygenase INDO 131.4 2.66E-04 1557557_at CDNA FLJ46197 fis, clone TESTI4007565 118.4 1.71E-03 241869_at apolipoprotein L, 6 APOL6 115.5 9.01E-04 204972_at 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 110.3 4.73E-03 233092_s_at DKFZP434B061 protein DKFZP434B061 105.8 1.43E-03 221766_s_at family with sequence similarity 46, member A C6orf37 95.1 2.36E-04 204439_at interferon-induced protein 44-like C1orf29 84.9 3.03E-06 207194_s_at intercellular adhesion molecule 4, Landsteiner-Wiener blood group ICAM4 83.8 1.16E-03 206932_at cholesterol 25-hydroxylase CH25H 80.3 3.47E-03 229802_at WNT1 inducible signaling pathway protein 1 WISP1 76.9 1.43E-04 219209_at interferon induced with helicase C domain 1 IFIH1 75.0 5.58E-07 235175_at guanylate binding protein 4 GBP4 73.0 4.12E-03 1565913_at Zinc finger CCCH type, antiviral 1 71.0 9.08E-03 219364_at likely ortholog of mouse D11lgp2 LGP2 69.1 3.60E-04 236156_at lipase A, lysosomal acid, cholesterol esterase (Wolman disease) LIPA 68.0 1.36E-04 219684_at 28kD interferon responsive protein IFRG28 58.6 1.62E-03 Viemann et al. Supplemental data 11

1559287_at CDNA FLJ35503 fis, clone SMINT2009216 57.4 4.15E-03 210147_at ADP-ribosyltransferase 3 ART3 56.8 1.11E-02 203687_at chemokine (C-X3-C motif) ligand 1 CX3CL1 56.1 2.30E-04 1561084_at Homo sapiens, clone IMAGE:5268539, mRNA 48.6 3.93E-04 220655_at TNFAIP3 interacting protein 3 TNIP3 46.4 5.44E-03 223501_at tumor necrosis factor (ligand) superfamily, member 13b TNFSF13B 46.0 5.70E-03 219593_at solute carrier family 15, member 3 SLC15A3 45.7 3.48E-03 219584_at phospholipase A1 member A PLA1A 45.1 5.04E-04 224225_s_at ets variant gene 7 (TEL2 oncogene) ETV7 43.9 7.95E-04 204698_at interferon stimulated gene 20kDa ISG20 43.2 1.85E-04 228439_at hypothetical protein BC012330 MGC20410 40.5 5.61E-05 202357_s_at B-factor, properdin BF 39.2 5.03E-03 205476_at chemokine (C-C motif) ligand 20 CCL20 35.6 1.25E-03 202869_at 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 35.5 7.61E-06 229625_at Guanylate binding protein 5 GBP5 34.9 1.20E-02 220316_at neuronal PAS domain protein 3 NPAS3 33.2 3.27E-03 228707_at claudin 23 CLDN23 33.1 5.19E-04 232593_at hypothetical protein BC012317 LOC93082 31.3 9.76E-05 217076_s_at homeo box D3 HOXD3 30.6 1.58E-03 238850_at CDNA clone IMAGE:5260726, partial cds 30.5 4.84E-03 1552917_at interleukin 29 (interferon, lambda 1) IL29 27.9 1.02E-03 1570197_at Homo sapiens, clone IMAGE:5111803, mRNA 27.1 6.38E-03 219011_at pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 4 PLEKHA4 27.1 1.02E-02 1557116_at CDNA FLJ39947 fis, clone SPLEN2024232 26.4 1.69E-03 229288_at EPH receptor A7 EPHA7 25.7 4.61E-03 221841_s_at Kruppel-like factor 4 (gut) 24.9 3.35E-05 214453_s_at interferon-induced protein 44 IFI44 24.9 1.53E-07 1562031_at 2 (a protein kinase) JAK2 24.8 3.09E-03 218400_at 2'-5'-oligoadenylate synthetase 3, 100kDa OAS3 24.7 1.39E-05 214022_s_at interferon induced transmembrane protein 1 (9-27) IFITM1 23.7 1.95E-06 1563076_x_at Homo sapiens clone IMAGE:110987 mRNA sequence 23.3 1.78E-02 202307_s_at transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) TAP1 23.2 1.80E-06 205681_at BCL2-related protein A1 BCL2A1 23.2 2.63E-02 231577_s_at guanylate binding protein 1, interferon-inducible, 67kDa GBP1 22.9 5.98E-05 208392_x_at SP110 nuclear body protein SP110 22.9 6.28E-03 Viemann et al. Supplemental data 12

202437_s_at cytochrome P450, family 1, subfamily B, polypeptide 1 CYP1B1 22.6 9.52E-03 204415_at interferon, alpha-inducible protein (clone IFI-6-16) G1P3 22.5 9.54E-05 204363_at coagulation factor III (thromboplastin, tissue factor) F3 22.0 7.02E-04 228230_at peroxisomal proliferator-activated receptor A interacting complex 285 PRIC285 21.9 4.95E-05 204279_at proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional protease 2) PSMB9 21.6 6.87E-04 218943_s_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 20.9 8.81E-06 gb:NM_022155.1 /DEF=Homo sapiens PP3227 protein (PP3227), mRNA. /FEA=mRNA 221200_at /GEN=PP3227 /PROD=PP3227 protein /DB_XREF=gi:11545901 /UG=Hs.302027 PP3227 protein 20.6 5.81E-04 /FL=gb:NM_022155.1 205483_s_at interferon, alpha-inducible protein (clone IFI-15K) G1P2 20.5 4.32E-06 204070_at retinoic acid receptor responder (tazarotene induced) 3 RARRES3 20.4 1.10E-04 206942_s_at pro-melanin-concentrating hormone PMCH 19.5 4.62E-03 228531_at sterile alpha motif domain containing 9 C7orf5 19.5 1.79E-05 226474_at nucleotide-binding oligomerization domains 27 NOD27 19.1 2.78E-04 207160_at interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35) IL12A 18.8 9.34E-03 202672_s_at activating transcription factor 3 ATF3 18.7 1.13E-04 213418_at heat shock 70kDa protein 6 (HSP70B') HSPA6 18.5 2.23E-02 235699_at hypothetical protein FLJ38964 FLJ38964 18.5 2.06E-04 210538_s_at baculoviral IAP repeat-containing 3 BIRC3 18.2 6.03E-03 209546_s_at apolipoprotein L, 1 APOL1 18.1 2.53E-04 1565915_at Full length insert cDNA clone YR04D03 17.6 2.10E-03 220302_at male germ cell-associated kinase MAK 16.8 3.53E-02 235157_at Transcribed sequences 16.7 2.68E-03 206271_at toll-like receptor 3 TLR3 16.2 4.36E-05 205890_s_at ubiquitin D UBD 15.9 6.67E-04 228617_at XIAP associated factor-1 HSXIAPAF1 15.7 8.69E-06 231956_at KIAA1618 KIAA1618 15.6 8.43E-04 219352_at hect domain and RLD 6 HERC6 15.5 6.30E-05 202644_s_at tumor necrosis factor, alpha-induced protein 3 TNFAIP3 15.0 9.18E-05 214038_at chemokine (C-C motif) ligand 8 CCL8 14.9 6.94E-04 207850_at chemokine (C-X-C motif) ligand 3 CXCL3 14.8 1.38E-02 1568915_at Homo sapiens, clone IMAGE:4822684, mRNA 14.7 9.08E-03 202687_s_at tumor necrosis factor (ligand) superfamily, member 10 TNFSF10 14.6 4.45E-04 233694_at CDNA FLJ12144 fis, clone MAMMA1000361 14.6 2.07E-02 210432_s_at , voltage-gated, type III, alpha SCN3A 14.3 1.59E-02 Viemann et al. Supplemental data 13

206408_at leucine rich repeat transmembrane neuronal 2 LRRTM2 14.3 3.51E-03 232792_at ring finger protein 36 RNF36 13.8 1.80E-03 204286_s_at phorbol-12-myristate-13-acetate-induced protein 1 PMAIP1 13.6 3.20E-06 213716_s_at secreted and transmembrane 1 SECTM1 13.6 1.57E-04 201422_at interferon, gamma-inducible protein 30 IFI30 13.5 4.54E-04 1569503_at KIAA1414 protein KIAA1414 13.5 9.44E-04 235116_at TNF receptor-associated factor 1 TRAF1 13.5 4.97E-02 230383_x_at Transcribed locus 13.4 2.67E-04 209841_s_at leucine rich repeat neuronal 3 LRRN3 12.7 4.95E-02 206432_at hyaluronan synthase 2 HAS2 12.7 3.86E-04 243271_at Chromosome 7 open reading frame 6 12.4 2.26E-04 208436_s_at interferon regulatory factor 7 IRF7 12.3 3.42E-05 244163_at Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A SEMA3A 12.2 1.13E-02 1553378_a_at hypothetical protein FLJ32830 FLJ32830 12.2 4.23E-02 1561313_at hypothetical gene supported by AK092630 12.1 4.71E-02 209498_at carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) CEACAM1 12.1 6.41E-05 232284_at Proteasome regulatory particle subunit p44S10 11.6 1.51E-02 205671_s_at major histocompatibility complex, class II, DO beta HLA-DOB 11.4 1.84E-02 204205_at apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G APOBEC3G 11.3 8.51E-04 203596_s_at interferon-induced protein with tetratricopeptide repeats 5 IFIT5 11.3 6.95E-04 241412_at betacellulin BTC 11.1 1.85E-02 209417_s_at interferon-induced protein 35 IFI35 11.1 1.30E-05 219211_at ubiquitin specific protease 18 USP18 11.0 1.09E-05 1565830_at KIAA1731 protein KIAA1731 10.9 9.65E-04 205027_s_at mitogen-activated protein kinase kinase kinase 8 MAP3K8 10.9 7.20E-03 243386_at Similar to cDNA sequence BC035954 10.8 3.11E-02 206026_s_at tumor necrosis factor, alpha-induced protein 6 TNFAIP6 10.5 2.56E-02 1557071_s_at NEDD8 ultimate buster-1 NYREN18 10.5 3.53E-02 1568706_s_at Pp12719 10.5 3.81E-02 221653_x_at apolipoprotein L, 2 APOL2 10.4 7.98E-05 208747_s_at complement component 1, s subcomponent C1S 10.2 1.78E-02 232787_at hypothetical protein LOC200213 LOC200213 10.0 8.95E-03 223220_s_at poly (ADP-ribose) polymerase family, member 9 BAL 10.0 1.41E-04 204224_s_at GTP cyclohydrolase 1 (dopa-responsive dystonia) GCH1 9.9 5.21E-04 243299_at Vaccinia related kinase 2 9.9 2.25E-06 Viemann et al. Supplemental data 14

233376_at interaction factor 2-like mRNA sequence 9.7 1.40E-02 Transcribed locus, moderately similar to NP_055301.1 neuronal thread protein AD7c-NTP [Homo 238725_at 9.7 7.17E-04 sapiens] 208529_at basic transcription factor 3, like 1 9.4 3.52E-02 230000_at chromosome 17 open reading frame 27 C17orf27 9.4 2.29E-03 242907_at guanylate binding protein 2, interferon-inducible GBP2 9.4 2.22E-05 225415_at deltex 3-like (Drosophila) BBAP 9.4 4.22E-06 1557840_at hypothetical LOC401057 9.3 4.21E-03 235964_x_at FLJ46365 protein FLJ46365 8.7 2.03E-05 1559883_s_at SAM domain and HD domain 1 SAMHD1 8.7 3.22E-02 1555912_at ST7 overlapping transcript 1 (antisense non-coding RNA) ST7 8.6 3.15E-05 1555852_at proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional protease 7) PSMB8 8.5 4.77E-04 240338_at Leukocyte-derived arginine aminopeptidase LRAP 8.4 1.47E-03 203148_s_at tripartite motif-containing 14 TRIM14 8.4 1.45E-04 201649_at ubiquitin-conjugating enzyme E2L 6 UBE2L6 8.3 5.01E-05 220358_at p21SNFT SNFT 8.3 3.05E-03 226218_at Interleukin 7 receptor IL7R 8.2 1.71E-03 211367_s_at caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase) CASP1 8.2 1.65E-02 224701_at poly (ADP-ribose) polymerase family, member 14 KIAA1268 8.1 2.75E-03 220703_at open reading frame 110 C10orf110 8.1 1.10E-02 220148_at aldehyde dehydrogenase 8 family, member A1 ALDH8A1 8.1 4.04E-03 235061_at protein phosphatase 1K (PP2C domain containing) DKFZp761G058 7.9 3.46E-04 205539_at advillin AVIL 7.8 1.21E-04 214409_at Clone IMAGE:5272895, mRNA [BLAST] 7.8 2.74E-03 232311_at Beta-2-microglobulin B2M 7.7 2.49E-02 213618_at centaurin, delta 1 CENTD1 7.7 1.40E-03 209829_at open reading frame 32 C6orf32 7.7 2.07E-02 230405_at hypothetical gene supported by AL713721 7.6 4.48E-03 203868_s_at vascular cell adhesion molecule 1 VCAM1 7.5 2.25E-04 226847_at follistatin FST 7.3 6.21E-03 230314_at Similar to hypothetical protein 628 7.2 2.48E-03 202531_at interferon regulatory factor 1 IRF1 7.2 8.39E-04 58916_at tetramerisation domain containing 14 7.2 7.15E-02 211959_at insulin-like growth factor binding protein 5 IGFBP5 7.1 1.08E-02 237083_at Transcribed locus, weakly similar to NP_055301.1 neuronal thread protein AD7c-NTP [Homo sapiens] 7.1 2.29E-02 Viemann et al. Supplemental data 15

228152_s_at hypothetical protein FLJ31033 FLJ31033 7.1 2.55E-04 1556427_s_at similar to hypothetical protein LOC221091 6.9 1.77E-03 1569263_at casein kinase 1, delta CSNK1D 6.8 4.68E-02 210056_at Rho family GTPase 1 RND1 6.7 4.04E-03 203236_s_at lectin, galactoside-binding, soluble, 9 (galectin 9) LGALS9 6.7 1.76E-03 202800_at solute carrier family 1 (glial high affinity glutamate transporter), member 3 SLC1A3 6.7 2.08E-02 214321_at nephroblastoma overexpressed gene NOV 6.7 4.69E-04 223192_at solute carrier family 25, member 28 SLC25A28 6.6 7.63E-05 219992_at tachykinin 3 (neuromedin K, neurokinin beta) TAC3 6.5 2.96E-02 1556814_a_at Full length insert cDNA clone ZA96G04 6.3 4.80E-02 204057_at Interferon regulatory factor 8 ICSBP1 6.3 3.96E-02 244511_at Phosphodiesterase 4D interacting protein (myomegalin) 6.3 2.06E-02 230300_at CDNA FLJ42315 fis, clone TRACH2019661 6.3 1.68E-05 Transcribed sequence with weak similarity to protein sp:P39194 (H.sapiens) ALU7_HUMAN Alu 241722_x_at 6.2 1.23E-02 subfamily SQ sequence contamination warning entry 203973_s_at CCAAT/enhancer binding protein (C/EBP), delta 6.2 1.05E-04 53720_at hypothetical protein FLJ11286 FLJ11286 6.2 3.15E-05 1564430_at HBxAg transactivated protein 2 XTP2 6.2 7.47E-04 239203_at hypothetical protein FLJ39575 FLJ39575 6.1 4.25E-02 217371_s_at interleukin 15 IL15 6.1 1.29E-04 1568615_a_at Homo sapiens, clone IMAGE:5295612, mRNA 6.1 3.14E-03 215223_s_at superoxide dismutase 2, mitochondrial SOD2 6.1 2.28E-05 242974_at CD47 antigen (Rh-related antigen, integrin-associated signal transducer) CD47 6.0 5.01E-03 200628_s_at tryptophanyl-tRNA synthetase WARS 5.9 7.51E-04 1557078_at hypothetical protein MGC19764 MGC19764 5.9 2.53E-02 228347_at Sine oculis homolog 1 (Drosophila) SIX1 5.9 9.89E-05 202638_s_at intercellular adhesion molecule 1 (CD54), human rhinovirus receptor ICAM1 5.8 1.03E-03 221477_s_at FLJ43505 protein SOD2 5.8 4.05E-03 222796_at pentatricopeptide repeat domain 1 PTCD1 5.8 7.15E-03 202510_s_at tumor necrosis factor, alpha-induced protein 2 TNFAIP2 5.7 1.80E-04 221087_s_at apolipoprotein L, 3 APOL3 5.6 7.34E-05 208498_s_at amylase, alpha 2A; pancreatic AMY2B 5.6 4.02E-02 222128_at , voltage-dependent, beta 2 subunit CACNB2 5.6 7.19E-03 223434_at guanylate binding protein 3 GBP3 5.6 2.89E-05 209774_x_at chemokine (C-X-C motif) ligand 2 CXCL2 5.5 4.05E-06 Viemann et al. Supplemental data 16

214933_at calcium channel, voltage-dependent, P/Q type, alpha 1A subunit CACNA1A 5.5 4.23E-03 1568634_a_at similar to Hypothetical protein MGC38937 5.5 3.34E-02 220066_at caspase recruitment domain family, member 15 CARD15 5.4 8.47E-03 218986_s_at hypothetical protein FLJ20035 FLJ20035 5.4 9.80E-05 230741_at Full length insert cDNA clone YX74D05 5.4 5.44E-04 205932_s_at msh homeo box homolog 1 (Drosophila) MSX1 5.3 3.62E-05 1568592_at hypothetical gene supported by BC031266 5.3 2.55E-04 210001_s_at suppressor of cytokine signaling 1 SOCS1 5.2 5.43E-05 229437_at BIC noncoding mRNA, complete sequence [BLAST] 5.1 1.36E-04 228532_at hypothetical protein MGC24133 MGC24133 5.1 2.71E-03 242752_at MRNA (clone ICRFp507I1077) 5.1 4.70E-02 241956_at Ring finger protein (C3HC4 type) 159 5.1 4.89E-04 1558938_at Clone IMAGE:5222953, mRNA 5.0 1.00E-02 243785_at Far upstream element (FUSE) binding protein 3 FUBP3 5.0 1.84E-02 230636_s_at basic transcription element binding protein 1 BTEB1 4.8 3.29E-03 213361_at tudor domain containing 7 TDRD7 4.8 2.83E-04 222139_at KIAA1466 gene 4.7 3.17E-04 230917_at CDNA clone IMAGE:4309350, partial cds 4.7 2.93E-04 238025_at mixed lineage kinase domain-like FLJ34389 4.7 8.68E-06 202411_at interferon, alpha-inducible protein 27 IFI27 4.7 2.95E-04 242803_at Cysteine-rich motor neuron 1 4.6 2.40E-01 232375_at Signal transducer and activator of transcription 1, 91kDa 4.6 3.95E-03 213415_at chloride intracellular channel 2 CLIC2 4.5 1.35E-04 227697_at suppressor of cytokine signaling 3 SOCS3 4.5 1.55E-04 216598_s_at chemokine (C-C motif) ligand 2 CCL2 4.5 1.66E-03 209545_s_at receptor-interacting - kinase 2 RIPK2 4.4 3.56E-04 225973_at transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) TAP2 4.3 2.46E-05 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 (H.sapiens) 241686_x_at 4.3 4.31E-03 hypothetical protein FLJ20378 [Homo sapiens] 212641_at human immunodeficiency virus type I enhancer binding protein 2 HIVEP2 4.3 1.19E-03 1561915_at Homo sapiens, clone IMAGE:5278237, mRNA, partial cds 4.3 1.89E-03 209716_at colony stimulating factor 1 (macrophage) CSF1 4.2 8.99E-04 228186_s_at thrombospondin, type I, domain containing 2 THSD2 4.1 3.64E-05 1552302_at hypothetical protein MGC20235 MGC20235 4.1 4.57E-03 230333_at Spermidine/spermine N1-acetyltransferase SAT 4.1 3.05E-04 Viemann et al. Supplemental data 17

1554283_at CCR4 carbon catabolite repression 4-like (S. cerevisiae) CCRN4L 4.1 4.09E-03 202430_s_at phospholipid scramblase 1 PLSCR1 4.1 1.80E-03 238736_at REV3-like, catalytic subunit of DNA polymerase zeta (yeast) REV3L 4.0 6.37E-03 202071_at syndecan 4 (amphiglycan, ryudocan) SDC4 4.0 1.74E-03 204748_at prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) PTGS2 4.0 5.51E-04 229548_at Unc-84 homolog B (C. elegans) UNC84B 3.9 1.99E-03 209795_at CD69 antigen (p60, early T-cell activation antigen) CD69 3.9 9.94E-03 202902_s_at cathepsin S CTSS 3.9 6.97E-03 206211_at selectin E (endothelial adhesion molecule 1) SELE 3.8 2.63E-03 241031_at Nuclear localized factor 1 LOC145741 3.8 1.68E-03 204804_at tripartite motif-containing 21 SSA1 3.8 3.21E-03 1559436_x_at MRNA; cDNA DKFZp313M2114 (from clone DKFZp313M2114) 3.8 4.31E-04 205656_at protocadherin 17 PCDH17 3.8 1.12E-04 227347_x_at hairy and enhancer of split 4 (Drosophila) Hes4 3.8 5.81E-03 204780_s_at Fas (TNF receptor superfamily, member 6) TNFRSF6 3.7 2.24E-03 217933_s_at leucine aminopeptidase 3 LAP3 3.7 1.93E-05 203304_at BMP and activin membrane-bound inhibitor homolog (Xenopus laevis) BAMBI 3.7 1.66E-03 242389_at Cisplatin resistance-associated overexpressed protein LUC7A 3.7 2.37E-03 201641_at bone marrow stromal cell antigen 2 BST2 3.7 1.14E-03 202837_at FLN29 gene product FLN29 3.7 4.01E-03 211911_x_at major histocompatibility complex, class I, B HLA-C 3.7 5.70E-03 243465_at Hypothetical protein FLJ11000 3.6 4.47E-03 227066_at MOB1, Mps One Binder kinase activator-like 2C (yeast) MOBKL2C 3.6 1.09E-05 237623_at Cystatin C (amyloid angiopathy and cerebral hemorrhage) CST3 3.6 2.06E-03 223217_s_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta MAIL 3.6 2.18E-03 205207_at interleukin 6 (interferon, beta 2) IL6 3.5 1.68E-04 244547_at Hypothetical protein FLJ25006 3.5 6.30E-02 1558560_s_at basic nuclear factor 1 (JEM-1) BLZF1 3.5 4.80E-03 225076_s_at KIAA1404 protein KIAA1404 3.5 1.76E-05 231769_at F-box protein 6 FBXO6 3.5 5.09E-04 225636_at signal transducer and activator of transcription 2, 113kDa STAT2 3.5 7.56E-04 224261_at PRO3098 mRNA, complete cds 3.4 1.98E-02 227458_at Programmed cell death 1 ligand 1 PDCD1LG1 3.4 3.70E-05 209640_at promyelocytic leukemia PML 3.4 7.62E-04 228967_at Putative translation initiation factor SUI1 3.2 1.24E-03 Viemann et al. Supplemental data 18

210230_at MRNA; cDNA DKFZp666D074 (from clone DKFZp666D074) 3.2 9.79E-03 228213_at H2A histone family, member J H2AFJ 3.2 2.38E-04 203964_at N- (and STAT) interactor NMI 3.2 9.13E-04 214808_at MRNA; cDNA DKFZp762N156 (from clone DKFZp762N156) 3.2 2.15E-03 244774_at Phosphatase and actin regulator 2 PHACTR2 3.1 4.18E-02 203851_at insulin-like growth factor binding protein 6 IGFBP6 3.1 1.70E-03 203882_at interferon-stimulated transcription factor 3, gamma 48kDa ISGF3G 3.1 2.13E-04 210785_s_at chromosome 1 open reading frame 38 C1orf38 3.1 7.29E-04 208966_x_at interferon, gamma-inducible protein 16 IFI16 3.1 5.90E-06 211512_s_at opioid growth factor receptor OGFR 3.1 5.34E-03 227289_at hypothetical protein LOC144997 PCDH17 3.1 1.66E-04 217995_at sulfide quinone reductase-like (yeast) SQRDL 3.1 1.26E-03 218273_s_at protein phosphatase 2C, magnesium-dependent, catalytic subunit PPM2C 3.0 3.94E-03 1560071_a_at thioesterase superfamily member 2 THEM2 3.0 6.15E-04 242814_at serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 9 SERPINB9 3.0 2.04E-02 214715_x_at zinc finger protein 160 ZNF160 3.0 4.40E-02 1552794_a_at zinc finger protein 547 ZNF547 2.9 7.52E-03 209706_at NK3 transcription factor related, locus 1 (Drosophila) NKX3-1 2.9 3.16E-03 200800_s_at heat shock 70kDa protein 1B HSPA1A 2.9 4.51E-02 207375_s_at interleukin 15 receptor, alpha IL15RA 2.9 5.03E-06 220987_s_at likely ortholog of rat SNF1/AMP-activated protein kinase SNARK 2.9 2.33E-03 227583_at processing of precursor 4, ribonuclease P/MRP subunit (S. cerevisiae) POP4 2.8 9.89E-03 227558_at chromobox homolog 4 (Pc class homolog, Drosophila) CBX4 2.8 5.65E-04 203889_at secretory granule, neuroendocrine protein 1 (7B2 protein) SGNE1 2.8 4.51E-03 201502_s_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha NFKBIA 2.8 2.08E-04 205535_s_at BH-protocadherin (brain-heart) PCDH7 2.8 8.83E-05 204601_at Nedd4 binding protein 1 N4BP1 2.8 2.75E-03 1562677_at Hyaluronan-mediated motility receptor (RHAMM) 2.8 2.41E-01 213293_s_at tripartite motif-containing 22 TRIM22 2.8 1.33E-02 225344_at coactivator 7 NCOA7 2.8 5.91E-04 38241_at butyrophilin, subfamily 3, member A3 BTN3A3 2.8 2.11E-03 211799_x_at major histocompatibility complex, class I, A HLA-C 2.7 2.94E-02 221816_s_at PHD finger protein 11 PHF11 2.7 4.80E-04 39248_at 3 AQP3 2.7 2.76E-03 209941_at receptor (TNFRSF)-interacting serine-threonine kinase 1 RIPK1 2.7 2.92E-02 Viemann et al. Supplemental data 19

239629_at CASP8 and FADD-like apoptosis regulator CFLAR 2.7 2.77E-02 220334_at regulator of G-protein signalling 17 RGS17 2.7 1.03E-03 212781_at retinoblastoma binding protein 6 RBBP6 2.7 1.00E-02 225291_at polyribonucleotide nucleotidyltransferase 1 PNPT1 2.7 5.68E-04 216526_x_at major histocompatibility complex, class I, C HLA-C 2.7 2.80E-04 208935_s_at lectin, galactoside-binding, soluble, 8 (galectin 8) LGALS8 2.7 9.66E-04 213138_at AT rich interactive domain 5A (MRF1-like) ARID5A 2.7 6.20E-04 205379_at carbonyl reductase 3 CBR3 2.6 1.16E-03 206529_x_at solute carrier family 26, member 4 SLC26A4 2.6 1.59E-02 212067_s_at complement component 1, r subcomponent C1R 2.6 9.13E-03 1552274_at PX domain containing serine/threonine kinase PXK 2.6 1.23E-04 211529_x_at HLA-G histocompatibility antigen, class I, G HLA-G 2.6 1.96E-02 201473_at jun B proto-oncogene JUNB 2.6 3.42E-03 228468_at microtubule associated serine/threonine kinase-like MASTL 2.6 6.62E-03 232794_at Hypothetical protein LOC153682 2.6 5.78E-03 241706_at copine VIII CPNE8 2.6 6.48E-03 210845_s_at plasminogen activator, urokinase receptor PLAUR 2.6 1.35E-04 230519_at Hypothetical protein FLJ30707 FLJ30707 2.6 4.20E-06 1554543_at sperm associated antigen 9 SPAG9 2.6 7.19E-03 201702_s_at protein phosphatase 1, regulatory subunit 10 PPP1R10 2.6 5.30E-03 202702_at tripartite motif-containing 26 TRIM26 2.5 2.65E-04 223647_x_at J-type co- HSC20 CHEK2 2.5 1.41E-03 208296_x_at tumor necrosis factor, alpha-induced protein 8 TNFAIP8 2.5 2.09E-03 203275_at interferon regulatory factor 2 IRF2 2.5 1.24E-02 212660_at PHD finger protein 15 PHF15 2.5 2.07E-03 212427_at KIAA0368 KIAA0368 2.5 5.79E-03 225571_at Leukemia inhibitory factor receptor LIFR 2.5 5.50E-05 209074_s_at TU3A protein TU3A 2.5 7.50E-05 232273_at RNA binding motif protein 21 EEF1G 2.5 1.19E-02 211998_at H3 histone, family 3B (H3.3B) H3F3B 2.5 1.01E-03 201694_s_at early growth response 1 EGR1 2.4 2.48E-02 228948_at EPH receptor A4 EPHA4 2.4 1.13E-02 1553407_at microtubule-actin crosslinking factor 1 2.4 1.95E-01 220351_at chemokine (C-C motif) receptor-like 1 CCRL1 2.4 5.61E-03 239186_at hypothetical protein MGC39372 MGC39372 2.4 5.66E-04 Viemann et al. Supplemental data 20

222870_s_at UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 B3GNT1 2.4 2.07E-04 209946_at vascular endothelial growth factor C VEGFC 2.4 9.80E-04 238706_at PAP associated domain containing 4 PAPD4 2.3 3.98E-03 203378_at pre-mRNA cleavage complex II protein Pcf11 PCF11 2.3 4.50E-03 242377_x_at THUMP domain containing 3 THUMPD3 2.3 1.88E-02 204422_s_at fibroblast growth factor 2 (basic) FGF2 2.3 7.36E-03 214844_s_at docking protein 5 DOK5 2.3 2.00E-03 237426_at nuclear antigen Sp100 SP100 2.3 4.82E-02 227877_at Similar to annexin II receptor 2.3 1.75E-03 216565_x_at similar to Interferon-induced transmembrane protein 3 (Interferon-inducible protein 1-8U) IFITM3 2.3 4.86E-04 209770_at butyrophilin, subfamily 3, member A1 BTN3A1 2.3 1.54E-03 Transcribed locus, weakly similar to XP_519878.1 similar to ubiquitin-conjugating enzyme E2 variant 230449_x_at 2.3 5.89E-04 1 isoform c; DNA-binding protein [Pan troglodytes] 221875_x_at major histocompatibility complex, class I, F HLA-F 2.3 5.33E-03 228700_at Chromosome X open reading frame 38 MGC39350 2.3 8.08E-04 218928_s_at solute carrier family 37 (glycerol-3-phosphate transporter), member 1 SLC37A1 2.3 4.36E-02 214290_s_at histone 2, H2aa HIST2H2AA 2.3 7.29E-03 205651_x_at Rap guanine nucleotide exchange factor (GEF) 4 RAPGEF4 2.3 2.05E-02 227014_at similar to aspartate beta hydroxylase (ASPH) LOC57168 2.2 1.16E-04 226814_at a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 9 ADAMTS9 2.2 1.42E-02 212274_at lipin 1 LPIN1 2.2 3.25E-02 217436_x_at CDNA clone MGC:71446 IMAGE:5420082, complete cds 2.2 2.08E-02 225222_at hippocampus abundant transcript 1 HIAT1 2.2 1.18E-03 205467_at caspase 10, apoptosis-related cysteine protease CASP10 2.2 3.30E-03 1563014_at Ribosomal protein S15 RPS15 2.2 1.50E-02 200867_at zinc finger protein 313 ZNF313 2.2 2.68E-03 236170_x_at hypothetical protein FLJ22313 FLJ22313 2.2 2.39E-02 208370_s_at Down syndrome critical region gene 1 DSCR1 2.2 7.30E-04 210138_at regulator of G-protein signalling 20 RGS20 2.2 1.55E-02 210145_at phospholipase A2, group IVA (cytosolic, calcium-dependent) PLA2G4A 2.2 2.97E-03 201744_s_at lumican LUM 2.2 2.80E-02 209505_at Nuclear receptor subfamily 2, group F, member 1 NR2F1 2.2 2.41E-02 229720_at CDNA clone IMAGE:5756190, partial cds 2.2 9.86E-04 235023_at Vacuolar protein sorting 13C (yeast) VPS13C 2.2 1.76E-02 209124_at myeloid differentiation primary response gene (88) MYD88 2.2 5.56E-05 Viemann et al. Supplemental data 21

1560792_at Immediate early response 5 IER5 2.2 1.80E-02 224648_at vasculin DKFZp761C169 2.2 3.88E-05 229123_at Zinc finger protein 224 ZNF224 2.2 1.73E-03 213004_at angiopoietin-like 2 ANGPTL2 2.2 1.67E-03 208912_s_at 2',3'-cyclic nucleotide 3' phosphodiesterase CNP 2.2 1.22E-02 230031_at heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) HSPA5 2.2 5.31E-03 235593_at Zinc finger homeobox 1b ZFHX1B 2.2 3.84E-03 227262_at hyaluronan and proteoglycan link protein 3 HAPLN3 2.2 3.64E-03 235084_x_at Tripartite motif-containing 38 TRIM38 2.2 1.98E-02 230511_at cAMP responsive element modulator CREM 2.2 3.03E-02 225706_at glucocorticoid induced transcript 1 GLCCI1 2.2 2.53E-02 200799_at heat shock 70kDa protein 1A HSPA1A 2.1 1.30E-03 235587_at hypothetical protein LOC202781 2.1 2.39E-02 242450_at RGM domain family, member B RGMB 2.1 3.14E-02 235539_at Nuclear mitotic apparatus protein 1 NUMA1 2.1 2.76E-02 220364_at hypothetical protein FLJ11235 FLJ11235 2.1 3.76E-02 228071_at GTPase, IMAP family member 7 hIAN7 2.1 6.18E-03 202948_at interleukin 1 receptor, type I IL1R1 2.1 6.84E-03 228835_at CDNA FLJ33090 fis, clone TRACH2000559 2.1 6.20E-03 240407_at Homo sapiens, clone IMAGE:5171705, mRNA 2.1 1.48E-02 Transcribed sequence with weak similarity to protein ref:NP_060312.1 (H.sapiens) hypothetical 217713_x_at 2.1 4.38E-02 protein FLJ20489 [Homo sapiens] 213261_at lupus brain antigen 1 KIAA0342 2.1 2.37E-02 227044_at Full-length cDNA clone CS0DI009YA14 of Placenta Cot 25-normalized of Homo sapiens (human) 2.1 5.89E-03 200880_at DnaJ (Hsp40) homolog, subfamily A, member 1 DNAJA1 2.1 2.08E-04 225767_at hypothetical protein LOC284801 2.1 4.49E-03 202659_at proteasome (prosome, macropain) subunit, beta type, 10 PSMB10 2.1 1.24E-02 238757_at Dbf4-related factor 1 DRF1 2.1 3.34E-02 202081_at immediate early response 2 IER2 2.1 9.34E-03 203765_at grancalcin, EF-hand calcium binding protein GCA 2.1 8.73E-03 232024_at GTPase, IMAP family member 2 HIMAP2 2.1 3.44E-03 1554026_a_at myosin X MYO10 2.1 2.27E-02 235086_at Thrombospondin 1 THBS1 2.1 1.67E-02 236449_at Cystatin B (stefin B) 2.1 5.81E-04 209822_s_at very low density lipoprotein receptor VLDLR 2.1 4.49E-02 Viemann et al. Supplemental data 22

220577_at very large inducible GTPase 1 FLJ13373 2.1 2.01E-02 235174_s_at CDNA clone IMAGE:5286843, partial cds 2.0 1.42E-04 231906_at homeo box D8 HOXD8 2.0 5.97E-04 200904_at major histocompatibility complex, class I, E HLA-E 2.0 5.41E-03 214994_at apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F APOBEC3G 2.0 1.39E-02 226275_at MAX dimerization protein 1 MAD 2.0 3.84E-03 227195_at zinc finger protein 503 ZNF503 2.0 7.74E-03 225847_at KIAA1363 protein KIAA1363 2.0 1.21E-02 a FC and p-value were computed from 3 independent experiments determining gene expression changes in 5h H5N1-infected HUVEC by microarray analysis (U133Plus2 microarray, Affymetrix; see Material and Methods).

Table IV Commonly PR8-, FPV- and H5N1-induced genes in HUVEC Affymetrix Description Gene Symbol FCa p-valuea ID 202869_at 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 35.5 7.61E-06 204972_at 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 110.3 4.73E-03 205660_at 2'-5'-oligoadenylate synthetase-like OASL 873.9 4.10E-04 230300_at CDNA FLJ42315 fis, clone TRACH2019661 6.3 1.68E-05 1405_i_at chemokine (C-C motif) ligand 5 CCL5 2281.0 3.31E-04 204533_at chemokine (C-X-C motif) ligand 10 CXCL10 3348.3 3.87E-04 211122_s_at chemokine (C-X-C motif) ligand 11 CXCL11 314.8 1.11E-04 203915_at chemokine (C-X-C motif) ligand 9 CXCL9 705.2 3.07E-04 243271_at Chromosome 7 open reading frame 6 12.4 2.26E-04 242389_at Cisplatin resistance-associated overexpressed protein LUC7A 3.7 2.37E-03 218943_s_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 20.9 8.81E-06 225415_at deltex 3-like (Drosophila) BBAP 9.4 4.22E-06 239979_at Epithelial stromal interaction 1 (breast) 136.9 1.45E-03 221766_s_at family with sequence similarity 46, member A C6orf37 95.1 2.36E-04 231577_s_at guanylate binding protein 1, interferon-inducible, 67kDa GBP1 22.9 5.98E-05 219863_at hect domain and RLD 5 HERC5 227.8 1.38E-07 228439_at hypothetical protein BC012330 MGC20410 40.5 5.61E-05 Viemann et al. Supplemental data 23

226702_at hypothetical protein LOC129607 LOC129607 141.3 7.09E-08 214022_s_at interferon induced transmembrane protein 1 (9-27) IFITM1 23.7 1.95E-06 219209_at interferon induced with helicase C domain 1 IFIH1 75.0 5.58E-07 202531_at interferon regulatory factor 1 IRF1 7.2 8.39E-04 208436_s_at interferon regulatory factor 7 IRF7 12.3 3.42E-05 204415_at interferon, alpha-inducible protein (clone IFI-6-16) G1P3 22.5 9.54E-05 208173_at interferon, beta 1, fibroblast IFNB1 1412.6 1.92E-07 209417_s_at interferon-induced protein 35 IFI35 11.1 1.30E-05 214453_s_at interferon-induced protein 44 IFI44 24.9 1.53E-07 204439_at interferon-induced protein 44-like C1orf29 84.9 3.03E-06 203153_at interferon-induced protein with tetratricopeptide repeats 1 IFIT1 6607.3 4.51E-05 226757_at interferon-induced protein with tetratricopeptide repeats 2 IFIT2 477.1 3.77E-07 229450_at interferon-induced protein with tetratricopeptide repeats 3 IFIT4 264.4 7.00E-07 203596_s_at interferon-induced protein with tetratricopeptide repeats 5 IFIT5 11.3 6.95E-04 203236_s_at lectin, galactoside-binding, soluble, 9 (galectin 9) LGALS9 6.7 1.76E-03 202086_at myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 412.3 1.09E-04 228230_at peroxisomal proliferator-activated receptor A interacting complex 285 PRIC285 21.9 4.95E-05 223220_s_at poly (ADP-ribose) polymerase family, member 9 BAL 10.0 1.41E-04 1555852_at proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional protease 7) PSMB8 8.5 4.77E-04 235061_at protein phosphatase 1K (PP2C domain containing) DKFZp761G058 7.9 3.46E-04 228967_at Putative translation initiation factor SUI1 3.2 1.24E-03 242625_at radical S-adenosyl methionine domain containing 2 cig5 582.8 2.76E-05 208392_x_at SP110 nuclear body protein SP110 22.9 6.28E-03 228531_at sterile alpha motif domain containing 9 C7orf5 19.5 1.79E-05 230383_x_at Transcribed locus 13.4 2.67E-04 235157_at Transcribed sequences 16.7 2.68E-03 202687_s_at tumor necrosis factor (ligand) superfamily, member 10 TNFSF10 14.6 4.45E-04 201649_at ubiquitin-conjugating enzyme E2L 6 UBE2L6 8.3 5.01E-05 228617_at XIAP associated factor-1 HSXIAPAF1 15.7 8.69E-06 1565913_at Zinc finger CCCH type, antiviral 1 71.0 9.08E-03 a FC and p-value were computed from 3 independent experiments determining gene expression changes in 5h PR8-, FPV- or H5N1-infected HUVEC by microarray analysis (U133Plus2 microarray, Affymetrix; see Material and Methods).

Viemann et al. Supplemental data 24

Table V Specifically H5N1-induced genes in HUVEC

Description Gene Symbol BH3 interacting domain death agonist BID CDNA clone IMAGE:4514712, partial cds PHLDA1 CDNA FLJ12885 fis, clone NT2RP2003988 chemokine (C-C motif) ligand 7 CCL7 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCL1 chemokine (C-X-C motif) ligand 5 CXCL5 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) CXCL6 chromosome 3 open reading frame 6 C3orf6 Cleavage and polyadenylation specific factor 6, 68kDa CPSF6 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 DDX5 Family with sequence similarity 49, member B Glycosyltransferase AD-017 AD-017 Homo sapiens, clone IMAGE:4797099, mRNA HSPB (heat shock 27kDa) associated protein 1 HSPBAP1 Hypothetical protein DKFZp313G1735 DKFZp313G1735 hypothetical protein LOC283012 LOC283012 hypothetical protein LOC284390 LOC284390 interferon, alpha-inducible protein (clone IFI-15K) (ISG15) G1P2 interleukin 18 receptor accessory protein IL18RAP interleukin 8 IL8 KIAA0146 protein KIAA0146 KIAA1272 protein KIAA1272 Mitochondrial tumor suppressor 1 MTUS1 MRNA; cDNA DKFZp434M054 (from clone DKFZp434M054) Nedd4 family interacting protein 2 NEDD4 Nuclear factor I/X (CCAAT-binding transcription factor) NFIX nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 NFATC4 nudix (nucleoside diphosphate linked moiety X)-type motif 4 NUDT4 Sarcoma antigen NY-SAR-79 mRNA, partial cds CCDC73 Suppression of tumorigenicity 7 like ST7L associated protein 1 NCOA3 Transcribed locus, strongly similar to NP_001055.1 transketolase Transcribed sequence with weak similarity to protein sp:P39192 (H.sapiens) Viemann et al. Supplemental data 25

Table VI Comparison of microarray and qRT-PCR data with respect to NF-κB dependence of influenza-induced genes

Array_NF-κB- qRT- qRT-PCR_ NF- Array_ NF-κB- qRT- qRT-PCR_NF- Array_NF-κB- qRT- qRT-PCR_NF- Array_FC Array_FC Array_FC dependence PCR_FC by κB-dependence dependence PCR_FC κB-dependence dependence PCR_FC κB-dependence by PR8a by FPVa by H5N1a PR8 (fold)b PR8c PR8 (fold)c FPV (fold)b by FPVc FPV (fold)c H5N1 (fold)b by H5N1c H5N1 (fold)c H5N1-specific genes IL8 2.5 NDd 0.2 3.8 NSe ND 0.2 ND 4.1 1.8 3.7 2.2 CCL2 4.5 3.2 0.0 1.9 NS ND 0.4 ND 8.8 4.0 9.3 4.7 CCL7 NS ND 0.8 ND NS ND 1.7 1.2 3.5 1.5 17.2 1.7 CXCL5 NS ND 0.6 ND NS ND 1.1 ND 4.5 ND 5.7 1.8 CXCL6 NS ND 0.8 ND NS ND 1.0 ND 2.7 ND 3.5 1.3 Generally influenza-inducible genes / NF-κB-dependent predominantly in H5N1 IRF7 12.3 ND 15.1 1.3 2.7 ND 6.6 2.6 3.6 2.1 4.6 2.2 SELE 3.8 ND 2.9 0.4 NS ND 2.3 1.8 8.4 4.0 8.9 3.3 IFNB1 1412.6 4.7 86.4 0.2 40.5 50.9 42.7 0.6 165.7 156.0 12.9 2.0 CXCL9 705.2 6.2 259.7 0.2 9.8 5.3 42.0 0.5 15.7 45.6 54.7 4.8 IFIH1 17.1 2.0 50.1 0.3 6.1 3.5 21.7 1.4 5.5 15.4 9.2 2.4 Generally influenza-inducible and NF-κB-dependent genes CCL5 2281.0 5.7 1113.8 5.7 35.6 3.2 7.5 3.3 56.8 4.3 761.8 2.0 CXCL10 3348.3 3.9 5931.3 5.7 74.6 21.0 1889.9 24.6 191.0 11.9 2513.6 30.9 CXCL11 314.8 ND 773.9 3.4 17.3 8.6 428.9 12.1 71.1 5.4 109.5 4.8 TNFSF10 14.6 ND 1.7 2.0 2.6 3.5 0.9 3.3 5.4 2.2 4.2 3.4 CXCL2 5.5 4.1 5.3 6.8 NS 6.5 3.3 8.0 16.5 3.0 9.0 2.1 TLR3 16.2 ND 3.0 3.0 NS 4.7 6.5 2.5 3.2 2.7 7.5 4.0 VCAM 7.5 3.6 6.1 6.8 NS 6.1 4.8 10.2 8.0 3.3 7.6 3.5 ICAM 5.6 ND 3.0 5.5 NS ND 1.8 2.1 3.8 ND 3.4 3.2 a The fold changes (FC) of gene expression were established by microarray analysis of 3 independent experiments of HUVECs infected for 5h with FPV, PR8 or H5N1. b NF-κB-dependence was quantified by determining the fold inhibition of virus-induced gene expression in HUVECs with blocked NF-κB pathway (IKK2kd- transfected HUVEC) compared to empty-vector transfected HUVEC. c FC and NF-κB-dependence determined by qRT-PCR were derived from 5 independent experiments using the same experimental settings for each influenza strain as in microarray analyses (see Material and Methods). d ND = no dependence on NF-κB or not determined due to lacking inducibility e NS = no significant FC

Viemann et al. Supplemental data 26

Table VII Transcription factor profile of PR8-induced genes in HUVECs

Transcription factor binding site p-valuea STAT5A 2.09E-19 ISRE 7.91E-16 STAT5B 1.20E-14 IRF 1.22E-14 ICSBP 6.41E-12 IRF1 3.23E-11 NF-kappaB 3.03E-09 GATA1 5.36E-09 IPF1 7.91E-09 IRF7 1.46E-08 Cart-1 3.62E-08 TEF 2.10E-07 AP-1 4.05E-07 Octamer binding factor 1 3.54E-06 cell division control protein 5 3.97E-06 HNF-4alpha 4.46E-06 58 KDA repressor protein 9.24E-06 a p-value indicates the significance of overrepresentation of binding sites in promoters of H5N1- regulated genes compared to unregulated genes computed by ROVER analysis (see Methods).

Table VIII Transcription factor profile of specifically PR8-induced genes in HUVECs

compared to H5N1 and FPV

Transcription factor binding site p-valuea STAT5A 8.44E-18 STAT5B 1.14E-13 NF-kappaB 5.83E-07 IPF1 7.62E-07 ISRE 2.22E-04 myoblast determining factor 1.76E-03 Octamer 5.14E-03 IRF 5.33E-03 tumor suppressor 8.05E-03 a p-value indicates the significance of overrepresentation of binding sites in promoters of H5N1- regulated genes compared to unregulated genes computed by ROVER analysis (see Methods). Viemann et al. Supplemental data 27

Table IX Transcription factor profile of FPV-induced genes in HUVECs

Transcription factor binding site p-valuea IRF 5.86E-14 ISRE 2.29E-10 ICSBP 4.67E-10 IRF7 6.00E-09 IRF1 3.18E-07 a p-value indicates the significance of overrepresentation of binding sites in promoters of H5N1- regulated genes compared to unregulated genes computed by ROVER analysis (see Methods).

Table X Transcription factor profile of specifically FPV-induced genes in HUVECs

compared to H5N1 and PR8

Transcription factor binding site (BS) p-valuea NF-kappaB (p50) 2.77E-05 a p-value indicates the significance of overrepresentation of binding sites in promoters of H5N1- regulated genes compared to unregulated genes computed by ROVER analysis (see Methods).

Table XI Primers used for quantitative real-time RT-PCR

Gene Forward Reverse RPL9 AGGTATGCTGCCCCACAAAAC TGTAGGCTTCAGACGCACGAC IL8 ACCACCGGAAGGAACCATTC TTCACACAGAGCTGCAGAAATCA CCL2 TCGCCTCCAGCATGAAAGTC TTGCATCTGGCTGAGCGAG SELE TGGTAGCTGGACTTTCTGCTGC TGCTTTCCGTAAGCATTTCCG IFNB1 TCTGGCACAACAGGTAGTAGGC GAGAAGCACAACAGGAGAGCAA CXCL9 CCACCCACCAGTTGAATTTCAT TTCCAGGCAGCTGTTGTGAGT IFIH1 GCTGGACTACCTGACCTTTCTGC AACTGCCTGCATGTTCCCG IRF7 CCACGCTATACCATCTACCTGG TGTCGTCATAGAGGCTGTTGG CXCL11 CAGAATTCCACTGCCCAAAGG GTAAACTCCGATGGTAACCAGCC TNFSF10A GTCTCTCTGTGTGGCTGTAACTTACG AAACAAGCAATGCCACTTTTGG CXCL2 ACATCCAAAGTGTGAAGGTGAAGTC AAGCTTTCTGCCCATTCTTGAGT TLR3 CCAAGCCTTCAACGACTGATG TACGAAGAGGCTGGAATGGTG VCAM1 ACATGGAATTCGAACCCAAACA GGCTGACCAAGACGGTTGTATC CCL5 GCCTGTTTCTGCTTGCTCTTGT TGCTCGTCGTGGTCAGAATCT IRF3 AGGCCACTGGTGCATATGTTC CCTCTGCTAAACGCAACCCTT Viemann et al. Supplemental data 28

GATA6 GCTGACAGAACGTGATTCTCGT TCCTAGTCCTGGCTTCTGGAA HMGA1 TCCATTCTTCGACATCCGTCAT CCCCTCTTCCCCACAAAGAGTA NFATC4 ACAACATGGCGGCCAACATT TGTGTTTTTGCGCCCGATG

Table XII Nucleotide sequence of used siRNAs

siRNA nucleotide sequence negative control 5’-UUCUCCGAACGUGUCACGU-3’ IRF3 5’-GGAAGACAUUCUGGAUGAG-3’ IRF7 5’-CGAGCUGCACGUUCCUAUA -3’ GATA6 5’-AGACUUGCUCUGGUAAUAG-3’ HMGA1 5’-GGACAAGGCUAACAUCCCA-3’ NFATC4 5’-CCGUAGGUACUGAGUACAA -3’