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Supplementary Figure 1: Datamining tools used for gene search. Several datamining tools, such as AliBaba (Top), Topic Map from HighWire (Bottom), were used to pool the host genes significantly associated to Kaposi’s Sarcoma Associated Herpes Virus (KSHV) infection. The found genes were further validated by comparing them to the microarray data (see text for detail).

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Supplementary Figure 1: Datamining tools used for gene search. Several datamining tools, such as AliBaba (Top), Topic Map from HighWire (Bottom), were used to pool the host genes significantly associated to Kaposi’s Sarcoma Associated Herpes Virus (KSHV) infection. The found genes were further validated by comparing them to the microarray data (see text for detail).

Supplementary Figure 3: Methodology of connecting viral ORF homologs. The viral ORF homologs were searched for their interaction partner using STRING9.0 server. Because the viral system is packed, small world property applied for the system. Hence a dense mesh-like network was found for each ORFs. Two modules in the Right figure connected here represent two ORF homologs. The apparently messy mesh was later cleared by joining the ORF homologs with their in-between interactors (Left).

Supplementary Figure 2: The host network. The merged network for the host proteins are visualized here. The nodes stand for proteins whereas the edges in blue stand for interaction between them. Node analysis by NetworkAnalyzer plugin revealed a total of 680 host protein was merged in the document in 4 different networks. Only one network has been represented here for simplicity. The bigger nodes stand for highly connected genes whereas the shorter ones are lowly connected. The red nodes stand for higher values for average shortest path of the nodes.

Supplementary Figure 4: 12 ORF homologues were brought into 7 different networks. The figure here shows pairwise merged networks. The ORFs for which no human homologues were found were brought to merge with ones for which human homologues were found.

Supplementary Figure 5: ORF homologues were merged together. The figure shows that 4 ORFs homologues were merged together in organism of ORF11. The network was further merged with the host network later on.

Supplementary Figure 6: Viral ORFs hijacks host cell division mechanisms. Host cell division proteins are represented in this figure. This faction of the whole network shows that several cellular proteins, such as MAP3K4, PCNA, CDK1, IL6R interacts with ORF10 (node0_929), ORF49, ORF35 and ORF55. Here square represents the edges and their evidence sources and circles represent nodes and their names.

A B C

D E F

G H I

Supplementary Figure 7: Parameters for the integrated network. The parameters revealed the robustness of the network. 733 proteins accommodated in the network. (A) Average clustering coefficient vs number of neighbors, (B) Betweenness centrality vs number of neighbors, (C) Closeness centrality vs number of neighbors, (D) Average neighborhood connectivity vs number of neighbors, (E) Number of nodes vs degree, (F) Frequency vs number of shared neighbors, (G) Frequency vs path length, (H) Number of nodes vs stress centrality and (I) Topological coefficient vs number of neighbors has been plotted. Clustering coefficient of the network is 0.62. Network diameter was 14 units. Network centralization valued 0.094. Shortest path covered 86% of the proteins. Average number of neighbors for the nodes was 10.35. Network heterogeneity was 0.868. The red line fits the line whereas the blue line shows the power law distribution.

Supplementary Figure 8: Interfering RNA structure. Predicted structure of the 3rd interfering RNA has been represented. A stable interfering RNA is supposed to contain 6-7 mismatches with 2-3 bulges. The structure fits well within the pre-requisites.

Supplementary Figure 9: The perplexity of the capsid proteins. Capsid proteins show both DNA binding regions and membrane binding potential. (Right) The structure shows extracellular localization in Gene Ontology and also shows DNA binding region (marked with # sign) (Left) DNA marked in golden circle (shown through the axis) binds to the protein in a groove which according to KyteDolittle plot is a hydrophobic one.

Supplementary Table 1: Summary of the GEO2R pooled microarray expression data for selected genes

ID_Ref Gene symbol

Gene title and Function Cluster no.

2008_s_at

MDM2 p53 binding protein; overexpression of this gene can result in excessive inactivation of tumor protein p53, diminishing its tumor suppressor function.

6

31962_at

RPL37A Ribosomal protein L37a; responsible for viral translation and translocation of membrane bound protein

6

33162_at INSR Insulin receptor 537154_at PCDH17 Protocadherin 17 732902_at SCTR Secretin receptor 634337_s_at MTF2 Metal response element binding transcription factor 2 437289_at CDH8 Cadherin 8 733237_at DDX46 DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 439140_at DHX29 DEAH box polypeptide 29 2

34593_g_atRPS17 Ribosomal protein S17; responsible for viral translation

and translocation of membrane bound protein6

38904_at SRC v-src Sarcoma 61533_at RAD50 DNA repair protein 439794_at USP8 Ubiquitin specific peptidase 8 434813_at E1F1AX Eukaryotic transcription factor E1 2847_at EIF5A Eukaryotic transcription factor E5 6

1607_atCDKN2B Cyclin-dependent kinase inhibitor 2B (p15, inhibits

CDK4)7

34326_at COPB1 Coatomer protein complex, subunit beta 1 31456_s_at IFI16 Interferon inducible protein 16 4

33983_at

CD101 Plays a role as inhibitor of T-cells proliferation induced by CD3. inhibits expression of IL2RA on activated T-cells and secretion of IL2. Inhibits tyrosine kinases that are required for IL2 production and cellular proliferation.

6

35842_atIL6ST Interleukin 6 signal transducer (gp130, oncostatin M

receptor)5

40637_at HSPA8 Heat Shock Protein 1

38462_atNDUFA5 NADH dehydrogenase (ubiquinone) 1 alpha

subcomplex, 5, 13kDa4

1101_atAP881 Amyloid beta (A4) precursor protein-binding, family B,

member 1 (Fe65)7

37625_at IRF4 Interferon Regulatory Factor 4 639742_at TANK TRAF family member-associated NFKB activator 4458_at CCR6 Chemokine Receptor 6 6

35811_at

RNF13 Ring finger protein 13, contains RING zinc finger, a motif known to be involved in protein-protein interactions.

3

37476_at RNF216L Ring finger protein 216-like 5

640_at RNF185 Ring finger protein 185-like 731880_at UBXN8 UBX domain protein 539007_at MMP2 Matrix metalloprotease 2 61144_at FGFR2 Fibroblast growth factor receptor 2 134751_at ZBTB1 Zinc finger and BTB domain containing 1 4

40217_s_atCDS1 CDP-diacylglycerol synthase (phosphatidate

cytidylyltransferase) 17

37617_at FAM190B Family with sequence similarity 190, member B 3

35570_atLST-3TM12

Solute carrier organic anion transporter family, member 1B7

6

34759_at SLC30A1 Solute carrier family 30 (zinc transporter), member 1 337440_at SLC30A4 Solute carrier family 30 (zinc transporter), member 4 635854_at SLC9A7 Solute carrier family 30 (zinc transporter), member 7 5

40248_atSLC9A7 Solute carrier family 9 (sodium/hydrogen exchanger),

member 76

39777_at MYCBP2 MYC binding protein 2 41471_at MYB v-myb myeloblastosis viral oncogene homolog 636323_at GABRA1 gamma-aminobutyric acid (GABA) A receptor, alpha 1 634775_at TSPAN1 Tetraspanin; located in Lysosome membrane protein 741557_at SKIV2L2 Superkiller viralicidic activity 2-like 2 233910_at PTPRD Protein tyrosine phosphatase, receptor type, D 536449_s_at PYY Peptide YY 639714_at SH3BGRL SH3 domain binding glutamic acid-rich protein like 441523_at RAB32 RAB32, member RAS oncogene family 740909_at RABL3 RAB, member of RAS oncogene family-like 3 536575_at RGS1 Regulator of G-protein signaling 1 140893_at SUCLA2 Succinate-CoA ligase, ADP-forming, beta subunit 2

34106_atGNA12 Guanine nucleotide binding protein (G protein) alpha

126

1139_atGNA13 Guanine nucleotide binding protein (G protein) alpha

136

39355_at ABCE1 ATP-binding cassette, sub-family E (OABP), member 1 437962_r_at STXBP3 Syntaxin binding protein 3 234753_at VAMP7 Vesicle-associated membrane protein 7 2339_at CAV2 Caveolin 2 734497_at TAF1B TATA box binding protein (TBP)-associated factor 4

40797_atADAM10 ADAM metallopeptidase domain 10, important for cell

differentiation2

34761_r_atADAM9 ADAM metallopeptidase domain 9, important for cell

differentiation3

1227_g_atADAM17 ADAM metallopeptidase domain 17, important for cell

differentiation2

35126_at WASF2 WAS protein family, member 2 641784_at SFRS18 Splicing factor, arginine/serine-rich 18 2

2026_atRUNX1 Runt-related transcription factor 1, needed for stemm

cell like property6

Supplementary Table 2: List of the viral homologues with their STRING Id.

ORF Homologue protein STRING Id

Protein title Organism String identifier

BLAST2SEQ score

ORF 10

Acel_1402 deoxyuridine 5'-triphosphate nucleotidohydrolase Dut

Acidothermus cellulolyticus

Ace Identity: 35%Length: 31 aa

ORF 11

ABSDF1895 ABC transporter membrane protein

Acinetobacter baumannii SDF

ABSDF Identity: 31%Length: 26 aa

ORF 23

Ent638_4000 UDP-N-acetylglucosamine 2-epimerase

Enterobacter sp. 638 Ent Identity: 26%Length: 102 aa

ORF 27

ATG1 DEHA2D12452p; Serine/threonine protein kinase probably involved in the cytoplasm to vacuole transport(Cvt) and in autophagy, where it may be required for the formation of autophagosomes

Debaryomyces hansenii

B5,Q6BH Identity: 30%Length: 53 aa

ORF 30

vdh vanillin-NAD+ oxidoreductase

Azotobacter vinelandii

Avin Identity: 44%Length: 33 aa

ORF 31

CBU_0621 NAD-dependent oxidoreductase

Coxiella burnetii Q212

CBU Identity: 32%Length: 72 aa

ORF 33

FP1864 glycosyl transferase, group 1 family protein

Flavobacterium psychrophilum

FP Identity: 30%Length: 46 aa

ORF 34

PGN_0933 probable transcriptional regulator

Porphyromonas gingivalis 33277

PGN Identity: 80%Length: 14 aa*The region was predicted as conserved binding site in I-Tasser and Motif Ffinder analysis

ORF 35

NT05HA_0957 DNA internalization-related competence protein ComEC/Rec2

Aggregatibacter aphrophilus

NT05HA Identity: 20%Length: 59 aa

ORF 49

yhjJ predicted zinc-dependent secreted peptidase

Bacillus licheniformis 14580

BL Identity: 30%Length: 57 aa

ORF 52

BT_1888 LuxR family transcriptional regulator

Bacteroides thetaiotaomicron

BT Identity: 60%Length: 16 aa*The region was predicted as conserved binding site in I-Tasser

and Motif Ffinderanalysis

ORF 55

ABAYE1243 putative Holliday-junction resolvase

Acinetobacter baumannii AYE

ABAYE Identity: 29%Length: 29 aa

Supplementary Table 3: Control experiment done for selecting bacterial homologues using PSIPRED for interactome study

Virus gene Bacteria gene References BLAST2SEQ parameter

Mimivirus nucleoside diphosphate

Columba livia nucleoside diphosphate

[1, 2] Identity: 36%Length: 130 aa

Positive control

Paramecium bursaria Chlorella virus SH6A dUTPase

Mycobacterium bovis AF2122/97 DUT protein

[3, 4] Identity: 35%Length: 102 aa

Myxomavirus TNFR

Sorangium cellulosum hypothetical TNFR

[5, 6] Identity: 28%Length: 54 aa

Mammary tumor virus superantigen

Staphylococcus Aureus superantigen

[7-9] Identity: 22%Length: 55 aa

Vaccinia virus Tyrosin Phosphatase

Yersinia pseudotuberculosis yopH

[10] Identity: 16%Length: 56 aa

Negative controlAdenovirus BCL-2

proteinCoraliomargarita akajimensis hypothetical Bcl-2 like

No significant similarity

Synechococcus phage peptidase

Nocardia brasiliensis peptidase

No similarity found

Vaccinia virus oxidoreductase

Mesorhizobium loti oxidoreductase

Identity: 31%Length: 39 aa

Ostreococcus tauri virus LuxR family protein

Microcystis aeruginosa LuxR protein

Identity: 46%Length:13 aa

Cafeteria roenbergensis virus family ATPase

Halanaerobium praevalens ATPase

Identity: 50%Length: 10 aa

Supplementary Table 4: Gene target name and corresponding RNAi against them.

Gene name Interfering IDPTPRS 1aFGF 2aSTX 2bRAB 2bSKI 2bHSP 2bADAM 3aTBP 4bUSP 4bCadherin 5a*a= sense strand, b= antisense strandSee supplementary file Interfering RNA for detail

Supplementary Table 5: Host proteins that interact with the viral ORFs.

Host protein name Function of the host proteinsADAMTS5 Metalloprotease type enzyme responsible for cleaving proteoglycan.SNX9 Sorting nexin 9; involved in intracellular traffickingACAN A member of the aggrecan proteoglycan family. The encoded protein is an

integral part of the extracellular matrix GAK Cyclin G associated kinase; Associates with cyclin G and CDK5. Act as an

auxilin homolog that is involved in the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

DnaJ ATP hydrolyzing chaperone protein upregulated in the times of extensive metabolism within the cell

CLTC Clathrin heavy chain; Clathrin is a major protein component of the cytoplasmic face of intracellular organelles, called coated vesicles and coated pits. These specialized organelles are involved in the intracellular trafficking of receptors and endocytosis of a variety of macromolecules

RAD51 Participates in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. BRCA1/BRCA2-containing complex is one of the most known RAD51 protein

HSP90AA1 Molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved for instance in cell cycle control and signal transduction

RSAD1 Radical S-adenosyl methionine domain containing 1; May be involved in porphyrin cofactor biosynthesis

EXOSC10 Putative catalytic component of the RNA exosome complex which has 3'->5' exoribonuclease activity and participates in a multitude of cellular RNA processing and degradation events

Dut Deoxyuridine triphosphatase; PCNA This gene encodes an essential enzyme of nucleotide metabolism. The encoded

protein forms a ubiquitous, homotetrameric enzyme that hydrolyzes dUTP to dUMP and pyrophosphate. This reaction serves two cellular purposes: providing a precursor (dUMP) for the synthesis of thymine nucleotides needed for DNA replication, and limiting intracellular pools f dUTP. Elevated levels of dUTP lead to increased incorporation of uracil into DNA, which induces extensive excision repair mediated by uracil glycosylase. This repair process, resulting in the removal and reincorporation of dUTP, is self-defeating and leads to DNA fragmentation and cell death

DTYMK Deoxythymidylate kinase (thymidylate kinase); Catalyzes the conversion of dTMP to dTDP

WRNIP1 Werner helicase interacting protein 1; Functions as a modulator for initiation or reinitiation events during DNA polymerase delta-mediated DNA synthesis. Has an intrinsic ATPase activity that functions as a sensor of DNA damage or of arrested replication forks and regulates the extent of DNA synthesis

Supplementary Table 6: ORF and their viral homologues

ORF Homologous EBV Protein name

Correlations Conclusion Faction of References

ORF 10 BZLF1 Early lytic gene; BZLF1 induces dUTPase; BZLF1 itself is analogous to bZIP

They may act in the same early lytic pathway; not homologue, may be partners

[11-14]

ORF 52 BLRF2 Same motif (BLRF2) sharing; Sequence homologue [15]ORF 50 (characterized)

Rta Activating human herpesvirus 8 lytic gene expression

Functional homologue

[16, 17]

ORF 50 (characterized)

ZEBRA Activating human herpesvirus 8 lytic gene expression

Functional homologue

[16, 17]

ORF 53 BLRF1 In early phase regulated by Rta. Herpesvirus UL49 motif in both of them;Sequence homologue

[18, 19]

ORF 31 LF2 In latent cycle; dUTPase function Functional Homologue

[20]

Supplementary Table 7: Host proteins that are downregulated by predicted antisense RNA.

Host protein name Function of the host proteinsAngioprotein like Angptl also acts as dual inhibitor of lipoprotein lipase (LPL) and endothelial

lipase (EL), and increases plasma triglyceride and HDL cholesterol in rodentsClaudin Caludin is a tight junction protein. Tight junctions represent one mode of cell-to-

cell adhesion in epithelial or endothelial cell sheets, forming continuous seals around cells and serving as a physical barrier to prevent solutes and water from passing freely through the paracellular space.

Glutathione peroxidase

Glutathione peroxidase functions in the detoxification of hydrogen peroxide, and is one of the most important antioxidant enzymes in humans

Ribonucleases Ribonucleases cuts RNA molecules Heat Shock Proteins (HSP)

HSPs are responsible for protein folding. HSP dysregulation causes cellular stress and adds to cell’s oncogenic poteintial

Solute carrier family member 6 (SLC6)

SLC6 works as an amino acid transporter.

CCL3L3 Chemokine ligand-3 like binds to several chemokine receptors including chemokine binding protein 2 and chemokine (C-C motif) receptor 5 (CCR5). CCR5 is a co-receptor for HIV, and binding of this protein to CCR5 inhibits HIV entry.

IGFBP3 Insulin Growth Factor Binding Protein 3 forms a ternary complex with insulin-like growth factor acid-labile subunit (IGFALS) and either insulin-like growth factor (IGF) I or II. In this form, it circulates in the plasma, prolonging the half-life of IGFs and altering their interaction with cell surface receptors

RAB5C Members of the Rab protein family are small GTPases of the Ras superfamily that are thought to ensure fidelity in the process of docking and/or fusion of vesicles with their correct acceptor compartment

TSKS Testis Specific Serine Kinase Substrate may play a role in testicular physiology, spermatogenesis or spermiogenesis. Expression of the encoded protein is highest in the testis and down-regulated in testicular cancer.

RAN B2-AS1 RAN (ras-related nuclear protein) is a small GTP binding protein belonging to the RAS superfamily that is essential for the translocation of RNA and proteins through the nuclear pore complex.

NFKB1 NF-kappa-B inhibitor family, which contain multiple ankrin repeat domains. Theencoded protein interacts with REL dimers to inhibit NF-kappa-B/REL complexes which are involved in inflammatory responses.

MAF Musculoaponeurotic fibrosarcoma oncogene homolog encoded by this gene is a DNA-binding, leucine zipper-containing transcription factor that acts as a homodimer or as a heterodimer. Depending on the binding site and binding partner, the encoded protein can be a transcriptional activator or repressor. This protein plays a role in the regulation of several cellular processes, including embryonic lens fiber cell development, increased T-cell susceptibility to apoptosis, and chondrocyte terminal differentiation.

GEMIN8 The protein encoded by this gene is part of the SMN complex, which is necessary for spliceosomal snRNP assembly in the cytoplasm and pre-mRNA splicing in the nucleus. The encoded protein binds to both SMN1 and the GEMIN6/GEMIN7

heterodimer, mediating their interaction. This protein is found in nuclear Gemini of Cajal bodies (gems) and in the cytoplasm. Three transcript variants encoding the same protein have been found for this gene.

RBL2 Retinoblastoma Like protein is directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV420H1 and SUV420H2, leading to epigenetic transcriptional repression.

Reference for Supplementary Figures and Table

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3. Morales, M., et al., Genome comparison of a nonpathogenic myxoma virus field strain with its ancestor, the virulent Lausanne strain. J Virol, 2009. 83(5): p. 2397-403.

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7. Geisbrecht, B.V., et al., The crystal structures of EAP domains from Staphylococcus aureus reveal an unexpected homology to bacterial superantigens. J Biol Chem, 2005. 280(17): p. 17243-50.

8. Crouse, C.A. and R.J. Pauley, Molecular cloning and sequencing of the MTV-1 LTR: evidence for a LTR sequence alteration. Virus Res, 1989. 12(2): p. 123-37.

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