Identification of common human host genes involved in pathogenesis of different rotavirus strains: An attempt to recognize probable antiviral targets

Virus Research 169 (2012) 144–153

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Identification of common human host genes involved in pathogenesis of different rotavirus strains: An attempt to recognize probable antiviral targets Parikshit Bagchi a , Satabdi Nandi a , Shiladitya Chattopadhyay a , Rahul Bhowmick a , Umesh Chandra Halder a , Mukti Kant Nayak b , Nobumichi Kobayashi c , Mamta Chawla-Sarkar a,∗ a

Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700010, India Department of Zoology, University of Calcutta, Ballygunge Circular Road, Kolkata 700019, India c Department of Hygeine, Sapporo Medical University, S-1 W-17, Chuo-ku, Sapporo 060-8556, Japan b

a r t i c l e

i n f o

Article history: Received 21 May 2012 Received in revised form 19 July 2012 Accepted 20 July 2012 Available online 28 July 2012 Keywords: Rotavirus strain Host gene expression regulation Microarray Drug target USP18 A20

a b s t r a c t Although two rotavirus vaccines have been licensed and approved by WHO and FDA; other parallel therapeutic strategies are needed to reduce the mortality and morbidity of rotavirus induced diarrhea worldwide. Since rotaviruses utilize the host cell machinery for their replication, study was initiated to identify host proteins which positively regulate rotavirus infection. To overcome the possible variations in host response due to existence of large variety of genotypes and human–animal reassortants, the total gene expression profile of HT29 cells infected with either simian (SA11) or bovine (A5-13) or human (Wa) rotavirus strains was analyzed using genome microarrays. Even though cells infected with human strain revealed some differences compared to the viruses of animal origin, 131 genes were similarly induced by all three strains. Genes involved in innate immune response, stress response, apoptosis and protein metabolism were induced by all viral strains. Results were validated by immunoblotting or RT-PCR. Role of some host genes in rotavirus infection was analyzed by using specific siRNAs. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Rotaviruses (RVs), member of the family Reoviridae, remain the number one cause of severe diarrhea in infants and young children aged <5 years worldwide (Estes and Kapikian, 2007; Kawai et al., 2011). In spite of significant mortality and morbidity, no effective RV-specific antivirals are available to date. At least 27 G and 35 P genotypes of rotavirus A have been described which gives RV a substantial diversity with a possible 132 separate G–P combinations (Mukherjee et al., 2010; Desai and Vaˇızquez, 2010; Matthijnssens et al., 2011). In addition to this, the existence of animal–human reassortant rotaviruses can further increase the complexity of RV strains. RV strains with G-type such as G3 (found commonly in species such as cats, dogs, monkeys pigs, mice, rabbits and horses), G5 (pigs and horses), G6 and G8 (cattle), G9 (pigs and lambs), and G10 (cattle) as well as human–animal reassortants viruses (like G12 RV in human) have been isolated from the human population throughout the world (Desselberger et al., 2001; Mukherjee et al., 2010; Matthijnssens et al., 2010; Rahman et al., 2007; Stupka et al.,

∗ Corresponding author at: Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700010, West Bengal, India. Tel.: +91 33 2353 7470; fax: +91 33 2370 5066. E-mail addresses: [email protected], [email protected] (M. Chawla-Sarkar). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.07.021

2012; Tra My et al., 2011; Iturriza-Gómara et al., 2011; Sharma et al., 2010). Two RV vaccines (Rotateq, Rotarix) have been licensed and approved by WHO and FDA (USA) and some reported protection against RV in developing countries (Madhi et al., 2010; Armah et al., 2012; Zaman et al., 2010; Lewis et al., 2012), but the protective effect of the vaccines in developing world is yet to be fully elucidated (Desai and Vaˇızquez, 2010). In vivo, the virus infects the mature, differentiated enterocyte of the small intestinal epithelium (Bishop et al., 1973; Blutt and Conner, 2007; Estes and Kapikian, 2007) but the majority of our knowledge about the virus replication cycle comes from the studies performed in cell culture, to which rotaviruses have been adapted to grow. Both in tissue culture or in vivo RV utilizes the host cell machinery for its replication; in contrast the host cells activate antiviral innate immunity to combat the viruses (Bagchi et al., 2010; Dutta et al., 2009; Broquet et al., 2011; Rollo et al., 1999). So the identification of cellular genes induced by the virus may help us to identify genes with either pro viral or antiviral function for better understanding the RV induced pathogenesis. Host genes which positively regulate RV replication can be targeted for the future therapeutics. In the last decade, DNA microarray has emerged as a powerful tool to study the global regulation of host gene expression after virus infection (Zhu et al., 1998; Geiss et al., 2000, 2001; Chang and Laimins, 2000; Morgan et al., 2001). Characterization of transcriptional response of human

P. Bagchi et al. / Virus Research 169 (2012) 144–153

intestinal Caco-2 cells following the infection with simian rotavirus strain RRV or comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus has also been reported previously (Cuadras et al., 2002; Aich et al., 2007). However, recent studies have highlighted the differences in host responses such as innate immunity and interferon signaling after infection with different strains of RV (Arnold and Patton, 2011; Sen et al., 2009). To address the question of strain specific differences, we have analyzed the total gene expression profile of human colon epithelial cells (HT29) infected with either simian (SA11) or bovine (A5-13) or human (Wa) RV strains to identify common as well as variable host response genes using microarrays of host cell genome. Our study has highlighted that though subset of activated genes are strain type specific, large number of induced cellular genes are common. It can be hypothesized that genes commonly regulated by both human and animal rotaviruses will probably have an important role in either virus replication process or in regulation of host innate immune response.

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2.4. Gel electrophoresis and immunoblot analyses Whole cell lysates were prepared and immunoblotting was done as per standard protocols (Bagchi et al., 2010). Polyclonal antibodies against rotavirus non structural proteins (NSP-3, NSP-5) were kindly provided by Dr. Koki Taniguchi, Dept. of Virology and Parasitology, Fujita Health University School of Medicine, Japan. Monoclonal mouse antirotavirus VP6 antibody (3C10) used in the study was received from HyTest Ltd., Turku, Finland. Antibodies against A20, USP18, STAT1, Hsp70, RSAD2 and Mx1 were from Santa Cruz Biotechnology, CA. Bound primary antibodies were detected using HRP-conjugated secondary antibodies (Pierce, Rockford, IL) and chemiluminescent substrate (Millipore, Billerica, MA). Blots were reprobed with anti-␤-actin or GAPDH (Santa Cruz Biotechnology, CA) to standardize for protein loading. The immunoblots shown here are representative of three independent experiments. Blots were scanned and quantitated using GelDoc XR system and Quantity One® software version 4.6.3 (BioRad, Hercules, CA). 2.5. Immunofluorescence microscopy

2. Materials and methods 2.1. Cell culture and virus infection In this study we have compared the host gene regulation after infection of human intestinal cell line (HT29) with four different wild type RV strains i.e. SA11 (simian, G3, P2), A5-13 (bovine, G8, P1), Wa (human, G1, P8) and KU (human, G1, P8). Viruses were purified as described previously (Jolly et al., 2000) and were titrated by plaque assay. HT29 cells were selected as a cell culture model for human intestinal cells where the RV infectivity is very good (Rollo et al., 1999). The human intestinal epithelial cells (HT29) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with US certified 10% fetal bovine serum and 1% antibiotic–antimycotic solution (Invitrogen, Carlsbad, CA). For infection, viruses were activated with acetylated trypsin (Invitrogen, Carlsbad, CA) (10 ␮g/ml) at 37 ◦ C for 30 min, diluted as per required multiplicity of infection (moi) and added to the cells for adsorption (45 min) at 37 ◦ C, followed by washing 3× with media to remove unbound virus. Infection was continued in fresh medium with acetylated trypsin (Invitrogen, Carlsbad, CA) (1 ␮g/ml). The time of virus removal was taken as 0 hour post infection (hpi) for all experiments. In this study we have used three wild type RV strains for infection; SA11 (simian), A5-13 (bovine) and Wa (human) at moi 2. Three biological repeats were set up for each infection.

2.2. Plaque assay Monolayer of MA104 cells in six well plates were infected with serial dilutions (102 –108 ) of viral supernatants. After 45 min of adsorption, inoculum was removed and cells were overlaid with 0.7% agar in 1× MEM with 1 ␮g/ml trypsin. After 36–48 hpi second agar overlay (0.7% agar in 1× MEM with 0.1% neutral red) was added and plates were incubated at 37 ◦ C until plaques were visualized. Viral infectivity in plaque forming units/ml was calculated as described previously (Rollo et al., 1999).

2.3. siRNA transfection Control siRNA (Santa Cruz Biotechnology, CA) and siRNA against A20, Ubiquitin Specific Peptidase 18 (USP18) (Santa Cruz Biotechnology, CA) were transfected in HT29 cells by siPORT NeoFX (Ambion, NY) transfection reagent using manufacturer’s protocol.

HT29 cells were seeded in 4 well chamber slides (BD Pharmingen, San Diego, CA) and infected with rotavirus SA11 or A5-13 or Wa strain or were mock infected. The presence of rotavirus protein NSP5 was analyzed by immunofluorescence microscopy using antiNSP5 primary antibody and rhodamine tagged secondary antibody as previously described (Bagchi et al., 2010). 2.6. RNA isolation and microarray hybridization In the present study, we performed global gene expression analyses using Affymetrix Human GeneChip® ST arrays. The sample preparation was performed according to the manufacturer’s instruction (Affymetrix). Total RNA was isolated from both mock infected and virus infected HT29 cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and purified by of RNeasy Mini Kit (Qiagen, Germany). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies), and quantity was determined by Nanodrop Spectrophotometer (Eppendorf, Germany). 6 ␮g of total RNA from each sample was converted to double-strand cDNA using oligo (dT) primer incorporating a T7 promoter. Amplified, biotinylated and fragmented sense strand DNA targets from the extracted RNA were generated by the Affymetrix GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Kit. Fragmentation was checked on 1% agarose gel stained with EtBr and fragments were hybridized to the gene containing over 22,500 probe sets, as described in the Gene Chip Expression Analysis Technical Manual (Affymetrix). After hybridization, the chips were stained and washed in a GeneChip Fluidics Station 450 (Affymetrix) and scanned by using a GeneChip Array scanner 3000 7G (Affymetrix). 2.7. Microarray data analysis Data was analyzed using Genespring GX 11 microarray and pathway analysis software (Agilent Technologies, CA, USA). For the normalization, AFFYMETRIX EXPRESSION CONSOLE 1.1 was used. The level of gene expression was calculated as fold change and only genes with more than 2 fold change in expression and p < 0.05 (corrected p-value after checking false discovery rate) calculated by one-way ANOVA, Genespring GX 11 software, Agilent were listed and further analyzed to get biological functions and interacting pathway analysis by Genespring GX 11 software (Agilent Technologies, CA, USA). The microarray dataset is deposited to EMBL-EBI ArrayExpress (Accession number E-MEXP-3670).

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Fig. 1. HT29 cells were equally infected by the three rotavirus strains. (A) HT29 cells were either mock infected or infected by any of the three rotavirus strains [SA11 and A5-13, Wa (moi 2)]. At 5 hpi rotavirus infection was checked by immunofluorescence by using anti rotavirus NSP5 antibody followed by rhodamine labeled secondary antibody. Nuclei were stained blue with DAPI and (B) level of two rotavirus proteins (NSP3 and VP6) was analyzed by western blotting. It showed similar amount of rotavirus protein in HT29 cells infected with any of the three rotavirus strains [SA11 and A5-13, Wa] at 5 hpi. ␤-Actin was used as internal control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2.8. Quantitative real-time RT-PCR The differential expression data was validated by quantitative real-time RT-PCR of some of the genes showing altered expression. Confluent monolayers of HT29 cells were mock-infected or infected with either rotavirus SA11 or A5-13 (at a moi of 2) or Wa (at a moi of 3.5). RNA was extracted with TRIZOLTM reagent (Invitrogen, USA) and quantitative PCR was done in triplicates with SYBR GreenTM Mastermix using ABI7500 (Applied Biosystems Inc., Foster city, CA). All primers were synthesized based on the sequence of corresponding human mRNAs in GenBank and the primer sequences of the selected genes are listed in Table S3. Relative gene expressions were normalized to GAPDH using the formula 2−CT [CT = CT (sample) − CT (untreated control)]. Here 2−CT denotes fold change compared to untreated control, CT (sample) = CT (sample) − CT (GAPDH), CT (untreated control) = CT (untreated control) − CT (GAPDH).

one of the aims of this study was to identify potential antiviral target genes for which early time point (5 hpi) had more relevance. The moi was adjusted (moi 2 for SA11 and A5-13 and moi 3.5 for Wa) in such a way that at 5 hpi > 80% cells are infected (Fig. 1A) and expression of two conserved RV protein VP6 and NSP3 was similar for all three strains at 5 hpi in RNA and protein level (Fig. 1B, Supplementary Fig. 2). Analysis of microarray data as shown in Fig. 2A revealed that of all differentially regulated genes, 131 genes were common for all three RV strains whereas 57 genes were modulated by both animal RV strains (SA11 and A5-13). Among strain specific genes, 147, 6 and 3 genes were differentially regulated only by Wa, SA11 and A5-13 strains respectively. Cluster analysis of differentially expressed genes was carried out using GENESPRING GX 11.0 software. Hierarchical clustering methods resulted in identification of 9 distinct patterns of gene expression in RV infected cells compared to mock infected cells, where cluster 3 represents similarly up regulated genes and cluster 8 represents similarly down regulated genes (Fig. 2B).

2.9. Statistical analysis Data are expressed as mean ± standard deviations of at least three independent experiments (n ≥ 3). In all tests, p < 0.05 was considered statistically significant.

3.2. Genes related to chromatin assembly and cell adhesions were regulated only in human rotavirus strain infected cells

3. Results 3.1. Host gene expression response to rotavirus strain SA11, A5-13 or Wa infection At 5 hpi, HT29 cells infected with any of the three strains [SA11(at a moi of 2), A5-13 (at a moi of 2), Wa (at a moi of 3.5)] of RV showed large number of differentially (±2.0 fold change; p < 0.05) expressed genes (494, 501, 816, respectively) compared to the mock infected cells. The host gene regulation was analyzed at 5 hpi, when virus replication had initiated but viral load was not high enough to shut down host translation as it is reported that RV NSP3 shut down host translation (Piron et al., 1998). In addition,

Our study showed that human RV strain Wa uniquely regulates number of genes which were not found to be altered by animal RV strains (SA11 or A5-13). Among these, most important were the genes involved in chromatin assembly. A 2.38 fold up regulation of histone cluster 2, H4a mRNA whereas 2.5–3 fold downregulation of histone cluster 1 and 4 transcripts was observed (Table 1). Cell adhesion molecules play an important role in immune response to virus infection. Some cell adhesion and cell cycle regulatory genes like transforming growth factor beta 2, transglutaminase 2, BCL6 (which is also a transcription repressor), trypsin 2 and trypsinogen C were down regulated and S100 calcium binding protein A4 was up regulated in Wa infected HT29 cells (Table 1).

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Table 1 Pathway analysis of some important genes differentially expressed by 3 RV strains with respect to control. Pathway

Gene description

Fold change with SA11 infection (p < 0.05, n = 3)

Fold change with A5-13 infection (p < 0.05, n = 3)

Fold change with Wa infection (p < 0.05, n = 3)

Immune response

Interferon, alpha-inducible protein 6 Interleukin 18 Chemokine (C C motif) ligand 5 Radical S-adenosyl methionine domain containing 2 (RSAD2) Interferon induced with helicase C domain 1 Tumor necrosis factor (ligand) superfamily, member 10 Interleukin 8 (IL8) Chemokine (C-X-C motif) ligand 10 Chemokine (C-X-C motif) ligand 11 TAP1 Retinoic acid early transcript 1L DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 Interleukin 29 CCAAT/enhancer binding protein (C/EBP), beta Interleukin 28A Interleukin 28B Secreted phosphoprotein 1 Interferon, alpha-inducible protein 6 DNA-damage-inducible transcript 3 Nuclear protein 1 CCAAT/enhancer binding protein (C/EBP), gamma Interferon induced with helicase C domain 1 STAT1 MX1 Hsp70 Vascular endothelial growth factor A Tumor necrosis factor, alpha-induced protein 3 (TNFAIP3) Superoxide dismutase 2 (SOD2) B-cell translocation gene 1, anti-proliferative CEBPB Interleukin 18 (interferon-gamma-inducing factor) Chemokine (C-C motif) ligand 5 Tumor necrosis factor (ligand) superfamily, member 10 Amphiregulin Chemokine (C-X-C motif) ligand 10 Chemokine (C-X-C motif) ligand 11 Vascular endothelial growth factor A Thioredoxin interacting protein DNA-damage-inducible transcript 3 Epiregulin Junction-mediating and regulatory protein Tubulin, epsilon1 Sestrin 2 Claspin homolog (Xenopus laevis) E2F transcription factor 8 Chromosome 13 open reading frame 15 UHRF1 Minichromosome maintenance complex component 6 Cyclin E2 DSCC1 Cyclin-dependent kinase inhibitor 1B Transforming growth factor, beta 2 (TGFB2) v-ets erythroblastosis virus E26 oncogene homolog 1 Epiregulin Interleukin 18 Chemokine (C-X-C motif) ligand 10 Vascular endothelial growth factor A S100 calcium binding protein A4 Transglutaminase 2 BCL6 Protease, serine, 2 (trypsin 2) Trypsinogen C Steroid sulfatase (microsomal), isozyme S Histone cluster 2, H4a Histone cluster 4, H4 Histone cluster 1, H3f Histone cluster 1, H4k Histone cluster 1, H3i DNA-damage-inducible transcript 3 Epiregulin ETS1

4.35 3.2 10.28 4.14 4.28 2.27 5.53 7.71 5.02 2.97 2.93 7.11 11.22 2.45 6.75 5.38 – 4.35 −4.8 −2.15 −2.2 4.28 2.88 4.21 2.03 −6.22 3.99 2.06 3.06 −2.45 3.21 10.28 2.27 −2.53 7.71 5.02 −6.22 −7.39 −4.8 −3.46 −2.86 −3.4 −2.62 2.29 2.21 3 2.55 2.5 2.8 2.3 2.26 – 2.53 −3.5 3.21 7.71 −6.22 – – – – – – – – – – – – −3.5 2.53

4.84 2.5 7.58 4.6 4.4 2.03 4.46 6.71 4.74 3.04 2.38 7.01 7.37 2.66 4.26 3.82 – 4.84 −5.7 −2.32 −2.37 4.4 3.14 4.39 2.13 −5.51 3.66 2.26 2.95 −2.66 2.5 7.58 2.04 −3.32 6.71 4.74 −5.51 −6.52 −5.7 −4.24 −2.63 −3.48 −3.14 2.45 2.4 2.1 3 2.57 2.77 2.41 – – 2.56 −4.2 2.5 6.71 −5.51 – – – – – – – – – – – −5.7 −4.2 2.56

7.28 3.39 2.71 2.2 5.32 2.1 7.97 5.28 2.2 4.38 2.42 9.94 – – – – 32.26 7.3 −4.54 −2.7 −2.76 5.32 3.41 5.04 3.08 −3 5.9 2.42 – – 3.39 2.72 2.11 −3.66 5.28 2.2 −3 −4.46 −4.54 −4.77 – −3.53 – – – – – – – – – −6.88 2.2 −4.8 3.39 5.28 −3 2.45 −7.09 −2.58 −7.56 −2.69 −2.64 2.38 −2.82 −2.52 −3.04 −2.52 −4.54 −4.8 2.2

Apoptosis regulation

Cytokine activity

Cell cycle

Regulation of cell proliferation

Regulation of cell adhesion

Chromatin assembly

Transcription regulation

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Table 1 (Continued) Pathway

DNA metabolic process

Regulation of protein metabolism

Response to virus

Gene description

Fold change with SA11 infection (p < 0.05, n = 3)

Fold change with A5-13 infection (p < 0.05, n = 3)

Fold change with Wa infection (p < 0.05, n = 3)

STAT1 SP100 SP110 BCL6 E2F transcription factor 8 Junction-mediating and regulatory protein Superoxide dismutase 2, mitochondrial MCM6 MCM5 MCM4 Cyclin E2 Claspin homolog (Xenopus laevis) MCM10 BRCA1 interacting protein C-terminal helicase 1 UHRF1 Ribonucleotide reductase M2 polypeptide Proliferating cell nuclear antigen Defective in sister chromatid cohesion 1 homolog Matrix metallopeptidase 13 (collagenase 3) Ubiquitin specific peptidase 18 Ubiquitin specific peptidase 41 Hect domain and RLD 6 Interferon-induced protein 44 2 ,5 -oligoadenylate synthetase 1 (OAS1) Chemokine (C-C motif) ligand 5 Radical S-adenosyl methionine domain containing 2 Interferon induced with helicase C domain 1 STAT1 MX2 MX1 ZC3HAV1 DDX58

2.88 2.3 4.21 – 2.21 −2.86 2.06 2.5 2.25 2.06 2.81 2.3 2.48 3.71 2.55 2.7 2.17 2.29 2.64 2.92 6.38 4.25 6.67 5.56 10.28 4.14 4.28 2.88 5.73 4.21 2.72 7.11

3.14 2.35 4.51 – 2.4 −2.63 2.26 2.57 2.21 2.48 2.77 2.45 2.64 4.08 3.01 3.13 2.16 2.41 2.87 2.81 7.21 4.25 7.23 5.68 7.58 4.61 4.41 3.14 6.03 4.39 2.88 7.02

3.41 2.4 5.65 −2.58 – – 2.42 – – – – – – – – – – – 2.58 4.94 13.25 3.57 5.05 10.74 2.72 2.22 5.32 3.41 3.05 5.04 3.31 9.94

3.3. Cell cycle and DNA metabolic process regulatory genes were modulated following A5-13 and SA11 infection Most of the cell cycle regulatory proteins were differentially regulated only following infection with SA11 and A5-13 but not

in Wa infected HT29 cells. Genes like junction mediating and regulatory protein, tubulin epsilon 1, sestrin 2 were down regulated whereas exonuclease1, E2F transcription factor 8, claspin homolog, UHRF1, cyclin E2, MCM4-6, MCM10, BRCA1 interacting protein C terminal helicase, ribonucleotide reductase M2 polypeptide which

Fig. 2. Comparison of the host cellular gene expression regulation by human and animal rotavirus strains. (A) Venn diagram showing commonly regulated host genes by all three rotavirus strains (SA11, A5-13 and Wa) and (B) hierarchical clustering of differentially expressed genes from HT29 cells infected with different rotavirus strains. Data presented were averaged gene expression changes for 3 different replicates for each virus as well as for mock infected. The clusters represents the type of or pattern of gene expression between three types of strains compared to mock e.g. cluster 3 represents similarly up regulated genes and cluster 8 represents similarly down regulated genes.

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Table 2 Validation of microarray data by qRT-PCR. Gene name

Human HIST4H4 Human TGFB2 Human Cyclin E2 Human TNFAIP3 Human VEGF A Human Epiregulin Human SOD2 Human CXCL10

Fold change in microarray data at 5 hpi in cells infected with Fold change (± error) in qRT PCR data at 5 hpi in cells infected with SA11

A5-13

Wa

SA11

A5-13

Wa

– – 2.81 fold 3.99 fold −6.22 fold −3.46 fold 2.06 fold 7.71 fold

– – 2.77 fold 3.66 fold −5.51 fold −4.24 fold 2.26 fold 6.71 fold

−2.82 fold −6.88 fold – 5.9 fold −3 fold −4.77 fold 2.42 fold 5.28 fold

– – 3.23 fold (±0.32) 4.11 fold (±0.14) −4.11 fold (±0.41) −3.37 fold (±0.06) 2.63 fold (±0.18) 11.63 fold (±1.04)

– – 2.83 fold (±0.21) 3.27 fold (±0.17) −4 fold (±0.08) −3.58 fold (±0.19) 3.06 fold (±0.06) 19.35 fold (±0.89)

−3.06 fold (±0.42) −4.26 fold (±0.34) – 4.52 fold (±0.28) −3.03 fold (±0.11) −6.36 fold (±0.23) 2.93 fold (±0.15) 13.47 fold (±1.73)

are also involved in DNA metabolism were regulated in SA11 and A5-13 infected cells (Table 1). In addition, immune response (IL29, CCAAT/enhancer binding protein beta), apoptosis regulation (B cell translocation gene 1, CEBPB) and cytokine activity (IL 28A, IL28B, IL29) related genes (Table 1) were induced only in cells infected with RV strains of animal origin (A5-13 or SA11). 3.4. Genes differentially regulated by all three RV strains are involved in immune and stress responses, apoptosis, protein metabolism and transcription regulation Immune response is an important host response against virus infection. Most of the interferon inducible genes like IL18, IFN␣ inducible protein 6, radical S-adenosyl methionine domain containing 2 (RSAD2), guanylate binding protein 1 and 2, interferon induced transmembrane protein 3, TAP1, DDX58 were up regulated after infection with any of the three RV strains (SA11, A5-13 and Wa) (Table 1). Several cytokines were found to be commonly up regulated (CCL5, CXCL10, CXCL11) or down regulated (growth differentiation factor 15, amphiregulin) by all three RV strains. One of the most important host innate immune responses during virus infection is induction of apoptosis. Our microarray data showed that apoptotic regulators like DDIT3 (DNA damage inducible transcript 3), nuclear protein 1, VEGF A were commonly down regulated whereas OAS1, STAT1, MX1, Hsp70 (also involved in stress response), TNFAIP3 or A20, SOD2 were commonly up regulated by all three RV strains (Table 1). Suppression and induction of host genes plays an important role in virus propagation. Down regulation of transcriptional regulators like DDIT3, epiregulin and up regulation of ETS1, STAT1, SP100 was observed in cells infected by all three strains. Up regulation of genes involved in protein metabolism e.g. collagenase 3, ubiquitin specific peptidase 18 (USP18) and 41(USP41), hect domain RLD6 was also observed (Table 1). 3.5. Validation of microarray data Expression of selected genes was validated by qPCR or immunoblotting, which correlated with the microarray results (Table 2, Fig. 3A). HT29 cells were infected with either SA11 or A5-13 or Wa strain (moi 2) in duplicate and after 5 hpi RNA was isolated from one set and protein lysates were prepared from second set. Real time PCR was done to confirm HIST4H4, TGFB2, Cyclin E2, TNFAIP3, VEGF A, Epiregulin, SOD2, CXCL10 genes (Table 2). Some commonly regulated genes in both human and animal RV strain infected cells like STAT1, Hsp70, Mx1, RSAD2, A20 and USP18 were validated by immunoblotting as shown in Fig. 3A. Time dependent expression kinetics (3–16 hpi) was analyzed for A20 and USP18. Fig. 3B shows time dependent (3–16 hpi) up-regulation of USP18 and A20 by all three virus strains compared to the mock infection. Expression of these proteins was quantitated with reference to housekeeping protein GAPDH.

3.6. Up regulation of USP18 and TNFAIP3 (A20) positively modulate RV propagation To assess whether induction of USP18 and A20 during RV infection has any functional significance in RV propagation, specific siRNAs were used to down regulate their expression. Briefly, HT29 cells were transfected with either non-specific control siRNA or USP18 or A20 siRNA (50 pmoles and 80 pmoles). After 12 h of siRNA transfection, cells are infected with either A5-13 or SA11 or Wa strain. After 24 h of infection, cells were lysed and plaque assays were done to measure viral titer. As shown in Fig. 4A, compared to nonspecific control siRNA, log 0.8–log 1.3 reduction in viral titer (pfu/ml) was observed in USP18 or A20 siRNA (50 pmoles) transfected cells. With 80 pmoles of siRNA (A20 or USP18), log 1.5–log 2 reduction in viral titer was observed (Fig. 4A). To validate activity of siRNAs used in the study, cell lysates prepared from HT29 cells transfected with either control siRNA or USP18 or A20 siRNA followed by infection with SA11 (4 hpi) were immunoblotted using specific antibodies. As shown in Fig. 4B, dose dependent inhibition in USP18 and A20 expression was observed. To further confirm strain independence, induction and role of USP18 and A20 in RV propagation was analyzed in another human wild type RV strain KU. Consistent with previous results, A20 and USP18 genes were also induced in KU infected HT29 cells and in presence of specific siRNAs, viral titers were also reduced (Supplementary Fig. 1). This suggested that USP18 and A20 are required for RV infection and can be targeted to develop future anti viral drug. 4. Discussion Studies of changes of the host cell transcriptome after virus infection are useful for better understanding of immune responses and viral pathogenesis. The global transcription regulation of Caco2 cells following RV infection was previously reported (Cuadras et al., 2002). Recent reports on the variation in host response by different RV strains as well as increasing evidence of animal–human reassortant strains suggested importance of identifying commonly regulated host genes by wide variety of RV strains to understand viral pathogenesis and host response (Mukherjee et al., 2010; Wani et al., 2004; Saif, 1999; Enriquez et al., 2001; Feng et al., 1997; Cook et al., 2004; Arnold and Patton, 2011; Sen et al., 2009). However since all RV strains depend on host cell machinery for their propagation, it can be hypothesized that host genes involved in activation of antiviral responses and genes which are required by virus for their propagation (pro-viral) should be induced commonly irrespective of strain variation. The genes which positively regulate virus can be targeted for developing antiviral drugs. In this study we have compared the host gene regulation in HT29 cells following infection with RV strains of animal (SA11 and A5-13) and human origin (Wa). The data in this study supported previous observation that modulation of host gene response by RV is strain dependent, though as hypothesized, 131 genes were identified to be commonly

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Fig. 3. Validation of microarray data at the protein level: (A) some of the commonly regulated genes in all three rotavirus infected cells were further validated at protein level. HT29 cells were either mock infected or infected with each of the three rotavirus strains. Then protein lysates were taken at 5 hpi and performed western blotting. Data showed induction of STAT1, Hsp70, RSAD2, Mx1, A20 and USP18 proteins in either animal or human rotavirus infected cells compared to the mock infected. GAPDH was used as internal control and (B) time course study showed gradual induction of A20 and USP18 in all three (SA11, Wa, A5-13) rotavirus strain infected cells compared to mock infection from 3 hpi to 16 hpi. The band intensities of induced proteins were normalized against that of housekeeping protein GAPDH.

modulated by all 3 RV strains, of which some have known antiviral or pro-viral function (Fig. 2A). To rule out any effect of slower propagation rate of the human RV strain (Arnold and Patton, 2011) on commonly induced genes, we performed time course study (3–14 hpi) by qPCR which confirmed that slow propagation rate of Wa strain cannot make any difference to commonly induced genes (Table S1). Among the antiviral innate immune response genes, the interferon inducible genes (viperin, IL18, TAP1, IFN alpha inducible protein 6, GBP1, GBP2, OAS1, etc.) were up-regulated significantly by all three strains. In case of RV infection, increase in IL-18 level in serum and stool samples has been previously reported (Gao et al.,

2006). DDX58 gene codes for a protein called RIG1 which functions as a pattern recognition receptor which is a sensor for viruses, and is involved in viral double-stranded (ds) RNA recognition and the regulation of immune response (Poeck et al., 2010). Previous report has shown that RIG-1 signaling is required for the activation of IFN-␤ production by RV infected intestinal epithelial cells and it has an important role in RV replication in intestinal epithelium (Broquet et al., 2011). Another antiviral gene is OAS1, which activates RNase L resulting viral RNA degradation and inhibition of viral replication. In this study too, we found 5–10 fold induction of OAS1 in all strains, which is consistent with the previous study in Caco2 cells (Arnold and Patton, 2011; Cuadras et al., 2002). In

Fig. 4. Pro-viral proteins like USP18 and A20 facilitate rotavirus infection: (A) plaque assay data showed gradual decrease (log 0.8–log 2) in viral titer irrespective of strain type with increasing amount (50 pmoles and 80 pmoles) of A20 or USP18 siRNA compared to control siRNA transfected cell which suggest that USP18 or A20 facilitate rotavirus infection. The data shown represent mean ± SD of three experiments. The decreases in titers were all statistically significant (p < 0.05) and (B) down regulation of A20 and USP18 in presence of A20 and USP18 siRNA respectively (at amount of 50 pmoles and 80 pmoles) after SA11 infection at 4 hpi analyzed by western blotting.

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addition to IFN and IFN induced genes, other cytokines and chemokines have also been shown to participate in controlling virus infection (Melchjorsen et al., 2003). All three RV strains (SA11, A5-13 and Wa) induce cytokines like CCL15, CCL5, CXCL10, and CXCL11 in HT-29 cells (Table 1). Among these cytokines CCL5 is the most studied cytokine with respect to molecular mechanisms governing virus-induced chemokine expression. CCL5 induction has also been previously reported in simian rotavirus SA11 infected HT29 and Caco2 cells (Casola et al., 1998). Among the stress responsive genes, Hsp70 was induced by SA11, A5-13 and Wa, which is consistent with observation in bovine RV infected Caco2 cells where Hsp70 has been shown to negatively regulate RV infection (Broquet et al., 2007). Apoptosis induction is another frontline mechanism of host cells to combat against virus and pathogens. In this study too, down regulation of antiapoptotic genes (VEGF A) and up regulation of some pro-apoptotic genes like MX1 and STAT1 (Table 1, Fig. 3A) (Gupta et al., 1999; Mibayashi et al., 2002; Sironi and Ouchi, 2004) was common in cells infected with any of the three RV strains suggesting that apoptosis regulation is independent of strain type. Delaying apoptosis, a host defense response can help virus in completing its replication cycle (Bagchi et al., 2010). Thus, viruses are known to modulate apoptosis. In this study too, down regulation of apoptotic genes like DDIT3 and nuclear protein 1 (NUPR1 or P8) and up regulation of anti apoptotic gene like SOD2 was observed in all 3 RV infected HT29 cells (Table 1). DDIT3 and NUPR1 are both endoplasmic reticulum stress related genes which lead to apoptosis (Oyadomari and Mori, 2004; Carracedo et al., 2006), whereas SOD2 resides in the mitochondria and converts the majority of superoxide anions on site into less reactive H2 O2 . SA11 induces SOD2 to avoid the oxidative stress to prolong cell survival and to allow the accumulation of viral particles before cell destruction and virus release in Caco2 cells (Gac et al., 2010). A20 (TNFAIP3) and USP18, genes involved in modulation of innate immunity by their antiapoptotic or ubiquitin activity were also found to be induced by all three RV strains. Overall although the results confirmed that most of the regulation of innate immunity to be strain independent, but type III interferon (interlukin 28A, B and interlukin 29) was observed to be induced only in animal rotavirus infected cells, not in human rotavirus infected cells (Table 1). This finding is interesting which need further study in future. We found 147 differentially regulated genes following infection with Wa strain at 5 hpi. Down regulation of the histone core proteins (Table 1) by Wa strain can be a viral strategy to allow host DNA repair system to find access to damaged host DNA due to viral stress and to avoid cellular apoptosis (Lilley et al., 2010). Consistent with previous studies in Human Papilloma Virus (HPV) (Nees et al., 2000), cell adhesion or cell cycle regulatory molecules like transforming growth factor beta 2 were down-regulated (Table 1). Viruses interact with the cell cycle to subvert host-cell function and increase the efficiency of virus replication. Majority of the studies have been conducted on DNA viruses whose primary replication site is nucleus, but recently some reports have suggested RNA virus interference with the cell cycle (Zhang et al., 2009; Izumiya et al., 2003; Honda et al., 2000; Lin and Lamb, 2000; Naniche et al., 1999). Surprisingly, in this study we observed transcriptional regulation of large number of cell cycle proteins only in case of animal rotaviral (RV) strains but not in human RV strain infected cells (Table 1). Cell cycle maintenance genes (UHRF1, cyclin E2) and inhibitors like E2F transcription factor 8 were up regulated (Maiti et al., 2005; Hopfner et al., 2000; Möröy and Geisen, 2004), whereas the positive regulator of cell cycle like tubulin epsilon 1 was down regulated (Dutcher, 2001). Minichromosome maintenance (MCM) complex acts as a replicative helicase to unwind the origin and initiate DNA replication (Forsburg, 2004). In this study, MCMs (MCM4-6, MCM10) were found to be up regulated following SA11 and A5-13 infection but not with Wa strain (Table 1). To rule out the possibility that

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these genes were up regulated at later time point in Wa infected cells, expression of MCM4-6 or MCM10 was analyzed by real time PCR at 3–14 hpi, however no up regulation of MCM4-6 or MCM10 was observed even at 14 hpi (Table S2). Here, our study showed some important differences in gene expression between human and animal rotavirus infected cells. It may be due to difference in interferon antagonism between human and animal rotavirus strains as previously reported (Arnold and Patton, 2011). Previous studies have reported pro-viral role of A20 by promoting resistance to TNF-␣ induced cell death as well as antagonists to the A20 protein have been found to block the replication of other viruses (Valck et al., 1996; Doukas and Sarnow, 2011; Neill et al., 2008; Zilliox et al., 2007; Nguyen et al., 2006; O’Donnell et al., 2006). In addition, A20 induces ubiquitination and degradation of the RIP kinase which is an important component of ligand dependent activation of NF␬B which has a key role in inflammation and immunity (Bedford et al., 2011). Similarly USP18 is an ubiquitinspecific protease that cleaves the ubiquitin-like (and IFN-induced) ISG15 protein from its cellular targets. It has been reported that ISG15 enhances the antiviral activity of IFN against human immunodeficiency virus and Sindbis virus replication in vitro (Kunzi and Pitha, 1996; Lenschow et al., 2005) and the augmentation of interferon induced antiviral response against hepatitis C was observed after silencing of USP18 (Randall et al., 2006). Since infections with SA11, A5-13, Wa and KU RV strains all increased USP18 and A20 expression (Fig. 3A and B, Supplementary Fig. 1), functional significance of these genes was analyzed during RV infection. Consistent with previous reports of their pro viral function (Valck et al., 1996; Randall et al., 2006), reduced viral titers were observed in presence of USP18 or A20 specific siRNAs (Fig. 4A, Supplementary Fig. 1) suggesting that these cellular genes can be potential antiviral targets. Targeting cellular genes for therapeutics has advantage since the mutation rate is lower in cellular genes compared to viral genes. Although, our study focused on identifying common gene induced by different RV strains (human and animal) as potential anti rotaviral targets, one of the major limitations is that transcriptional level studies cannot give idea about post translational changes of all these genes in cells. We did not observe any changes in Hsp90 or Akt genes at transcriptional level though our previous studies have shown regulation of both Hsp90 protein and Akt phosphorylation following RV infection (Bagchi et al., 2010; Dutta et al., 2009). In conclusion, this study provides comprehensive information on both strain independent as well as strain specific host responses against RV. A large number of commonly regulated pro-viral genes were identified, which can be further studied for delineating their role in RV infection. Of these, two genes (A20 and USP18) were shown here to have positive role during RV infection and these can be probable anti-rotaviral drug targets. Further in depth studies are required to understand function of these cellular genes during RV replication.

Acknowledgements The study was supported by financial assistance from Indian Council of Medical Research (ICMR), New Delhi and the Program of funding research centers for emerging and reemerging infectious diseases (Okayama University-NICED, India) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. P. Bagchi (SRF), U.C. Halder (SRF), S. Nandi (JRF) and R. Bhowmick (JRF) are supported by fellowships from CSIR, Govt. of India and M.K. Nayak supported by Dr. D.S. Kothari Postdoctoral Fellowship from UGC, Govt. of India.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2012.07.021.

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