Mutating the putative binding sites abolishes Sp1 and NF-κB transcriptional activation of the <i>TINF2</i> promoter.

<p><b>A</b>: Mutating core consensus nucleotides in predicted Sp1 or NF-κB binding sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021333#s4" target="_blank">Materials and Methods</a>) results in reduced <i>TINF2</i> promoter-driven luciferase activity in a minimal promoter context. For each transfection, the firefly luciferase activity was normalized to the <i>Renilla</i> activity expressed from the co-transfected pRL-CMV expression vector. The means from three independent experiments are shown; <i>bars</i>, SD. (p<0.001). <b>B</b>: Mutating core consensus nucleotides in the predicted Sp1 binding site at position −(88−74) results in reduced <i>TINF2</i> promoter-driven luciferase activity as compared to wild-type control. Results are shown as in panel A. (***p<0.001).</p

Sp1 binds to putative binding sequences in the <i>TIN2</i> promoter <i>in vitro</i> and <i>in vivo.</i>

<p><b>A</b>: EMSA showing the ability of Sp1 to bind to its two predicted sites <i>in vitro.</i> Sp1 protein was <i>in vitro</i> translated using the rabbit reticulocyte lysate (RRL) system and incubated with end-labeled DNA oligos encompassing the putative binding sites. These complexes (lanes 3 and 9) could be competed away with unlabeled wild-type oligos (lanes 4 and 10), but not with a mutant form (lanes 5 and 11). Furthermore, the complexes could be super-shifted using an anti-Sp1 antibody (lanes 6 and 12). <b>B</b>: The ability of Sp1 to bind to the endogenous <i>TINF2</i> promoter <i>in vivo</i> was shown using ChIP. The 407 base pair fragment could be amplified from a reaction including the anti-Sp1 antibody (lane 7), but not from reactions containing anti-c-Myc, normal IgG, or no antibody (lanes 4–6). <b>C</b>: Sp1 is the major Sp transcription factor that can activate the <i>TINF2</i> promoter. <i>Drosophila melanogaster</i> SL2 cells were co-transfected with the minimal promoter reporter construct pGL3-P406 and various amounts of expression vectors encoding either Sp1 or Sp3. For each transfection, the firefly luciferase activity was normalized to the total protein concentration. The means from three independent experiments are shown; <i>bars</i>, SD.</p

Luciferase assay analysis of <i>TINF2</i> promoter truncations defines two significant drops in promoter activity.

<p><b>A</b>: The name of each TIN2 reporter construct was assigned according to the 5′-end nucleotide numbers of the promoter sequences inserted upstream of the ATG initiation codon. Basic refers to the pGL3-Basic vector. For each transfection, the firefly luciferase activity was normalized to the β-galactosidase activity expressed from a co-transfected β-galactosidase expression vector. The means from three independent experiments are shown for each construct; <i>bars</i>, SD. (***p<0.001). <b>B</b>: Finer promoter mapping of the region between −450 and −351, expressed as in panel A. <b>C</b>: Finer promoter mapping of the region between −148 and −24, expressed as in panel A. <b>D</b>: Verification of the functionality of the <i>TINF2</i> promoter construct in various cell lines. For each transfection, the firefly luciferase activity was normalized to the <i>Renilla</i> activity expressed from the co-transfected pRL-CMV expression vector and expressed as in panel A. <b>E</b>: Schematic of the core <i>TIN2</i> promoter construct showing putative Sp1 and NF-κB binding sites predicted using bioinformatic tools. “Drop 1” and “Drop 2” refer to the drops in luciferase reporter activity shown in panel A.</p

Pharmacological inhibitors can interfere with Sp1- and NF-κB-mediated <i>TINF2</i> promoter activation.

<p><b>A</b>: Mithramycin A, an Sp1 inhibitor, reduces <i>TINF2</i> promoter-driven luciferase activity. 293T cells are transfected with either the promoter-less pGL3-Basic plasmid, the NFAT-responsive pNFAT-Luc plasmid, the SV40 promoter-containing pGL3-Control plasmid, or the minimal TIN2 promoter P406 plasmid and incubated with the indicated concentrations of drug for 24 hours. For each transfection, the firefly luciferase activity was normalized to <i>Renilla</i> luciferase activity expressed from a co-transfected <i>Renilla</i> luciferase expression vector. The means from three independent experiments are shown; <i>bars</i>, SD. (**p<0.01, ***p<0.001). <b>B</b>: Bay11-7082, an NF-κB inhibitor, reduces <i>TINF2</i> promoter-driven luciferase reporter activity. Cells are processed and values expressed as in panel A. (***p<0.001). <b>C</b>: PDTC, an NF-κB inhibitor, reduces <i>TINF2</i> promoter-driven luciferase reporter activity. Cells are processed and values expressed as in panel A. (***p<0.001). <b>D</b>. Mithramycin A and Bay11-7082 (Bay11) reduce endogenous <i>TINF2</i> gene expression. The levels of <i>TINF2</i> mRNA were normalized to those of the housekeeping <i>GAPDH</i> gene. The means from three independent experiments are shown.</p

Representative 2-D gel image obtained from muscle tissue of mock-infected and CHIKV-infected mice.

<p>Equal amount of protein sample (750 μg) was first separated in a linear gradient of pH 4–7, followed by separation in SDS-PAGE (12%) and coomassie staining. A total of 27 protein spots were found to be significantly altered. Fold changes (mean values± SD) of the identified proteins are illustrated graphically (** signifies p≤0.05). Spots M6, M23 and M24 were found to be affected qualitatively and hence were not presented graphically.</p

Chikungunya virus (CHIKV) induced disease signs.

<p>A. Pictures of the hind limbs of mock- and CHIKV-infected animals. Chikungunya virus infection induced severe hind limb disease in new born mice. 2–3 day old mice inoculated with 10⁶ PFU (50 ul) of CHIKV by subcutaneous injection in the loose fold of skin on the back of the animal developed peak clinical signs on 8 day post inoculation whereas mock-infected group injected with uninfected tissue culture supernatant remained healthy. B. Mice were scored for the development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, mild hind limb weakness; 3, moderate hind limb weakness; 4, severe hind limb weakness and dragging and 5, moribund. Each data point represents the arithmetic mean ± SD for eight animals. Data is representative of three independent experiments.</p

Skin, spleen and muscle pathology of CHIKV infected mice.

<p>Representative picture showing the pathology staining of the tissues (skin, spleen and hind limb muscle) from mock-infected and CHIKV-infected mice on 8th day of post infection. Characteristic histological features are indicated by arrows. Skin of CHIKV-infected mice showed hyperplasia of basal keratinocytes and hyperkeratinisation. Hair follicles showed atrophy with no dividing cells in the matrix. Spleen of CHIKV-infected mice showed considerable lymphoid proliferation and haemorrhage. Muscle sections showed degenerative changes with dark pink stained, atrophied and necrotic muscle fibres, infiltration of neutrophiles and monocytoid cells between the muscle fibres.</p

Validation of proteomic results using Q-PCR and immunoblotting.

<p>A. Transcript alteration of the differentially expressed proteins in muscle tissue upon CHIKV infection. Total RNA of muscle tissue (infected/uninfected) was analysed by real time RT-PCR. House-keeping GAPDH gene was used for normalization purpose. RNA expression changes of vimentin, hemopexin, haptoglobin, Rho GDP, PKM2 and kininogen were in concordance with protein expression changes and were determined to be statistically significant (p≤0.05). *Genes were considered to be significantly up-regulated if the change in their relative expression levels was ≥2 fold. No significant difference in the RNA expression of ApoA1 and peroxiredoxin 6 was found. B. Immunoblot of representative proteins showing increased expressions in muscle tissue upon CHIKV infection.</p

Gross pathology, viral replication and profile of inflammatory cytokines.

<p>A. Surgically removed hind limb muscles from mock- and CHIKV-infected mice showing gross pathology of the muscles (in terms of swelling) along with the 4 μm sections showing the localization of CHIKV antigen in the hind limb skeletal muscle on day 9 post infection. B. Virus titre in the hind limb muscles. C. Real time analysis showing relative fold change in inflammatory genes (MCP-1, MCP-3, IL-6, TNF-α, Rantes) in muscle tissue on day 9 post infection. ** Genes were considered significantly up-regulated if the change in their relative expression level was ≥2 fold at p<0.05 by student's <i>t</i> test.</p

Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.

<p>Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.</p