Release of CHIKV upon drug treatment to elucidate host cell factors involved in apical sorting of CHIKV.

<p>The infectious virus titer remained higher in the apical chamber than in the basolateral chamber upon treatment with (<b>A</b>) cytochalasin B, (<b>B</b>) nocodazole and (<b>C</b>) blebbistatin. One-tailed Student's <i>t</i>-test: ** p<0.005, *** p<0.001. Vertical bars represent one standard deviation from the mean of three readings. Apically-infected Vero C1008 cells were treated with (<b>D</b>) cytochalasin B, (<b>E</b>) nocodazole and (<b>F</b>) blebbistatin and co-labeled with antibodies against CHIKV E2 glycoprotein (green, arrows) and ZO-1 tight junction proteins apical markers (red, arrowheads). Cell nuclei were stained with DAPI (blue). Z-stacked images show the polarized release of CHIKV towards the apical plasma membrane of Vero C1008 cells upon treatment with cytochalasin B, nocodazole and blebbistatin.</p

Polarized entry of CHIKV at apical plasma membrane domain.

<p>(<b>A</b>) The total virus yield at 24 h.p.i. of non-polarized Vero, polarized Vero C1008 and polarized HBMEC cells at an MOI of 10 was quantified by viral plaque assay. Entry of CHIKV is bi-directional in non-polarized Vero cells but occurs preferentially at the apical domain of polarized Vero C1008 and HBMEC cells. Two-tailed Student's <i>t</i>-test: * p<0.05, ** p<0.005. Vertical bars represent one standard deviation from the mean of three readings. (<b>B</b>) Virus binding assays show higher amount of CHIKV binding upon apical infection as compared to upon basolateral infection of polarized Vero C1008 cells. In contrast, the amount of CHIKV binding upon apical and basolateral infection of non-polarized Vero cells is similar. Two-tailed Student's <i>t</i>-test: *** p<0.001.</p

Growth kinetics of CHIKV in HBMEC at an MOI of 10.

<p>(<b>A</b>) CHIKV-infected HBMEC cells were viewed under DIC microscopy to observe for morphological changes. The cell density was lower in CHIKV-infected cells as compared to mock-infected cells from 48 h.p.i. onwards. (<b>B</b>) CHIKV-infected HBMEC cells were fixed at various intervals post-infection and immunofluorescence assay was performed to detect for CHIKV protein expression (green). Cell nuclei were stained with DAPI (blue). Moderate amounts of CHIKV protein expression were observed at 24 h.p.i. of HBMEC. (<b>C</b>) Quantification of infectious virus titer by plaque assay showed an increasing trend, with a peak in infectious virus titer at 36 h.p.i.. Vertical bars represent one standard deviation from the mean of three readings.</p

Cell monolayer integrity post CHIKV infection at an MOI of 10.

<p>(<b>A</b>) TEER measurements of Vero, Vero C1008 and HBMEC cell monolayers were taken at 24 h.p.i.. The TEER measurements post-apical and basolateral infection were comparable to that of mock-infected cells. Vertical bars represent one standard deviation from the mean of three readings. (<b>B</b>) Immunofluorescence assays demonstrated the expression of ZO-1 tight junction proteins (green) in apically-infected and basolaterally-infected Vero, Vero C1008 and HBMEC cells at 24 h.p.i.. The expression of ZO-1 proteins in infected cells is comparable to that of mock-infected cells. (C) The FITC-dextran permeability assays demonstrated that the integrity of Vero and Vero C1008 cell monolayers remained intact at 24 h.p.i., where the permeability of the cell monolayers to FITC-dextran remained low as compared to the TNF-treated cells.</p

Small interfering (siRNA) sequences used in siRNA based experiments.

<p>Small interfering (siRNA) sequences used in siRNA based experiments.</p

2DE analysis of CHIKV-infected C6/36 cells.

<p>(A) Twenty-three protein spots of mock-infected samples that showed 2-folds changes with <i>p</i><0.05 (Student’s <i>t</i>-test) as compared to (B) CHIKV-infected samples were annotated. All the SDS-PAGE gels from the second dimension separation were analyzed using PDQuest 2-D Analysis Software (BioRad). Two biological replicates of the CHIKV-infected samples from each time-point infection were grouped and compared against to the corresponding biological replicates of the mock-infected samples. Representative gels are from 24h mock-infected and 24h CHIKV-infected lysates.</p

Mean change in spot intensities and mass spectrometry data of 23 differentially regulated proteins from CHIKV-infected C6/36 cells at M.O.I. 10.

<p>Out of the 23 selected protein spots, 3 protein spots were identified from other species with unknown functions. The functional grouping of the proteins is based on the most significant function of each protein shown in Uniprot protein knowledge base, UniprotKB database (Swiss-Prot/TrEMBL; <a href="http://www.uniprot.org/uniprot" target="_blank">http://www.uniprot.org/uniprot</a>).</p><p>Mean change in spot intensities and mass spectrometry data of 23 differentially regulated proteins from CHIKV-infected C6/36 cells at M.O.I. 10.</p

2-DE dynamic profiles of the selected host proteins upon CHIKV-infection at M.O.I. 10.

<p>Yellow box indicates the differentially expressed proteins. Mock-infection for each time-point served as a control, as shown in panel 24M, 48M and 96M. CHIKV-infection profiles analyzed at 24, 48 and 96 h.p.i., as shown in the panel of 24I, 48I and 96I, respectively. The functional grouping of each protein is shown in UniprotKB database.</p

STRING analysis of protein-protein interactions between differentially expressed proteins identified through 2DE.

<p>The protein network analysis was performed using STRING v9.05. The interactions between expressed proteins are indicated by the connecting line. The thickness of the connecting line represents the strength of the associations. The interactions network is significantly enriched (<i>p</i>< 0.05).</p

RNA levels on selected cellular genes.

<p>Scrambled siRNA-transfected cells (represented by white bars) against (A) Spermatogenesis-associated factor, (B) Enolase phosphatase e-1 and (C) Chaperonin-60kD show no knockdown of gene expression when compared to transfection control (TC). Cells transfected with targeted cellular siRNAs (represented by shaded bars) against (A) Spermatogenesis-associated factor, (B) Enolase phosphatase e-1 and (C) Chaperonin-60kD showed significant knockdown across all genes tested compared to transfection control (TC). The <i>asterisk</i> indicates *<i>p</i> values <0.05, **<i>p</i> values of <0.01 and ***<i>p</i> values <0.0001 by Student’s <i>t</i> test. Asterisks indicate statistically significant results relative to control group (■).</p