Host Cell Transcriptome Profile during Wild-Type and Attenuated Dengue Virus Infection

10.1371/journal.pntd.0002107PLoS Neglected Tropical Diseases73

Comparison of differential regulation of genes, isoforms and splicing in DENV1-16007 and DENV1-PDK13.

<p>Comparison of differential regulation of genes, isoforms and splicing in DENV1-16007 and DENV1-PDK13.</p

Cross-platform analysis of DENV host factors.

<p>The number of host factors identified in each of the RNAi screens listed compared to the differentially regulated isoforms and differentially spliced genes for both the wild-type DENV1-16007 and vaccine strain DENV1-PDK13. Percentages of overlap are indicated in parenthesis. The unpublished DENV screen by Jamison SF, Garcia-Blanco, M.A. utilize the same siRNA library, cell line, general methodology and statistical analysis described in Le Sommer et al 2012. The unpublished hits from the WNV screen are the full, unannotated results from the published Krishnan et al 2008 study.</p

Validation of single exon skipping events predicted by both Cuffdiff and MISO.

<p>Values graphed are the log2 transformed fold change differences between inclusion/skipping of the alternatively spliced exon in DENV1-16007 infected cells (red bars) or DENV1-PDK13 infected cells (blue bars) compared to uninfected control cells for 0, 12, 20 and 30 hours post infection. Asterisks indicate a p-value of less than or equal to 0.05.</p

Ingenuity pathway analysis.

<p>Pathway (<b>A, C, E</b>) and molecular and cellular function (<b>B, D, F</b>) analysis of common DENV1 response (<b>A, B</b>), unique DENV1-16007 response (<b>C, D</b>) and unique DENV1-PDK13 response (<b>E, F</b>). The numbers within the pie-slices in panels B, D and F are the number of isoforms belonging to the indicated molecular and cellular function.</p

Differential regulation of transcripts in response to DENV1 infection.

<p>Number of events in each category (Rows) predicted by Cuffdiff <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Trapnell1" target="_blank">[40]</a> (Top) and MISO <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Katz1" target="_blank">[41]</a> (Bottom) for Uninfected cells vs. DENV1-16007 infected cells (First column), Uninfected cells vs. DENV1-PDK13 infected cells (Second column). The number of up- and down-regulated events is also described for the gene and isoform categories. Superscript “a” indicates biological triplicates while superscript “b” indicates single measurement of the sample. Abbreviations used: differentially expressed genes (Genes exp), differentially expressed isoforms (Isoforms exp), differentially expressed transcriptional start site group (TSS group exp), differentially expressed coding sequence (CDS exp), differential coding output (CDS), differential promoter use (Promoters), differential splicing (Splicing), skipped exon (SE), mutually exclusive exons (MXE), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), alternative first exon (AFE), retained intron (RI) and tandem untranslated region (Tandem UTR).</p

Experimental design and analysis of infection.

<p><b>A</b>. Timeline of experimental procedure and data analysis steps. <b>B</b>. Quantification of viral copy number normalized to the cellular B2M gene expression at 0, 6, 12, 20 and 30 hours post infection. Asterisks indicate a p-value of less than or equal to 0.05. <b>C</b>. Immunofluorescence analysis of Huh7 cells either mock-infected (top panel), infected with DENV1-16007 (middle panel) or DENV1- PDK13 (bottom panel) and probed with antibody against prM protein (2H2) followed by FITC-conjugated goat anti-mouse IgG at 20 h post-infection. The cell nuclei were counter-stained with Evan's Blue.</p

Cross-platform analysis of human-Flaviviridae interactions.

<p>Members of eleven canonical pathways that have been previously implicated to be important for DENV propagation were interrogated for their appearance in an RNAi screen for Flaviviridae host factors, direct interaction with DENV gene products, differential regulation in a microarray study, differential isoform regulation following infection with DENV1-16007 and/or differential isoform regulation following infection with DENV1-PDK13. Host factors identified in published and/or unpublished Flaviviridae RNAi screens <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Krishnan1" target="_blank">[10]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Sessions1" target="_blank">[11]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Li1" target="_blank">[48]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Tai1" target="_blank">[49]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-LeSommer1" target="_blank">[51]</a> (Jamison and Garcia-Blanco, unpublished data) were combined and are indicated by a black bar. Host proteins found to interact DENV gene products <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-LeBreton1" target="_blank">[47]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Ward1" target="_blank">[50]</a> were combined and are indicated by a grey bar. Host genes found to be differentially regulated in microarray studies <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Balas1" target="_blank">[12]</a>–<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Warke1" target="_blank">[29]</a> are indicated by the striped bar. A red bar indicates host isoforms found to be differentially regulated during infection with DENV1-16007. A blue bar indicates host isoforms found to be differentially regulated during infection with DENV1-PDK13.</p