Journal: Journal of Virology

Article Title: Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1

doi: 10.1128/JVI.00559-19

Figure Lengend Snippet: MOI-dependent temporal expression dynamics of the NS1 protein (PR8-GFP virus) in human respiratory epithelial cells (A549 H2B-mCherry) using time-lapse microscopy. Green indicates NS1 expression (GFP). The cell nuclei (H2B-mcherry) are depicted in red. (A) Time course from 3 to 12 hpi. One representative field is shown for each MOI. (B) Quantification of the number of GFP-positive cells for the four MOIs used (approximately 25,000 cells/well). For MOI of 0.2, 0.6, and 2, averages of two replicate wells ± SDs are shown. For infection at an MOI of 10 and mock infection, data from one single well are shown. (C) Heat map showing the median of fluorescence intensity of the infected cells (GFP+ cells). (D) Table showing the measured percentages of infected cells at 12 hpi, as well as the theoretical percentages of cells infected either with ≥1 virion (all infected cells) or >1 virion (multiple infections).

Article Snippet: A549 H2B-mCherry-infected or mock-infected cells were detached from the cell culture plates using trypsin-0.05% EDTA (Gibco), washed with cell culture media, and passed through a 40-μm cell strainer to prepare single-cell suspensions, and the final concentration was set at 1,000 cells/μl.

Techniques: Expressing, Virus, Time-lapse Microscopy, Infection, Fluorescence

Journal: Journal of Virology

Article Title: Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1

doi: 10.1128/JVI.00559-19

Figure Lengend Snippet: Stimulation of A549 cells with TNF-α, IFN beta, IFNL1 (100 U/ml), or combinations of those cytokines or infected with PR8-GFP (MOI, 2) for 8 h. Induction of IFNL1 was not detected upon stimulation with those cytokines by qRT-PCR. Gene expression of the indicated genes (IFNs, with Mx1 and CCL5 to confirm the effectiveness of the treatments) was analyzed. Averages of biological triplicates ± SDs are shown.

Article Snippet: A549 H2B-mCherry-infected or mock-infected cells were detached from the cell culture plates using trypsin-0.05% EDTA (Gibco), washed with cell culture media, and passed through a 40-μm cell strainer to prepare single-cell suspensions, and the final concentration was set at 1,000 cells/μl.

Techniques: Infection, Quantitative RT-PCR, Gene Expression

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: (A) Parental A549, PAF1 KO/rescue and STAT2 KO/rescue cells were stimulated with poly(I:C) for 3 hours and subjected to RNA-seq and DESeq2 differential gene expression analysis. Results are based on three independent biological replicates. (B) Principal component analysis performed on all samples showed a clear separation between principal components (PC) describing untreated/treated cells and cell genotype. (C) GSEA was performed on genes differentially expressed in KO cells compared to parental A549 cells following poly(I:C) treatment. Up to the top 5 positively and negatively enriched Reactome pathways were plotted for each comparison (p adj < 0.1). A full list of GSEA results is available in . (D) Changes in gene expression caused by poly(I:C) treatment are shown for the subset of immune response genes (GO:0006955) significantly upregulated for poly(I:C)-treated parental A549 cells relative to mock-treated A549 cells (log2 fold change > 0.5, padj < 0.05). A Wilcoxon signed rank test with Bonferroni correction was performed to identify significant changes caused by PAF1 or STAT2 KO. (E) All genes significantly upregulated for poly(I:C)-treated parental A549 cells relative to mock-treated A549 cells (log2 fold change > 0.5, padj < 0.05) were plotted based on log2 fold change of PAF1 and STAT2 KO cells relative to parental A549 cells following poly(I:C) treatment. Parental A549 cells were used as a normalization so that it was identical for both comparisons. Unsupervised K-means clustering was also performed to identify genes with similar behavior (triangles, circles and squares). P values were adjusted for false discovery rate using the Benjamini Hochberg method. Significant changes in gene expression are plotted for PAF1 KO (cyan), STAT2 KO (magenta), both (yellow) or neither (grey). Immune response genes (GO:0006955) are highlighted with larger markers and opaque coloring.

Article Snippet: A549 PAF1 and STAT2 rescue cells were created by cloning PAF1 and STAT2 cDNA into pLenti6 plasmid (Addgene #89766, [ ]).

Techniques: RNA Sequencing, Gene Expression, Comparison

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: (A) The protein interaction between flavivirus NS5s and PAF1 was tested biochemically. Plasmids encoding NS5s and GFP were transfected and affinity purified in HEK293T cells via a 2xStrep II tag. Immunoblot analysis of lysate and purified (Strep-AP) fractions were performed against PAF1, Strep, and GAPDH (loading/negative control). (B) Logo analysis of amino acid conservation for flavivirus NS5s from (A) in the PAF1-interacting region. The conserved stretch from amino acids 258–263 (magenta) was targeted for alanine scanning leading to the generation of 2 NS5 mutants: LGS258AAA (NS5 LGS ) and GTR261AAA (NS5 GTR ). (C) DENV2 NS5 structure from PDB (5ZQK) with PAF1-interacting region highlighted (box). The PAF1-interacting region overlaps with the C-terminal end of the MTase (yellow) and the flexible linker domain (cyan), but not the RdRP (grey). The PAF1-interacting region includes a stretch of amino acids conserved in PAF1-interacting flaviviruses tested in (A). (D) Mutants from (B) were tested for an interaction with PAF1C biochemically. Purification and immunoblot analysis were conducted as in (A). PAF1C complex members CTR9, LEO1, CDC73 and PAF1 were probed. STAT2 was used as a positive control for an NS5 interaction outside of the PAF1C-interacting region. Abbreviations: Zika virus (ZIKV), West Nile virus (WNV), Powassan virus (POWV), Langat virus (LGTV), tick-borne encephalitis virus (TBEV), Strep-affinity purified (Strep-AP).

Article Snippet: A549 PAF1 and STAT2 rescue cells were created by cloning PAF1 and STAT2 cDNA into pLenti6 plasmid (Addgene #89766, [ ]).

Techniques: Transfection, Affinity Purification, Western Blot, Purification, Negative Control, Positive Control, Virus

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: Relative gene expression was plotted as log2 fold change for (A) PAF1 KO versus parental A549 cells or (B) STAT2 KO versus parental A549 (same as ), and NS5 mutant versus WT. Unsupervised K-means clustering was also performed to identify genes with similar behavior (triangles, circles and squares). P values were adjusted for false discovery rate using the Benjamini Hochberg method. Significant changes in gene expression are plotted for: PAF1 KO (cyan), both (yellow), neither (grey), NS5 LGS (orange), NS5 GTR (red) and NS5 2xNLS (purple). Immune response genes (GO:0006955) are highlight with larger markers and opaque coloring.

Article Snippet: A549 PAF1 and STAT2 rescue cells were created by cloning PAF1 and STAT2 cDNA into pLenti6 plasmid (Addgene #89766, [ ]).

Techniques: Gene Expression, Mutagenesis