george stark  (Addgene inc)

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

Techniques: Gene Expression, Mutagenesis

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) Immunoblot analysis of PAF1 expression in PAF1 KO and rescue A549 cells. GAPDH is a loading control. (B) DENV2 replication in PAF1 KO and rescue cells, MOI 0.1. Data from three independent biological replicates are plotted as mean values +/- standard deviation. P values were calculated using a paired, one-tailed Student’s t-test. Abbreviations: plaque forming units (pfu), not statistically significant (ns).

Article Snippet: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

Techniques: Western Blot, Expressing, Control, Standard Deviation, One-tailed Test

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

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) NLS mutants, including single mutant NS5 central (REE396-398AAA) and NS5 C-term (RR890-891AA) and the double mutant NS5 2xNLS (REE396-398AAA and RR890-891AA). (B) Subcellular localization of 2xStrep II tagged NS5s (yellow) and colocalization with PAF1 (cyan) was determined by immunostaining and confocal microscopy. Nuclei were stained with Hoechst (magenta). Scale bar represents 10 μm. (C) The protein interaction between NS5 2xNLS 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 was performed against PAF1, Strep and GAPDH (loading/negative control). Abbreviations: Strep-affinity purified (Strep-AP).

Article Snippet: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

Techniques: Mutagenesis, Immunostaining, Confocal Microscopy, Staining, Transfection, Affinity Purification, Western Blot, Purification, Negative Control

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) NS5 truncations constructed to test NS5-PAF1 interaction. Each truncation corresponds to ~100 amino acid deletions from the N- (T1, T2, T3) or C-terminus (T4, T5, T6, T7) of NS5. (B) Subcellular localization of 2xStrep II tagged truncations and NS5 full-length (yellow) and colocalization with PAF1 (cyan) were determined by immunostaining and confocal microscopy. Nuclei were stained with Hoechst (magenta). Scale bar represents 10 μm. (C) The protein interaction between NS5 truncations and PAF1 were tested biochemically. Plasmids encoding NS5 truncations, NS5 full-length 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). (D) Refined truncations in the T2-T3 region (T2, T2A, T2B and T3) were tested for an interaction with PAF1 biochemically. Purification and immunoblot analysis were conducted as in (C). Abbreviations: Strep-affinity purified (Strep-AP).

Article Snippet: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

Techniques: Construct, Immunostaining, Confocal Microscopy, Staining, Transfection, Affinity Purification, Western Blot, Purification, Negative Control

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

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: PAF1 and STAT2 (Addgene #71451, [ ]) were inserted into pLenti6 via Gibson assembly.

Techniques: Gene Expression, Mutagenesis