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(A) Parental A549, PAF1 KO/rescue and <t>STAT2</t> 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.

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1) Product Images from "Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1010100

(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.

Figure Legend 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.

Techniques Used: RNA Sequencing, Gene Expression, Comparison

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

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

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.

Figure Legend 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.

Techniques Used: Gene Expression, Mutagenesis

Image Search Results

a Relative IFN signaling (mean ± s.d., n = 6) (defined as the ratio of activities of the two luciferases normalized to naïve HEK-293T’s transfected with reporters only) in naïve and STAT2 KO (confirmed via immunoblot with molecular weight marker positions labeled) HEK-293T cells transfected with reporter plasmids only (dark grey), plus STAT2 plasmid (light grey), or plus STAT2 and ZIKV NS5 plasmids (pink). b Structure of the complex between ZIKV NS5 (grayscale space filling representation) and H. sapiens STAT2 (ribbon representation with domain colored as in ( c ) (composite of PDB:6WCZ, 6UX2). c Schematic of the strategy for the cloning the ND and CCD of various STAT2 orthologs (red fill) as chimeras with the rest of H. sapiens STAT2. d Node dated molecular phylogeny showing species relatedness of 38 mammalian species. Branch length corresponds to divergence time in millions of years with a scale included below. Grey-scale background coloring corresponds to order classification. e STAT2 ortholog ND/CCD chimera expression was compared by immunoblot at 48 h post transient transfection in STAT2 KO HEK-293T cells. Molecular weight marker positions are labeled on the top. f – h Relative IFN signaling (defined in this and all subsequent experiments as defined as the ratio of activities of the two luciferases normalized to H. sapiens STAT2 in the absence of an antagonist) mediated by each of ND/CCD chimeras in the absence of a viral antagonist (grey) and in the presence of f ZIKV NS5 (pink) (mean ± s.d., for Homo sapiens, Mus musculus, and No STAT2, n = 12, all others n = 6). g SPOV NS5 (yellow) (mean ± s.d., n = 6). h DENV2 NS5 (blue) (mean ± s.d., for Homo sapiens, Mus musculus, and No STAT2, n = 12, all others n = 6). Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Journal: Nature Communications

Article Title: Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

doi: 10.1038/s41467-024-49758-0

Figure Lengend Snippet: a Relative IFN signaling (mean ± s.d., n = 6) (defined as the ratio of activities of the two luciferases normalized to naïve HEK-293T’s transfected with reporters only) in naïve and STAT2 KO (confirmed via immunoblot with molecular weight marker positions labeled) HEK-293T cells transfected with reporter plasmids only (dark grey), plus STAT2 plasmid (light grey), or plus STAT2 and ZIKV NS5 plasmids (pink). b Structure of the complex between ZIKV NS5 (grayscale space filling representation) and H. sapiens STAT2 (ribbon representation with domain colored as in ( c ) (composite of PDB:6WCZ, 6UX2). c Schematic of the strategy for the cloning the ND and CCD of various STAT2 orthologs (red fill) as chimeras with the rest of H. sapiens STAT2. d Node dated molecular phylogeny showing species relatedness of 38 mammalian species. Branch length corresponds to divergence time in millions of years with a scale included below. Grey-scale background coloring corresponds to order classification. e STAT2 ortholog ND/CCD chimera expression was compared by immunoblot at 48 h post transient transfection in STAT2 KO HEK-293T cells. Molecular weight marker positions are labeled on the top. f – h Relative IFN signaling (defined in this and all subsequent experiments as defined as the ratio of activities of the two luciferases normalized to H. sapiens STAT2 in the absence of an antagonist) mediated by each of ND/CCD chimeras in the absence of a viral antagonist (grey) and in the presence of f ZIKV NS5 (pink) (mean ± s.d., for Homo sapiens, Mus musculus, and No STAT2, n = 12, all others n = 6). g SPOV NS5 (yellow) (mean ± s.d., n = 6). h DENV2 NS5 (blue) (mean ± s.d., for Homo sapiens, Mus musculus, and No STAT2, n = 12, all others n = 6). Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Article Snippet: To generate STAT2 KO HEK-293T cells, approximately 400,000 HEK-293T(ATCC, CRL-3216) cells were seeded into 6-well plates in complete DMEM.

Techniques: Transfection, Western Blot, Molecular Weight, Marker, Labeling, Plasmid Preparation, Cloning, Expressing, Derivative Assay

a STAT2 ND/CCD schematic showing the locations of the M. musculus resistance determinants, IRF9 contact sites, and the residue found to be rapidly evolving in rodents. Numbering is relative to H. sapiens STAT2. b Relative expression level in absence of antagonist, determined by immunoblot (with molecular weight marker positions on the top) and relative IFN signaling (mean ± s.d., n = 6) mediated by each of indicated STAT2 mutant in the absence of a viral antagonist (grey) and in the presence of ZIKV NS5 (pink). c Immunoblots with the antibodies labeled on the right of STAT2 KO HEK-293T cell whole cell extracts (WCE) and FLAG-antibody immunoprecipitations of the indicated STAT2 proteins with FLAG-tagged ZIKV NS5. To avoid degradation, STAT2 proteins and NS5 were expressed separately, and lysates were mixed prior to immunoprecipitation. Molecular weight marker positions are labeled on the left. Co-immunoprecipitation repeated one other time yielded similar results. d Structure of ZIKV NS5 and STAT2, as shown in Fig. , with residues in zoomed images colored in red to indicate IRF9 contact sites and M. musculus resistance determinants. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Journal: Nature Communications

Article Title: Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

doi: 10.1038/s41467-024-49758-0

Figure Lengend Snippet: a STAT2 ND/CCD schematic showing the locations of the M. musculus resistance determinants, IRF9 contact sites, and the residue found to be rapidly evolving in rodents. Numbering is relative to H. sapiens STAT2. b Relative expression level in absence of antagonist, determined by immunoblot (with molecular weight marker positions on the top) and relative IFN signaling (mean ± s.d., n = 6) mediated by each of indicated STAT2 mutant in the absence of a viral antagonist (grey) and in the presence of ZIKV NS5 (pink). c Immunoblots with the antibodies labeled on the right of STAT2 KO HEK-293T cell whole cell extracts (WCE) and FLAG-antibody immunoprecipitations of the indicated STAT2 proteins with FLAG-tagged ZIKV NS5. To avoid degradation, STAT2 proteins and NS5 were expressed separately, and lysates were mixed prior to immunoprecipitation. Molecular weight marker positions are labeled on the left. Co-immunoprecipitation repeated one other time yielded similar results. d Structure of ZIKV NS5 and STAT2, as shown in Fig. , with residues in zoomed images colored in red to indicate IRF9 contact sites and M. musculus resistance determinants. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Article Snippet: To generate STAT2 KO HEK-293T cells, approximately 400,000 HEK-293T(ATCC, CRL-3216) cells were seeded into 6-well plates in complete DMEM.

Techniques: Residue, Expressing, Western Blot, Molecular Weight, Marker, Mutagenesis, Labeling, Immunoprecipitation, Derivative Assay

a Node dated molecular phylogeny on the left showing species relatedness of H. sapiens and nine rodents species with those possessing a STAT2 resistant to ZIKV NS5 marked in green (*). Arrows mark the roots of two separate acquisitions of resistance to ZIKV NS5 antagonism. To the right is an amino acid alignment of these species spanning the region encoding the previously mapped M. musculus resistance determinants. The numbering on the top and bottom of the alignment corresponds to H. sapiens and M. musculus STAT2 respectively. b Relative IFN signaling (Mean ± s.d, n = 6) of the indicated STAT2 ND/CCD chimeras in the absence (grey) and presence of ZIKV NS5 (pink). c Top, space filling representation of ZIKV NS5 in complex with ribbon representation of H. sapiens STAT2 with a zoomed in view of the region encoding the C. porcellus resistance determinants (red) to the right. Below is a schematic of the STAT2 ND/CCD showing the C. porcellus resistance determinants with numbering corresponding to H. sapiens STAT2. d Relative IFN signaling (Mean ± s.d, n = 6) of the indicate STAT2 chimeras in the absence of an antagonist (grey), or in the presence of ZIKV MR766 NS5 (pink), SPOV NS5 (yellow), DENV1 NS5 (turquoise), DENV2 NS5 (blue), DENV3 NS5 (light purple), and DENV4 NS5 (purple). Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Journal: Nature Communications

Article Title: Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

doi: 10.1038/s41467-024-49758-0

Figure Lengend Snippet: a Node dated molecular phylogeny on the left showing species relatedness of H. sapiens and nine rodents species with those possessing a STAT2 resistant to ZIKV NS5 marked in green (*). Arrows mark the roots of two separate acquisitions of resistance to ZIKV NS5 antagonism. To the right is an amino acid alignment of these species spanning the region encoding the previously mapped M. musculus resistance determinants. The numbering on the top and bottom of the alignment corresponds to H. sapiens and M. musculus STAT2 respectively. b Relative IFN signaling (Mean ± s.d, n = 6) of the indicated STAT2 ND/CCD chimeras in the absence (grey) and presence of ZIKV NS5 (pink). c Top, space filling representation of ZIKV NS5 in complex with ribbon representation of H. sapiens STAT2 with a zoomed in view of the region encoding the C. porcellus resistance determinants (red) to the right. Below is a schematic of the STAT2 ND/CCD showing the C. porcellus resistance determinants with numbering corresponding to H. sapiens STAT2. d Relative IFN signaling (Mean ± s.d, n = 6) of the indicate STAT2 chimeras in the absence of an antagonist (grey), or in the presence of ZIKV MR766 NS5 (pink), SPOV NS5 (yellow), DENV1 NS5 (turquoise), DENV2 NS5 (blue), DENV3 NS5 (light purple), and DENV4 NS5 (purple). Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Article Snippet: To generate STAT2 KO HEK-293T cells, approximately 400,000 HEK-293T(ATCC, CRL-3216) cells were seeded into 6-well plates in complete DMEM.

Techniques: Derivative Assay

a Node dated molecular phylogeny on the left showing species relatedness of H. sapiens and the 15 bat species, with those that have a STAT2 that is resistant to DENV NS5 antagonism marked in green(*). The black arrow indicates the family Vespertilionidae branch point where resistance to DENV NS5 antagonism occurred. On the right is a graph corresponding to the Relative IFN signaling (Mean ± s.d, n = 6) of each species ND and CCD chimeras in the absence (gray) and presence of DENV2 NS5 (blue) b Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the M. natalensis and E. fuscus ND/CCD chimeras and chimeras with reciprocal changes in the resistance determinates mapped in (Supplementary Fig. ). c Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the M. natalensis ND/CCD chimera with each of the eight mapped resistance determinants swapped individually to the E. fuscus sequence. d Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the E. fuscus ND/CCD chimera with each of the eight mapped resistance determinants swapped individually to the M. natalensis sequence. e Top, space filling representation of ZIKV NS5 (as a proxy for DENV NS5) in complex with ribbon representation of H. sapiens STAT2. Zoomed region shows E. fuscus STAT2 resistance determinants (red). Below, schematic of the STAT2 ND/CCD showing the E. fuscus STAT2 resistance determinants, and the residues found to be rapidly evolving using PAML/FUBAR. Underlined numbers indicated resistance determinants that are also rapidly evolving. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Journal: Nature Communications

Article Title: Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

doi: 10.1038/s41467-024-49758-0

Figure Lengend Snippet: a Node dated molecular phylogeny on the left showing species relatedness of H. sapiens and the 15 bat species, with those that have a STAT2 that is resistant to DENV NS5 antagonism marked in green(*). The black arrow indicates the family Vespertilionidae branch point where resistance to DENV NS5 antagonism occurred. On the right is a graph corresponding to the Relative IFN signaling (Mean ± s.d, n = 6) of each species ND and CCD chimeras in the absence (gray) and presence of DENV2 NS5 (blue) b Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the M. natalensis and E. fuscus ND/CCD chimeras and chimeras with reciprocal changes in the resistance determinates mapped in (Supplementary Fig. ). c Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the M. natalensis ND/CCD chimera with each of the eight mapped resistance determinants swapped individually to the E. fuscus sequence. d Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 for the E. fuscus ND/CCD chimera with each of the eight mapped resistance determinants swapped individually to the M. natalensis sequence. e Top, space filling representation of ZIKV NS5 (as a proxy for DENV NS5) in complex with ribbon representation of H. sapiens STAT2. Zoomed region shows E. fuscus STAT2 resistance determinants (red). Below, schematic of the STAT2 ND/CCD showing the E. fuscus STAT2 resistance determinants, and the residues found to be rapidly evolving using PAML/FUBAR. Underlined numbers indicated resistance determinants that are also rapidly evolving. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Article Snippet: To generate STAT2 KO HEK-293T cells, approximately 400,000 HEK-293T(ATCC, CRL-3216) cells were seeded into 6-well plates in complete DMEM.

Techniques: Sequencing, Derivative Assay

a Left, STAT2 ND/CCD schematic with resistance (red), susceptibility (pink), and rapidly evolving residues via PAML/FUBAR (black) marked. Right, space filling representation of ZIKV NS5 (as a proxy for DENV NS5) in complex with ribbon representation of H. sapiens STAT2. Zoomed in regions show two views of the DENV NS5 resistance determinants mapped in P. coquereli STAT2 (red) and the residues involved in the reversion to susceptibility (pink). Underlined numbers indicated resistance determinants that are also rapidly evolving. Note: STAT2 residue 127 is not resolved in this structure. b Relative IFN signaling (Mean ± s.d, n = 6) of STAT2 chimeras in the absence (grey) and presence (blue) of DENV2 NS5. c Species level node dated molecular phylogeny with alignments of the STAT2 sequence at residues defined as resistance and susceptibility determinants. Species with STAT2 susceptible to DENV NS5 antagonism are marked (*). Arrows (1) and (2) indicate the acquisition of resistance to the reversion to susceptibility, respectively. d Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 of (i) P. coquereli STAT2 with L. catta amino acids (ii) L. catta STAT2 with P. coquereli amino acids (iii) L. catta STAT2 with E. flavifrons amino acids (iv) E. flavifrons STAT2 with L. catta amino acids. e Relative STAT2 abundance (Mean ± s.d, n = 3) following DENV (blue) or ZIKV (pink) infection compared to the uninfected condition for each cell line. Values are normalized to the percent infection for each virus in each cell line (quantification of Supplementary Fig. , gating strategy and quantification in Supplementary Fig. ). Data is generated from the quantification of three independent experiments. f Relative IFN signaling (Mean ± s.d, n = 6) of STAT2 chimeras with increasing amounts of DENV2 NS5. Values are normalized to signaling in absence of DENV2 NS5 and the baseline is set to reporter levels in the absence of STAT2. Curves are fit using least-squares fit method of non-linear regression to calculate IC50 values for each STAT2 in units of ng of DENV2 NS5 plasmid. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Journal: Nature Communications

Article Title: Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints

doi: 10.1038/s41467-024-49758-0

Figure Lengend Snippet: a Left, STAT2 ND/CCD schematic with resistance (red), susceptibility (pink), and rapidly evolving residues via PAML/FUBAR (black) marked. Right, space filling representation of ZIKV NS5 (as a proxy for DENV NS5) in complex with ribbon representation of H. sapiens STAT2. Zoomed in regions show two views of the DENV NS5 resistance determinants mapped in P. coquereli STAT2 (red) and the residues involved in the reversion to susceptibility (pink). Underlined numbers indicated resistance determinants that are also rapidly evolving. Note: STAT2 residue 127 is not resolved in this structure. b Relative IFN signaling (Mean ± s.d, n = 6) of STAT2 chimeras in the absence (grey) and presence (blue) of DENV2 NS5. c Species level node dated molecular phylogeny with alignments of the STAT2 sequence at residues defined as resistance and susceptibility determinants. Species with STAT2 susceptible to DENV NS5 antagonism are marked (*). Arrows (1) and (2) indicate the acquisition of resistance to the reversion to susceptibility, respectively. d Relative IFN signaling (Mean ± s.d, n = 6) in the absence (grey) and presence (blue) of DENV2 NS5 of (i) P. coquereli STAT2 with L. catta amino acids (ii) L. catta STAT2 with P. coquereli amino acids (iii) L. catta STAT2 with E. flavifrons amino acids (iv) E. flavifrons STAT2 with L. catta amino acids. e Relative STAT2 abundance (Mean ± s.d, n = 3) following DENV (blue) or ZIKV (pink) infection compared to the uninfected condition for each cell line. Values are normalized to the percent infection for each virus in each cell line (quantification of Supplementary Fig. , gating strategy and quantification in Supplementary Fig. ). Data is generated from the quantification of three independent experiments. f Relative IFN signaling (Mean ± s.d, n = 6) of STAT2 chimeras with increasing amounts of DENV2 NS5. Values are normalized to signaling in absence of DENV2 NS5 and the baseline is set to reporter levels in the absence of STAT2. Curves are fit using least-squares fit method of non-linear regression to calculate IC50 values for each STAT2 in units of ng of DENV2 NS5 plasmid. Source data are provided as a file. All IFN signaling values are derived from at least two independent experiments each with three technical replicates.

Article Snippet: To generate STAT2 KO HEK-293T cells, approximately 400,000 HEK-293T(ATCC, CRL-3216) cells were seeded into 6-well plates in complete DMEM.

Techniques: Residue, Sequencing, Infection, Virus, Generated, Plasmid Preparation, Derivative Assay

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 KO were verified by Sanger sequencing and immunoblotting using antibodies against PAF1 (1:1000, Bethyl Labs, A300-173A) or STAT2 (1:200, Santa Cruz Biotechnology) ( ).

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).

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.

Techniques: Gene Expression, Mutagenesis

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