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. 2021 Nov 19;17(11):e1010100.
doi: 10.1371/journal.ppat.1010100. eCollection 2021 Nov.

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

Marine J Petit  1   2 , Matthew W Kenaston  1 , Oanh H Pham  1 , Ariana A Nagainis  1   2 , Adam T Fishburn  1 , Priya S Shah  1   2
Affiliations

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

Marine J Petit et al. PLoS Pathog. .

Abstract

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. PAF1 restricts DENV2 infectious virion production.
(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).
Fig 2
Fig 2. PAF1 is required for expression of a subset of genes activated by poly(I:C).
(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 S2 Table. (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.
Fig 3
Fig 3. Nuclear localization is required for NS5-PAF1C interaction.
(A) NLS mutants, including single mutant NS5central (REE396-398AAA) and NS5C-term (RR890-891AA) and the double mutant NS52xNLS (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 NS52xNLS 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).
Fig 4
Fig 4. A C-terminal region of MTase domain is responsible for interaction with PAF1C.
(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 NS5full-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, NS5full-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).
Fig 5
Fig 5. A highly conserved region of the MTase C-terminus is required for PAF1C binding.
(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 (NS5LGS) and GTR261AAA (NS5GTR). (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).
Fig 6
Fig 6. NS5 mutants have shared and distinct impacts on gene expression.
(A) Parental A549 cells were transfected with NS5WT (grey), NS52xNLS (purple), NS5LGS (orange), NS5GTR (red), or GFP as a negative control. Transfected cells were stimulated with poly(I:C) for three hours and subjected to RNA-seq. Results are based on three independent biological replicates. (B) GSEA was conducted on genes differentially expressed by cells expressing WT versus mutant NS5s. 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 S2 Table. (C) Genes significantly upregulated in NS52xNLS versus WT NS5 (log2 fold change > 0.5, p adj < 0.05) were subjected to hierarchical clustering for each mutant and displayed as a heat map. Genes of interest are highlighted by zoom panels.
Fig 7
Fig 7. NS5 mutants rescue expression of PAF1-dependent genes.
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 Fig 2E), 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), NS5LGS (orange), NS5GTR (red) and NS52xNLS (purple). Immune response genes (GO:0006955) are highlight with larger markers and opaque coloring.
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