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

['Marine J. Petit', 'Department Of Microbiology', 'Molecular Genetics', 'University Of California', 'Davis', 'California', 'United States Of America', 'Department Of Chemical Engineering', 'Matthew W. Kenaston', 'Oanh H. Pham']

Date: 2021-12

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.

Dengue virus (DENV) is a pathogen that infects nearly 400 million people a year and thus represents a major challenge for public health. Productive infection by DENV relies on the effective evasion of intrinsic antiviral defenses and is often accomplished through virus-host protein interactions. Here, we investigate the recently discovered interaction between DENV non-structural protein 5 (NS5) and the transcriptional regulator Polymerase associated factor 1 complex (PAF1C). Our work demonstrates PAF1C member PAF1 acts as an antiviral factor and inhibits DENV replication. In parallel, we identified immune response genes involved in intrinsic antiviral defense that depend on PAF1 for expression. We further identified the regions of NS5 required for the protein interaction with PAF1C. Breaking the NS5-PAF1C protein interaction restores the expression of PAF1-dependent immune response genes. Together, our work establishes the antiviral role of PAF1C in DENV infection and NS5 antagonism of PAF1-dependent gene expression through a virus-host protein interaction.

Funding: Funding was provided by University of California, Davis and the W. M. Keck foundation to PSS. MJP was partially supported by the Philippe Foundation Inc. MWK was partially supported by the UC Davis Provost’s Undergraduate Fellowship. ATF was supported by a NIH T32 fellowship (2T32AI060555-16). The sequencing was carried out at the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01. The Olympus FV1000 confocal used in this study was purchased using NIH Shared Instrumentation Grant 1S10RR019266-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Petit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we investigate if NS5 antagonism of PAF1C may be complementary to the antagonism of STAT2. We find that PAF1 restricts DENV2 infectious virion production. We also find that PAF1 is required for the expression of genes that are primarily STAT2-independent following innate immune stimulation. We further map the viral determinants of the NS5-PAF1C interaction using affinity purification and immunoblot of NS5 mutants to show that NS5 nuclear localization and the C-terminal region of the MTase domain are required for its interaction with PAF1C. NS5 mutants with reduced binding to PAF1C also have reduced antagonism of PAF1-dependent and STAT2-independent genes. Taken together, PAF1C-mediated expression of immune response genes is STAT2-independent and breaking the NS5-PAF1C interaction rescues PAF1-dependent gene expression.

Using a comprehensive global proteomics approach that defined the DENV-host protein interaction landscape, we recently found DENV2 16681 NS5 interacts with and antagonizes Polymerase associated factor 1 complex (PAF1C) [ 14 ]. This nuclear complex, whose primary members are PAF1, LEO1, CTR9, and CDC73, has emerging significance in immunology. The complex generally promotes the expression of many genes, including genes related to the antiviral, antimicrobial, and inflammatory responses [ 15 , 16 ]. We previously showed that NS5 antagonizes PAF1C by inhibiting its recruitment to immune response genes [ 14 ]. However, it is currently unknown if NS5 inhibits expression of immune response genes distinct from STAT2-dependent ISGs through this protein interaction. Such antagonism may also explain a possible synergistic role for nuclear NS5. Yet, these mechanistic details have not been explored.

Interestingly, NS5 also has a nuclear role. DENV serotype 2 (DENV2) NS5 steady-state localization to the nucleus during infection has been known for over 25 years [ 7 ] and observed by several other groups independently [ 8 – 11 ]. For other DENV serotypes, NS5 has shown varying steady-state distribution between the nucleus and cytoplasm. For example, DENV1 NS5 is equally distributed across the cell while DENV2 NS5 is predominantly nuclear [ 9 , 11 ]. Despite these variations in NS5 nuclear localization, the consistent detection of NS5 in the nucleus suggests the conservation of NS5 nuclear-cytoplasmic shuttle in mammalian cells. DENV2 mutants that retain some NS5 nuclear localization are viable and can even replicate at near-wild-type (WT) levels. On the other hand, mutants that essentially eliminate NS5 nuclear localization are lethal [ 8 – 10 ]. These defects in replication are not due to decreased intrinsic enzyme activity of NS5 [ 8 , 9 ]. We and others have shown that NS5 of DENV2 16681 interacts with many nuclear proteins [ 12 – 14 ] and inhibits antiviral gene expression, potentially through these interactions [ 13 , 14 ]. Thus, some nuclear localization of DENV2 16681 NS5 is essential for replication and rewiring host gene expression, but the mechanisms by which nuclear DENV2 NS5 accomplishes these tasks are not completely understood.

Dengue virus (DENV) is a major source of human disease and is responsible for nearly 400 million infections annually [ 1 ]. DENV belongs to the Flavivirus genus of viruses, which are positive sense, capped single-stranded RNA viruses that rely on multifunctional proteins to replicate. Flavivirus non-structural protein 5 (NS5) functions as both the RNA-dependent RNA polymerase (RdRp) [ 2 ] and the methyltransferase (MTase) domain, displaying N7 and 2’-O methylation [ 3 – 5 ]. Genome replication is the main enzymatic activity of NS5 and occurs on the cytosolic side of ER-associated viral replication factories. In addition to playing a central role in genome replication, NS5 is a key player in disabling the innate immune response during DENV infection. Importantly, host protein interactions with NS5 are critical to its role in immune evasion. DENV NS5 binds human STAT2 and targets it for ubiquitin-mediated proteasomal degradation. This inhibits the expression of interferon-stimulated genes (ISGs) through interferon type 1 (IFN-I) signaling [ 6 ].

Results

PAF1C member PAF1 restricts DENV2 replication PAF1C is an emergent antiviral factor in mammals [15,16]. We previously showed that PAF1C restricts DENV2 replication by quantifying DENV antigen-positive cells by immunofluorescence microscopy [14]. To determine the impact of PAF1C on the full viral replication cycle, we measured infectious virion production in PAF1 knockout (PAF1 KO) A549 cells at several time points. Since the entire complex can be destabilized with depletion of PAF1 only [17], we chose this member as a target for knockout. Generating PAF1 KO cells required isolating single clones. Therefore, we generated PAF1 rescue cells to control for clonal and CRISPR/Cas9 off-target effects (Figs 1A and S1A). Infectious virion production increased approximately 3-fold at 48- and 72-hours post-infection in PAF1 KO cells (Fig 1B). This replication advantage in PAF1 KO cells was significant (p ≤ 0.05) but disappeared at 96 hours post-infection. To account for the two rounds of lentiviral transduction the PAF1 rescue cells experienced, we created PAF1 KO cells transduced with a GFP-expressing lentivirus. We also created a non-targeting gRNA from parental A549 cells, though this cell line is not clonally selected like the PAF1 KO cells. While overall replication kinetics were slightly different in this experiment, overall trends remained the same. Infectious virion production was approximately 5- and 14-fold higher at 72 and 96 hours post-infection, respectively in GFP-expressing PAF1 KO cells compared to PAF1 rescue cells (S1C Fig). The replication advantage in PAF1 KO cells was significant (p < 0.05 and p < 0.005). The cell line with non-targeting gRNA did display different replication kinetics, likely due to differences arising from clonal selection of the PAF1 KO cells, which were used to generate the PAF1 rescue cells (S1D Fig). An immunoblot comparing PAF1 expression in the cell lysate of the PAF1 rescue cells and non-targeting gRNA cell line shows lower expression in the PAF1 rescue cells (S1A Fig). Immunofluorescence microscopy analysis revealed slightly higher fluorescence intensity in PAF1 rescue cells, possibly resulting from a different antibody used for immunofluorescence compared to immunoblot. Notably, this difference in intensity was not significant (p = 0.98), and the heterogeneity of PAF1 expression in rescue cells was similar to parental cells (S1E and S1F Fig). These results suggest that the double transduction does not affect the overall behavior of PAF1 rescue cells compared to PAF1 KO cells. Based on these results, we conclude that PAF1 restricts DENV2 infectious virion production in human cells. PPT PowerPoint slide

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TIFF original image Download: 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). https://doi.org/10.1371/journal.ppat.1010100.g001

PAF1 is required for the expression of STAT2-independent genes PAF1C regulates the expression of stress response genes [14], including ISGs and inflammatory genes [15,16]. Since NS5 interacts with STAT2 and PAF1C, we hypothesized that PAF1C could play a unique role upstream or independent of STAT2-mediated IFN-I signaling. For this reason, we studied gene expression following activation of the immune response using a three-hour treatment with poly(I:C), a dsRNA mimic that can stimulate RIG-I, MDA5 and TLR3 signaling [18–21]. Importantly, our treatment was long enough to induce IFNBI expression in A549 cells (log2 fold change 11.9, p adj < 10−19) (S1 Table) and could thus capture events upstream and downstream of IFN-I signaling. To distinguish the PAF1- and STAT2-dependent responses, we performed experiments in PAF1 and STAT2 KO and rescue cells (Fig 2A). We assessed data quality on a global level using principal component analysis (PCA) (Fig 2B). Strong clustering of parental A549, PAF1 rescue, and STAT2 rescue cells suggests the effects observed on gene expression are not a result of clonal or CRISPR/Cas9 off-target effects, and we can compare the different KO cells to the same parental control for gene expression. It also suggests that the replication difference between non-targeting gRNA cells (bulk population) and PAF1 rescue cells (created from a clonal PAF1 KO line) (S1C Fig) is driven by differences not captured in the PCA, such as changes in cell metabolism or survival. Additionally, we show that PAF1 and STAT2 KO form distinct clusters for both poly(I:C) treated and control samples, suggesting that PAF1 and STAT2 have unique roles in basal and immune gene expression. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010100.g002 To gain additional insight into PAF1- and STAT2-mediated gene expression, we analyzed global changes using Gene Set Enrichment Analysis (GSEA) [22,23]. Several Reactome pathways were significantly enriched among PAF1 and STAT2 KO cells compared to parental A549 cells (Fig 2C and S1 and S2 Tables). Distinct enriched pathways were observed for each genotype, corroborating our PCA results. As expected, pathways related to IFN-I signaling were significantly downregulated in STAT2 KO cells (p adj < 0.01). Extracellular matrix organization (ECM) pathways were also significantly upregulated in STAT2 KO cells, indicating an additional role for STAT2 in repressing the expression of these genes (p adj < 0.01). Top downregulated categories in PAF1 KO cells were related to PAF1C roles in chromatin modification (p adj < 0.005) or regulation of TP53 and DNA double-strand break repair pathways (p adj < 0.1). One significantly downregulated category, the DDX58-IFIH1 (RIG-I-MDA5) induction of IFN-I response (p adj < 0.05), is important for the production of IFN-I during DENV infection [24–29]. Therefore, PAF1 may regulate the immune response upstream of IFN-I signaling. In yeast, the homolog of human PAF1C is associated with histone modifications that affect gene expression [30]. However, we did not observe any clear differences in global histone methylation in our PAF1 KO cells compared to parental A549 cells (S2 Fig), suggesting that these changes are not a result of global changes in PAF1 activity, but more gene specific. Surprisingly, significantly upregulated pathways in PAF1 KO cells revealed several categories that may be pro-flaviviral in nature (Fig 2C). While generally thought of as a transcription elongation factor that promotes gene expression, PAF1C can repress expression in some contexts, such as genes with highly active super-enhancers in cancer cells [31]. Given the context-dependent nature of PAF1C in promoting or repressing gene expression, we hypothesized that PAF1C-mediated repression of pro-flaviviral genes may contribute to PAF1C restriction of DENV2 replication. As such, we examined genes composing the upregulated pathways in PAF1 KO cells and found many were related to known flavivirus host dependency factors. To rigorously test if PAF1 was repressing the expression of pro-flaviviral factors, we performed GSEA on flavivirus host dependency factors identified through genetic screens [32–37] (S3 Table). We found that PAF1 KO cells had significant upregulation of expression of these genes (p adj < 0.005) (S3 Fig). This effect is PAF1-specific as STAT2 KO cells did not show a significant change in expression for this group of genes. We next explored the role of PAF1 in the expression of specific genes. We analyzed immune response genes (GO:0006955) whose expression was significantly induced in parental A549 cells following poly(I:C) treatment (log2 fold change > 0.5, p adj < 0.05) (S4 Table). This subset of genes allows the unbiased comparison of PAF1 and STAT2 KO cells to parental A549 cells in the context of the immune response. Both PAF1 and STAT2 KO resulted in the significant inhibition of expression of these immune response genes relative to parental A549 cells (Fig 2D). We also looked at genes that were significantly downregulated in PAF1 or STAT2 KO cells compared to A549 cells following poly(I:C) treatment (Fig 2E). While a few genes were significantly affected by both PAF1 and STAT2 KO, many genes displayed PAF1- and STAT2-specific responses. The overlap between the PAF1- and STAT2-dependent gene sets was significantly lower than expected by chance (p < 0.05). Unbiased k-means clustering formed unique clusters comprising PAF1- and STAT2-dependent genes, further underlining these differences. Our results indicate PAF1 and STAT2 have distinct but complementary roles and might function in synergy following poly(I:C) stimulation.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010100

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