Oncogenic virus hijacks SOX18 pioneer function to enhance viral persistence

Abstract

Kaposi's sarcoma herpesvirus (KSHV) establishes lifelong oncogenic infection in lymphatic endothelial cells (LECs) by ensuring episomal maintenance of its genome via the viral protein LANA. Efficient viral genome maintenance typically involves host DNA replication and episome tethering, but the extent of cell-type-specific regulation remains unclear. Here, we identify that KSHV hijacks the pioneering function of the endothelial-specific transcription factor SOX18 to facilitate persistence of viral episomes. Upon infection, LANA co-opts SOX18 to recruit the SWI/SNF chromatin-remodeling complex via its ATPase subunit BRG1, enhancing chromatin accessibility and enabling efficient viral genome persistence. Disruption of SOX18 or BRG1-genetically or pharmacologically-leads to reduced episome load and attenuated hallmarks of virus infection. This work highlights how viruses can harness lineage-specific transcriptional regulators to establish persistent nuclear retention of their episome into the host genome.

Keywords: BRG1; KSHV; SOX18; Transcription factor; chromatin; endothelial cell; herpesvirus; pioneer; small molecule inhibitor.

Figure 1.. SOX18 recruits SWI/SNF chromatin remodeling complex upon KSHV infection.

A-B. Bio-ID proximity-based protein-protein interaction screen using a BirA*-fusion of SOX18 (BirA*SOX18) or Cherry (BirA*Cherry) as a control in A. stably KSHV-infected iSLK.219 cells or B. parental, uninfected SLK cells. The strength of the interaction of SOX18 with the indicated proteins is shown as ≥ 2 log2 fold change FC (x-axis) and BirA*Cherry bait-normalized PSM = peptide spectral matches (y-axis). All shown proteins have ≥2 unique peptides. C. Heatmap of the canonical SWI/SNF (cBAF) complex subunits in all conditions. D. A schematic of the cBAF complex; ARID1A and BRG1, the top interactors of SOX18, are highlighted in purple and blue, respectively. E-H. Validation of the selected interactions by PLA with the indicated antibodies. E. PLA images of uninfected LECs (LEC) and rKSHV.219 -infected LECs (KLEC) at 72 h.p.i (hours post infection) treated with Sm4 or DMSO for 72h and imaged with Opera Phenix 40x, nuclei were counterstained with Hoechst (33342), scale bar is 20μm. F-G. Quantification of nuclear PLA puncta from 10 fields (n=100 nuclei) for F. SOX18-ARID1A and G. SOX18-BRG1 interactions in all conditions. H. LANA-BRG1 and LANA-ARID1A PLA images and I-J. quantification of nuclear PLA puncta from 10 fields (n=100 nuclei) in KLECs. Statistical significance was determined by ordinary one-way ANOVA with Tukey correction for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant.

Figure 2.. Perturbations to SOX18 causes changes in chromatin accessibility.

A. Representative images of HUVECs treated with DMSO or Sm4 and stained with SiR-DNA, scale bar is 50μm for confocal images and 5μm for STED images. B. Mean intensity of SiR-DNA from cells in panel A, n ≥ 137 cells/condition. Statistical significance was determined by Mann-Whitney test ****p < 0.0001. C. Line intensity profile of STED images from panel A. Solid (N1) and dashed (N2) lines represent individual nuclei (N) line profiles. D-E. Quadratic discriminant MIEL analysis using texture features derived from images of HUVECs ± SOX18 over-expression and ± Sm4 treatment stained with DAPI. D. Scatter plot depicts the first two discriminant factors for each cell population: each point is a pool of 60 cells. E. Matrix showing results for the discriminant analysis. Numbers represent the percent classified correctly (diagonal) and incorrectly (off the diagonal). F-G. LECs treated with DMSO or Sm4 for 24h and subjected for ATAC-seq. F. Data presented as volcano plot showing the human genomic regions with significant loss (turquoise) or gain (red) of accessibility and as number of differentially accessible regions (DAR) that have changed and G. as TF differential binding score volcano plot.

Figure 3.. Chromatin compaction state feedback on SOX18 mobility and oligomeric states.

A. Representative images and maps of SOX18 oligomeric states (monomer – dark green, dimer – light green, and oligomer – red) of HeLa cells transfected with SOX18 and treated with either DMSO, Trichostatin A (TSA), or Actinomycin D (ActD) and measured by N&B. B-D. Quantification of oligomeric states by N&B. For panels B-D, n > 5 cells, statistical significance was determined by one-way ANOVA with a Dunnett correction for multiple comparisons, *p < 0.05, **p < 0.01, ****p < 0.0001. E. Diffusion mobility graph from single molecule tracking (SMT) acquisition comparing SOX18 mobility in HeLa cells that are transfected with Halo-SOX18 and treated either with DMSO or TSA. Pie charts represent the proportion of the trajectory population that is found in either the confined or non-confined states based on its diffusion coefficient. F. Ratio of non-confined to confined molecules per cell from F. G-I. Temporal occupancy characteristics for HeLa cells transfected with Halo-SOX18 and treated with TSA as G) short occupancy time, H) long occupancy time, I) ratio of long to short occupancy times. For panels G-I, n > 13 cells, statistical significance was determined by Welch’s t-test, **p < 0.01, ***p < 0.001.

Figure 4.. KSHV hijacks SOX18 pioneer activity to increase chromatin accessibility in LECs.

A-G. Uninfected LECs (LEC) or LECs infected with KSHV-BAC16-ΔORF50 for 48h (ΔORF50-KLEC) were treated with DMSO or Sm4 for 24h and subjected for ATAC-seq. A-B. Volcano plots showing the human genomic regions with significant loss (turquoise) or gain (red) of accessibility upon A) KSHV infection and B) Sm4 treatment. C. Heatmap of the accessibility changes in the top 1000 sites upon KSHV infection (red line; KSHV gain), sites with accessibility loss after Sm4 treatment in ΔORF50-KLECs (light turquoise line; Sm4 loss) and sites with both accessibility gain upon infection and accessibility loss after Sm4 treatment (dark blue; shared). D-E. Volcano plots showing TF differential binding score prediction upon D) KSHV infection and E) Sm4 treatment in ΔORF50-KLECs. TFs with significant binding loss (turquoise), binding gain (red), and SOX family of TFs are marked (yellow). F. Top HOMER de novo transcription factor family motif enrichment gains upon infection and loss upon Sm4 treatment in ΔORF50-KLECs. G. Analysis of ATAC-seq peaks to show representative enhancer region with differential motifs and accessibility upon KSHV infection and SOX18 inhibition by Sm4 treatment in LECs and ΔORF50-KLECs. H-I. LECs infected with rKSHV.219 for 72h (KLEC) and treated with DMSO or Sm4 for 24h were H) labeled with anti- HP1α antibodies and imaged with Zeiss LSM880 confocal 63x for heterochromatin regions, nuclei were counterstained with DAPI, scale is bar 10μm. I) The mean nuclear intensity a.u. (arbitrary units) of HP1α signal quantified (n=200). Statistical significance was determined by one-way ANOVA with a Tukey correction for multiple comparisons, ***p < 0.001. J-K. Quadratic discriminant MIEL analysis using texture features derived from images of LECs and KLECs treated with DMSO or Sm4 24h and stained with DAPI and anti-HP1α antibodies. J. Scatter plot depicts the first two discriminant factors for each cell population. Each point is a pool of 60 cells. K. Matrix showing results for the discriminant analysis. Numbers represent the percent classified correctly (diagonal) and incorrectly (off the diagonal). L. Average distance matrix calculated from the distance between each point per condition, with blue as farthest distances and red as closest distances.

Figure 5.. SWI/SNF ATPase activity is required for the hallmarks of KSHV infection in LECs.

A-C. LECs were transfected with siRNAs targeting ARID1A, BRG1 or scramble (siScr) as a control for 24h, and thereafter infected with rKSHV.219 for 72h (KLEC). A. Immunoblotting for ARID1A and BRG1, and β-actin as a loading control. B. GFP-expressing KLECs imaged with Opera Phenix 20x for changes in the cell spindling phenotype. Nuclei were counterstained with Hoechst (33342), scale bar 100 μm, in magnification 30μm. C. KLECs quantified for normalized KSHV episome genome copies by qPCR (n=5). D-H. LECs infected with rKSHV.219 for 72h were treated with SWI/SNF inhibitors ACBI1, FHT-1015 and PFI-3 for 72h. D. Immunoblotting of KLECs for the indicated host proteins and LANA, and β-actin as a loading control. E. GFP-expressing KLECs imaged with Opera Phenix 20x for changes in spindling phenotype. Nuclei were counterstained with Hoechst (33342), scale bar is 100μm, in magnification 30μm. F. KLECs quantified for normalized KSHV genome copies by qPCR (n=3). G. Inhibitor-treated KLECs were labeled forHP1α and imaged with LSM 880 confocal 63x. Nuclei were counterstained with Hoechst (33342), scale bar is 10μm. H. The mean a.u. (arbitrary units) nuclear intensity of quantified HP1α signal (n=100 nuclei). Statistical significance was determined by one-way ANOVA with a Dunnett or Tukey correction for multiple comparisons, **p < 0.01, ***p < 0.001, ns = non-significant.

Figure 6.. KSHV episome maintenance relies on a functional SOX18-BRG1 axis to increase LANA occupancy to KSHV TR.

A-C. HeLa cells expressing SOX18wt, mutants C240X (dominant negative transactivation deficient) or HMGdel (DNA-binding deficient), or mCherry as a control, and thereafter infected with rKSHV.219 for 72h and A) measured for normalized KSHV genome copies by qPCR, B) subjected to ChIP-PCR with anti-LANA and IgG antibodies and analyzed for LANA binding at TR (n=3) or C) treated with BrdU for 4h and subjected to BrdU pulldown assay for nascent KSHV genome synthesis (n=3). D-E. KLECs treated with DMSO or Sm4 subjected to PLA assay using anti-SOX18 and anti-LANA antibodies, nuclei were counterstained with Hoechst (33342), D) imaged with Opera Phenix 40x, scale bar is 20μm and E) quantified as number of nuclear (n=100) PLA puncta (right panel). F. KLECs treated with DMSO, Sm4 or FHT-1015 for 24h were subjected to ChIP-PCR as described in B. (n=2). Two TR primers were used, and mean was taken for each replicate. G. KLECs treated with indicated inhibitors for 72h were subjected to BrdU pulldown assay as described in C (n=3). H. KLECs stained with anti-LANA and anti-H2A antibodies and imaged with LSM880 63x confocal, nuclei were counterstained with Hoechst (33342), scale bar is 10μm. I-J. Quantification of mean nuclear number of LANA speckles (n=100) and mean a.u. (arbitrary units) of H2A signal intensity (n=100). Statistical significance was determined by one-way ANOVA with Dunnett or Tukey correction for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant.

Figure 7.. Graphical abstract.

Top panel: The SOX18 transcription factor exhibits a pioneering role through its interaction with the SWI/SNF complex, shaping chromatin accessibility and genome organization in LECs. Middle panel: Upon KSHV infection, SOX18 is upregulated, and the viral LANA protein hijacks the SOX18/BRG1 pioneer complex to anchor viral episomes onto the host genome. This LANA–SOX18–BRG1 axis establishes a chromatin environment conducive to latent viral genome replication. Bottom panel: Pharmacological disruption of the host chromatin machinery impairs SOX18 or BRG1 function and consequently inhibits viral genome duplication. This highlights previously unrecognized host-derived therapeutic targets for the treatment of KSHV infection and the associated diseases.