Influence of ND10 components on epigenetic determinants of early KSHV latency establishment

Abstract

We have previously demonstrated that acquisition of intricate patterns of activating (H3K4me3, H3K9/K14ac) and repressive (H3K27me3) histone modifications is a hallmark of KSHV latency establishment. The precise molecular mechanisms that shape the latent histone modification landscape, however, remain unknown. Promyelocytic leukemia nuclear bodies (PML-NB), also called nuclear domain 10 (ND10), have emerged as mediators of innate immune responses that can limit viral gene expression via chromatin based mechanisms. Consequently, although ND10 functions thus far have been almost exclusively investigated in models of productive herpesvirus infection, it has been proposed that they also may contribute to the establishment of viral latency. Here, we report the first systematic study of the role of ND10 during KSHV latency establishment, and link alterations in the subcellular distribution of ND10 components to a temporal analysis of histone modification acquisition and host cell gene expression during the early infection phase. Our study demonstrates that KSHV infection results in a transient interferon response that leads to induction of the ND10 components PML and Sp100, but that repression by ND10 bodies is unlikely to contribute to KSHV latency establishment. Instead, we uncover an unexpected role for soluble Sp100 protein, which is efficiently and permanently relocalized from nucleoplasmic and chromatin-associated fractions into the insoluble matrix. We show that LANA expression is sufficient to induce Sp100 relocalization, likely via mediating SUMOylation of Sp100. Furthermore, we demonstrate that depletion of soluble Sp100 occurs precisely when repressive H3K27me3 marks first accumulate on viral genomes, and that knock-down of Sp100 (but not PML or Daxx) facilitates H3K27me3 acquisition. Collectively, our data support a model in which non-ND10 resident Sp100 acts as a negative regulator of polycomb repressive complex-2 (PRC2) recruitment, and suggest that KSHV may actively escape ND10 silencing mechanisms to promote establishment of latent chromatin.

Figure 1. Histone modification profiles acquired by transfected KSHV bacmids and de novo infecting episomes.

(A) SLK cells were transfected with KSHV BAC16 DNA and selected with hygromycin for 24 days to select for bacmid-carrying cells. Histone modifications were analyzed by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). (B) SLK cells were infected with KSHV and chromatin was prepared at indicated time points, and histone modification profiles were investigated by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). Arrows above H3K4me3 profiles denote peaks that are prominent at 24 h post infection, but were diminished upon acquisition of repressive H3K27me3 marks at later time points. Normalized signal intensity values from the profiles shown in A and B as well as from previously investigated SLK cells at 5 d post infection (5 dpi) were used to generate the heat maps shown in (C). The heat maps indicate the chromatin status as being either naïve (black), dominated by H3K4me3 (green) or H3K27me3 (red), or characterized by the presence of both modifications (yellow). The latter state is designated as ‘putative bivalent’ since co-occurrence of both modifications on the same nucleosome has formally been proven only for the ORF50/Rta promoter .

Figure 2. LANA foci do not co-localize with ND10/PML.

SLK cells were infected with KSHV, fixed at the indicated time point, and analyzed for LANA and PML using standard IF staining and confocal microscopy procedures. DAPI images represent one confocal plane, whereas LANA and PML are depicted as maximum intensity projections to demonstrate separate localization of both proteins in all three dimensions.

Figure 3. Soluble Sp100 levels are reduced upon KSHV infection of SLK cells.

(A) Analysis of low-salt soluble RIPA extracts prepared from mock infected or KSHV infected SLK cells after 48 hours. (B) Analysis of soluble low-salt RIPA extracts prepared from long-term KSHV infected SLK cells (SLKp, lane 6), mock infected SLK cells (lane 1), or SLK cells that had been infected with KSHV for the indicated time points (lanes 2–5). (C) Analysis of total protein extracts prepared from mock infected SLK cells (lane 1) or SLK cells infected with KSHV for the indicated time points. (D) Analysis of soluble low-salt extractable (left panel) or total (right panel) protein fractions in uninfected (SLK) or long-term KSHV infected SLK cells (SLKp). β-actin served as a loading control in all panels.

Figure 4. Soluble Sp100 levels are reduced upon KSHV infection of HDF, EA.hy and HUVEC cells.

Western blot analysis of low-salt soluble RIPA extracts prepared from: (A) HDF cells that had been infected with KSHV for the indicated time points (left panel, lanes 1–6) or mock infected cells at 0 h (left panel, lane 7) or between 8 h and 10 days post treatment (right panel, lanes 1–5), (B) mock infected HDF cells (lane 8) or HDF cultures exposed to UV-irradiated KSHV supernatants (lanes 1–7), (C) KSHV or mock infected EA.hy cells and (D) KSHV or mock infected HUVEC cells. β-actin served as a loading control in all panels.

Figure 5. Proteasomal degradation and transcriptional silencing are not responsible for loss of soluble Sp100.

(A) KSHV negative SLK cells or long-term infected SLK_P cells were treated with either the proteasome inhibitor MG-132 or DMSO for 8 h. Subsequently, soluble RIPA extracts were prepared and analyzed for Sp100 and LANA protein levels by immunoblotting. (B) Transcript levels of genes encoding the ND10 core components (PML, SP100, DAXX) and the three housekeeping genes GAPDH, ADH5 and VPS29. Transcript levels were analyzed by RNAseq (see complete dataset in Dataset S1) in mock infected (0 h value) or KSHV infected SLK cells after 2, 4, 8, 12, 16, 24, 48 or 96 hours of infection and are given as RPKM (reads per kilobase and million mapped reads) values. Baseline expression levels as observed in mock infected cells are marked across plots by a dashed gray line. The fold range (FR) of maximum up- or down-regulation across the entire time course is indicated in each panel.

Figure 6. LANA does not co-localize with Sp100, and KSHV infection does not disperse Sp100 from ND10s.

(A) SLK cells were infected with KSHV and analyzed for LANA and Sp100 using standard IF staining procedures. LANA and Sp100 are depicted as maximum intensity projections from z-stacks to demonstrate separate localization of both proteins in all three dimensions. (B) The number of Sp100 containing dots per nucleus (left panel) and total volume of Sp100 dots (sum of volume of all dots within each individual nucleus; right panel) were determined in >60 cells in mock infected or KSHV infected cells at 72 h post infection using the Volocity software (see material and methods for details). Each dot represents a single cell. Bars represent mean and SEM values.

Figure 7. Sp100 accumulates in the insoluble matrix upon de novo infection and in PEL-derived B-cell lines.

Western-blot analysis of sub-fractionated cellular extracts (cyto: cytoplasmic; memb: membrane-associated; nucpl: nucleoplasmic; chrom: chromatin-associated; matrix: associated with the insoluble matrix) from: (A) EA.hy cells de novo infected with KSHV and harvested 72 h after infection or (B) PEL-derived (BCBL1, HBL6, AP2) or BL-derived (BJAB, Jijoye, Raji) B cell lines. For each B cell line, infection status for KSHV and EBV is indicated to the right of the panel. Successful fractionation was confirmed by detection of specific marker proteins (Hsp70, Sp1, HDAC2 and Vimentin).

Figure 8. LANA induces relocalization and SUMOylation of Sp100.

(A) EA.hy cells were transduced with an YFP labeled retroviral LANA expression construct (right panel) or a control retrovirus (left panel). YFP positive cells were enriched by FACS to reduce background of non-transduced cells. After subcellular fractionation, the localization of Sp100 in LANA was analyzed by western blotting. Successful fractionation was confirmed by detection of specific marker proteins (Hsp70, Sp1 and Vimentin). Abbreviations above each lane indicate the following fractions: cyto: cytoplasmic; memb: membrane-associated; nucpl: nucleoplasmic; chrom: chromatin-associated; matrix: insoluble matrix-associated. (B) Stable HIS-tagged SUMO-1 or SUMO-2 expressing and parental HeLa cells were transfected with pcDNA3-LANA or a control vector for 72 hours. Protein lysates were purified via Ni-NTA affinity chromatography and SUMOylated Sp100 or all precipitated SUMOylated proteins were detected by western blot analysis using Sp100 or HIS-specific antibodies (right panel). Input protein levels of Sp100 and HIS-tagged SUMO-1/-2 are shown in the left panel.

Figure 9. Depletion of ND10 components does not interfere with latency establishment in SLK or EA.hy cells.

Cell lines depleted for Sp100, Daxx, PML or GFP (control) were generated by transduction with lentiviruses expressing specific shRNAs. After antibiotic selection, stable SLK (A) and EA.hy (B) cultures were analyzed by western blotting to confirm successful knock down. (C): FACS analysis to establish the frequency of ORF59 positive cells in SLK knockdown cultures. The right panel shows a positive staining control employing TPA (20 nM) and sodium butyrate (0.3 mM) induced) BCBL1 cultures 48 h after treatment. Columns 1 to 4 of the left panel show SLK knockdown cultures at 36 h of infection with KSHV. As an additional positive control for ORF59 staining, the rightmost column of the panel shows lytically reactivated cells from long-term infected and overconfluently grown SLKp cultures (see text for details). (D): FACS analysis of stably shRNA-expressing EA.hy cultures analysed at 48 hours post infection with KSHV. The rightmost columns show cells which were treated with sodium butyrate (2.5 mM) immediately after infection. Mock infected cells were used to correct for background staining levels in all experiments. Error bars represent SEM of at least two and up to four biological replicates. (E) Transcript levels of ORF50, ORF59, ORF64 and ORF73 in EA.hy cells at 48 hours post infection (see Table S1 for RT-qPCR primers). Expression was calculated by normalization to GAPDH and is shown relative to shGFP controls (set to 1). Error bars represent SEM of at least three data sets.

Figure 10. Depletion of PML or Sp100 does not interfere with latency establishment in HUVEC cells.

(A) Western blot analysis of HUVEC cells transduced with shRNA-expressing lentiviruses directed against PML, Daxx, Sp100 or GFP. shDaxx transduced cells did not show a reduction of Daxx protein levels and were thus not included in further analyses. Although residual levels of PML and Sp100 are still visible in bulk protein extracts, immunofluorescence analysis suggests that a considerable number of cells is devoid of PML or Sp100 positive nuclear bodies (Figure S9). (B) FACS analysis of stably shRNA-expressing HUVEC cultures analysed 48 h ater infection with KSHV. The rightmost columns show cells which were treated with sodium butyrate (5 mM) immediately after infection. Mock infected cells were used to correct for background staining levels. Error bars represent SEM of three biological replicates. (C) Transcript levels of ORF50, ORF59, ORF64 and ORF73 in EA.hy cells at 48 hours post infection. Expression was calculated by normalization to GAPDH and is shown relative to shGFP controls (set to 1). Error bars represent SEM of at least three data sets.

Figure 11. Depletion of Sp100 but not Daxx or PML accelerates acquisition of H3K27me3.

(A+B) SLK-shSp100, SLK-shDaxx, SLK-shPML and SLK-shGFP cells were de novo infected with KSHV and chromatin was prepared at 36 h post infection. Deposition of activating H3K4me3 (A) or repressive H3K27me3 (B) marks was evaluated by ChIP-qPCR using specific primers as given in Table S1. (C+D) ChIP-qPCR analysis of selected loci in de novo KSHV infected shSp100 (red squares) or shGFP (control; blue diamonds) SLK (C) or EA.hy (D) cells was performed to analyze deposition of H3K27me3 marks at the indicated time points.

Figure 12. Model of events during early KSHV latency establishment.

(A) Immediately after infection, MLL/SET containing complexes are recruited to viral episomes by as of yet unknown cellular and/or viral factors, resulting in the establishment of the early activation marks and transcription of a subset of KSHV genes that includes ORF73/LANA. Recruitment or activity of PRC2 to KSHV episomes is inhibited by the presence of soluble Sp100. (B) After accumulation of de novo expressed LANA protein, LANA induces SUMOylation and relocalization of Sp100 into insoluble fractions which may be ND10s, but could also represent another matrix-associated fraction. Depletion of soluble nucleoplasmic or chromatin-associated Sp100 allows widespread recruitment of PRC2 complexes via molecular mechanisms that remain to be determined. PRC2 recruitment establishes H3K27me3 patterns, allowing repression of lytic genes after extinction of activation marks (e.g., on the K2 promoter) or establishment of bivalent chromatin (e.g. at the ORF50/Rta promoter). The major latency promoter upstream of ORF73/LANA is protected from H3K27me3 acquisition and remains active throughout latency.