The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage

Abstract

The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5' end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.

Fig 1. MVM NS1 localizes to induced cellular sites of DNA damage during infection and overexpression.

(A) Laser micro-irradiation followed by immunofluorescence analysis of U2OS osteosarcoma cells was performed during infection with MVMp (MVM^WT; panels 1,2,4,5) and MVMp1989 variant (MVM^ΔNS2; panel 3, [66]) of MVM for 16 hours. The induced DDR site was monitored by γH2AX staining (green) and the viral non-structural protein was visualized by NS1 staining (red). The host transcription factors FOXP1 and NR5A2, which do not have strong binding sites on the MVM genome, were monitored as controls (red). The cell nuclei were visualized by DAPI staining and the nuclear periphery was demarcated by dashed white line. White bars in figure inset represent 10 microns. Data is representative of 3 independent experiments, each imaging at least five fields of view containing 4–5 nuclei. (B) U2OS cells were transfected with the indicated NS1 expressing vector with LipoD293 for 16 hours, DDR induced by laser micro-irradiation and processed for immunofluorescence by co-staining for γH2AX (green) and NS1 protein (red). The cell nuclei were visualized by DAPI staining and the nuclear periphery was demarcated by dashed white line. White bars in figure inset represent 10 microns. Data is representative of 3 independent experiments, each imaging at least five fields of view containing 4–5 nuclei. (C) Murine A9 fibroblasts which stably express NS1^WT were induced with doxycycline for 24 hours before focused DDR induction by laser micro-irradiation. Samples were then co-stained for γH2AX (green) and the indicated proteins (red). The cell nuclei were visualized by DAPI staining and the nuclear periphery was demarcated by dashed white line. White bars in figure inset represent 7.5 microns. Data is representative of 3 independent experiments, each imaging at least five fields of view containing 4–5 nuclei. (D) U2OS cells were transfected with NS1 expressing vector for 12 hours and induced with 1 mM Hydroxyurea or 200 nM Doxorubicin for 4 hours to chemically induce DNA damage. Samples were collected by CSK pre-extraction at 16 hours post-expression and processed for immunofluorescence by co-staining for DNA damage sites, visualized by γH2AX (green) and NS1 (red). The cell nuclei were visualized by DAPI staining and the nuclear periphery was demarcated by dashed white line. White bars in figure inset indicate 10 microns. (E) The distance between the center of mass of the fluorescence of γH2AX foci and NS1 foci were quantified for multiple nuclei imaged across 3 independent experiments using Leica LAS X image processing software. (F) The number of γH2AX foci were quantified in NS1 expressing cells over multiple fields of view in 3 independent experiments. Statistical analysis was performed using One-way ANOVA, multiple comparisons test, with statistical significance designated by ****, which represents p <0.0001. ns denotes not statistically significant.

Fig 2. Analysis of NS1 localization and relocalization to DDR sites at the genomic level.

(A) Representative NS1 and γH2AX ChIP-seq data on mouse chromosome 17 (top) and chromosome 19 (bottom) in murine A9 fibroblasts inducibly expressing NS1 for 24 hours. Cells were pulsed with 1 mM Hydroxyurea for 16 hours starting at 8 hours post-expression in the bottom 2 panels (histograms in blue and purple). Y-axis represents quantile normalized reads per million values of ChIP-seq reads for each sample. The left panels represent whole chromosome views of Chr17 and Chr19. The right panels represent zoomed-in views of the respective regions indicated by red rectangle on the left. (B) The total number of called NS1 peaks which were shared between two biological replicates of NS1 ChIP-seq experiments were compared between doxycycline induction (left) and HU pulse during doxycycline induction (right) in A9 cells containing an integrated NS1-pINDUCER20 cassette. (C,D) Venn diagrams representing whole-genome comparison of the overlap of NS1 and γH2AX ChIP-seq peaks in A9 cells expressing NS1 alone (C, left panel) and NS1 followed by HU pulse (D, left panel). The statistical significance of the overlap is shown via Jaccard analysis in the respective right panels (labelled as “Observed”; red cross). The bioinformatically-called NS1 ChIP-seq peaks common to two independent experiments were intersected with an equivalent number of randomly generated locations on the mouse genome (labelled as “Permuted”; black square). (E) The overlap of NS1 peaks throughout the genome in A9 cells expressing ectopic NS1 in the absence or presence of HU is shown via Venn diagram (E, top left) with the statistical significance of the overlap calculated by Jaccard analysis (E, top right). The intersection of the NS1 peaks with respective γH2AX peaks from two independent experiments were quantified and presented in the bar graphs (E, bottom). The subsets of NS1 peaks from the Venn diagram were represented according to the corresponding highlighted colors in the bottom panel of E. White fractions of the bars represent γH2AX-negative fraction and black bars represent γH2AX -positive fraction of identified NS1 peaks. (F) The relative position of NS1 ChIP-seq peaks with-respect-to γH2AX peaks 1 Mb upstream and downstream in the absence of HU (red; left) and the presence of HU (blue; right) were visualized using deepTools bioinformatic resource on the Galaxy project platform [63,67]. (G) Focused NS1 ChIP-qPCR assays were performed at a subset of cellular sites identified by ChIP-seq in cells inducibly expressing wild-type NS1 (NS1^WT, blue bars), and the dimerization mutant of NS1 (NS1^K405S, red bars) for 24 hours. Data is presented as mean ± SEM of percent input pulldown efficiencies across 2 independent experiments.

Fig 3. Ectopically expressed NS1 localizes with Virus Associated Domains identified during infection.

(A) Representative NS1 ChIP-seq tracks on mouse chromosome 17 comparing the localization sites of ectopically expressed NS1 in the absence or presence of HU (blue and green histograms respectively) with that of NS1 during infection (red histogram; third genome browser track). The NS1 binding sites are compared with MVM localization sites previously identified using V3C-seq (red histogram; fourth genome browser track). NS1 ChIP-seq and MVM localization V3C-seq were carried out at 16 hpi and have been described previously [21]. The resulting Virus Associated Domain (VAD) has been demarcated in blue rectangle. (B) The NS1 ChIP-seq peaks identified in the presence and absence of HU were compared with MVM genome localization sites at 12 hours post-infection (hpi), 16 hpi and 20 hpi previously identified by V3C-seq [21]. The statistical significance of this overlap was determined by Jaccard analysis (red crosses), and compared with a randomly generated set of cellular sites with approximately the same number of genomic sites as the identified NS1 ChIP-seq peaks (black squares). The extent of separation between red X and black squares reflect increased correlation of ChIP-seq and V3C-seq peaks. The overlap of NS1 ChIP-seq peaks previously identified at 16 hpi of MVM infection (C, D; red, [21]) was compared with NS1 peaks identified during expression (C, blue) and expression in the presence of HU treatment (D, green). The statistical significance of the respective overlap was evaluated by Jaccard analysis (C,D; right).

Fig 4. NS1 directs the localization of MVM genome to cellular DDR sites.

(A) Representative immuno-FISH data of U2OS cells infected with MVMp virus for 16 hours (panels 1,2) and MVM infectious clone lacking the replication origin (MVMp FD, panels 3,4) or pUC18 vector (panel 5) transfected for 36 hours. DNA damage was induced using laser micro-irradiation (panels 2–5) and monitored by γH2AX staining (green). The MVM genome or pUC18 DNA were visualized by AF-555 labelled FISH probes (red). The white bar in the figure inset represents 10 microns. (B) The corresponding NS1 localization during infection (panels 1,2) and MVMp FD transfection (panels 3,4) were monitored using immunofluorescence assays for NS1 (red). DNA damage was induced using laser micro-irradiation (panels 2–4) and monitored by γH2AX staining (green). The white bar in the figure inset represents 10 microns. Data is representative of 3 independent experiments, each imaging at least five fields of view containing 4–5 nuclei. (C) Schematic of the selection of laser-micro-irradiated ROI (left) for the quantification of Immuno-FISH assays in multiple nuclei (right). The average FISH signal intensity of AF-555 labelled MVMp and pUC18 (red) over the laser micro-irradiated sites (green) were calculated in over 10 nuclei in the indicated conditions in Fig 4A. Data is presented as mean ± SEM of signal intensity at 72 nm intervals along ROIs that are 8.4 μm in length. Statistical analysis was performed by One-way ANOVA, multiple comparisons test. Statistical significance is designated by **** < 0.0001 for all samples relative to MVMp NS1^WT FD. (D) Schematic of ACCAACCA consensus site and scrambled sequence (CACACACA) on MVMP38 with 100 bp of the genome sequence on either side, which were cloned into the HindIII site of the pUC18 vector. NS1 binding to this site on the pUC18 vector was monitored by NS1 ChIP-qPCR assay (bottom), with the qPCR primers located on the pUC18 backbone flanking the insert site (black arrows). NS1 expression was induced with doxycycline for 16 hours in stable A9 cells described above before transfecting with the modified pUC18 vectors for 8 hours, followed by ChIP assays at 24 hours post-induction. Data is represented as mean ± SEM for percent input of NS1 pulldowns in three independent experiments. Statistical significance was determined by unpaired t-test. Statistical significance is shown by ***, which reflects p < 0.05. (E) Immuno-FISH assays of U2OS cells transiently expressing wild-type (NS1^WT, panels 2,3) or dimerization mutant (NS1^K405S, panel 4) variants of NS1, transfected with the ACCAACCA–pUC18 (panels 1,2,4) or scrambled–pUC18 (panel 3) vectors for 16 hours and damaged by laser micro-irradiation which were monitored by γH2AX staining (left panel). Plasmid localization was monitored by AF-555 labelled probe (middle panels) and their colocalization was visualized in the right panel (labelled “Merge”). (F) Quantification of the average FISH signal intensity of AF-555 labelled pUC18 (red) over the laser micro-irradiated sites (green) calculated in over 10 nuclei in the indicated conditions in Fig 4E. Data is presented as mean ± SEM of signal intensity at 72 nm intervals along ROIs that are 8.4 μm in length. Statistical analysis was performed by One-way ANOVA- multiple comparisons test. Statistical significance is designated by **** < 0.0001 for all the samples relative to NS1^WT ACCAACCA-pUC18. (G) ChIP-loop assays with pUC18 viewpoint in NS1-expressing A9 fibroblasts (induced for 24 hours with doxycycline) transfected with the ACCAACCA–pUC18 / scrambled–pUC18 plasmids at 12 hours post-induction followed by DDR induction with hydroxyurea at 18 hours post-induction. Chromatin was processed using standard 3C methods before performing NS1-ChIP, and intramolecular ligation was subsequently performed. The localization of the pUC18 vector with cellular DDR sites (identified by γH2AX ChIP-seq, Fig 2) was assayed by Taqman qPCR where the probe viewpoint is complementary to the plasmid genome while the assay sites are on the mouse genome are at chromosome 17qA1, 17qA3.3 and the previously identified gene desert site 9qE1 [21]. Statistical analysis was performed by one-way ANOVA- multiple comparisons test. Statistical significance is designated by **** < 0.0001.