Fig 1.
CPV infection leads to simultaneous expression of NS1 and NS2 genes and nucleolar accumulation of NS2.
(A) Relative expression levels of NS1 (blue) and NS2 (red) measured by RT-qPCR in infected NLFK cells between 4 and 24 hours post infection (hpi). The blue and red shadings around the lines indicate the standard error of the mean (SEM, n = 3). (B) Representative confocal images of HeLa cells transfected with NS2-EGFP (green) and (C) BirA*-tagged NS2 (green) at 24 hpt in noninfected and infected cells at 24 hpi. The localization of NS1 (red) is shown in cells with DAPI-stained nucleus (gray). (D) Representative confocal images of noninfected and infected NLFK cells at 24 hpi stained with antibodies against NS2 (green) and nucleolin (red). Gray corresponds to DAPI staining of DNA. Scale bars, 3 μm.
Fig 2.
Identification of NS2 proximity interactome using BioID. (A) Schematic picture of 48 high-confidence BioID NS2 interactors related to chromatin modification and DNA damage response cellular processes. Interactors are presented by their gene names. Association of NS2 (HA-BirA*-NS2) is shown either in the presence (24 hpi, dark blue dashed line) or in the absence (red dashed line) of infection or in both cases (black solid line). (B) Table showing the GO functional annotation clusters for the NS2 BioID dataset. The functional annotation chart of NS2 high confidence interactors was created by PANTHER classification system for GO Biological process overrepresentation (http://www.pantherdb.org/) using the default BFDR < 0.01 (yellow circle) and < 0.05 (white circle) filters. The GO terms are grouped hierarchically by PANTHER to illustrate relations between functional classes and are colored based on relatedness to the two identified functional association groups: chromatin modification (blue) and DNA damage response (gray).
Fig 3.
Molecular context of NS2 varies in noninfected and infected cells.
The interactome-based localization of CPV NS2 was analyzed with mass spectrometry (MS) microscopy in (A) noninfected and (B) infected stably HA-BirA*-NS2 expressing Flp-In T-REx 293 cells. The polar plots show the location of NS2 protein associations based on cellular marker proteins. Each sector represents one subcellular location defined by our reference database. The color assigned to each of the localization is based on the annotation frequency (light green: 0–0.25; green: 0.25–0.5; dark green: 0.5–1). The markers used for nucleoli (NUC), chromatin (CHR) and nuclear envelope (NE) were rRNA 2′-O-methyltransferase fibrillarin, histone H3.1 and prelamin-A/C, respectively.
Fig 4.
NS2-associated proteins play important roles in chromatin remodeling and DDR machineries.
The CPV NS2 protein is linked with certain key factors that control chromatin remodeling and DDR pathways to optimize cellular conditions for viral replication. This schematic picture shows the major associations of NS2 (red) with cellular proteins (shades of green). Dashed arrows represent key factors of chromatin binding and DDR machineries interacting with NS2. (A) The cellular chromatin modification machinery includes chromatin remodeling complexes such as NORC, WICH, ACF and RSF. (B) DNA damage response (DDR) factors contain major upstream mediators of ATM response such as DNA damage checkpoint 1 (MDC1) protein and proteins included in chromatin and nucleosome remodeling complexes. MDC1 interacts with phosphorylated γ-H2AX and mediates the recruitment of DDR response proteins to the damage site. The DDR downstream FACT complex functions as a nucleosome remodeler facilitating transcription. During replication stress, FACT orchestrates the replacement of γ-H2AX with macro-H2A.1 to the damage site. The location of parvoviral genomes and the viral replication protein NS1 (red) adjacent to DNA damage sites of the cellular chromatin are shown. Figure was created with BioRender.com.
Fig 5.
Close proximity analysis verifies the association of NS2 with key BioID interactors.
Proximity Ligation Assay (PLA) assay was performed in HeLa cells transfected with NS2-EGFP and representative cellular proteins identified by BioID. The proteins were chromatin remodeling factor (SMARCA5), transcriptional repressor (macro-H2A.1), and histone chaperon of the FACT complex (SSRP1). (A) Representative single confocal cross-sections showing the localization of PLA signals (green) and the nuclei stained with DAPI (gray). (B) Positive and negative controls include PLA with antibodies against VP2 capsid protein and intact capsids in infected cells at 20 hpi and in noninfected cells. Scale bars, 3 μm. (C) The graph shows the average number of PLA signal foci per nucleus (n = 15) analyzed from 3D reconstructions of confocal images. The error bars show the standard error of the mean (SEM), and the samples having a significantly higher number of nuclear PLA foci compared to the negative control are denoted as ** (p<0.01). The significance was assessed using Poisson regression model.
Fig 6.
NS2 interacts with host proteins identified by BioID in infected cells.
Analyses of NS2 interaction with SMARCA5, MDC1, macro-H2A.1, and SSRP1 in infected NLFK cells at 24 hpi. (A) Representative single confocal cross-sections of PLA signals (green) in the DAPI-stained nuclei (gray). (B) Images of positive and negative PLA controls prepared by using antibodies against VP2 and intact capsids at 24 hpi and in noninfected cells. Scale bars, 3 μm. (C) The analyses of 3D reconstruction of confocal PLA data showing the average number of PLA signal foci/nucleus (n = 15). The error bars show the standard error of the mean (SEM), and the samples with a significantly higher number of nuclear PLA foci compared to the negative control are denoted as ** (p<0.01). The significance was assessed using Poisson regression model.
Fig 7.
NS2 mutation induce changes in the amount and distribution of euchromatin.
(A) Representative confocal images show the nuclear localization of euchromatin marker H3K27ac (magenta), NS1 (green), and DAPI staining (gray) in nontransfected HeLa cells, and cells transfected with wt, NS2 donor and acceptor mutants at 24 hpt. (B) Total fluorescence intensities of euchromatin. (C) Nuclear distribution of H3K27ac as a function of increasing distance from the NE in nontransfected, wt and NS2 mutants transfected cells (n = 29). The error bars show the standard error of the mean. Statistical significances were determined using Dunnett’s multiple comparison test. The significance values shown are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). Scale bars, 5 μm.
Fig 8.
Mutation of NS2 induce alteration in the amount and distribution of DDR proteins.
Representative confocal images show the nuclear localization of NS1 (green), (A) γ-H2AX (magenta), (B) MDC1 (magenta) and DAPI staining (gray) in HeLa cells without transfection or at 24 hpt with wt, NS2 splice donor and acceptor mutants. Scale bars, 5 μm. (C) Total fluorescence intensities of γ-H2AX together with (D) its nuclear distribution as a function of increasing distance from the NE in nontransfected, wt and NS2 mutants transfected cells (n = 29). (E) Intensity line profiles of γ-H2AX (purple) and NS1 (green) measured through the nucleoli. (F) Fluorescent intensities and (G) nuclear localization of MDC1 from the NE (n = 28). The error bars show the standard error of the mean. (H) Intensity line profiles of MDC1 (purple) and NS1 (green) measured through the nucleoli. Statistical significances were determined using Dunnett’s multiple comparison test. The significance values shown are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). Fluorescent intensity profiles of (G) γ-H2AX (magenta) with NS1 (green), and (H) MDC1 (magenta) with NS1 (green) in zoomed areas.
Fig 9.
NS2 mutation induce changes in the number and volume of replication centers.
(A) Representative pseudocolored sum projections of confocal image slices show the amount and distribution of nuclear NS1 in nontransfected, wt, splice donor and acceptor mutants transfected HeLa cells at 24 hpt. Calibration bar for pseudo coloring is shown. Scale bars, 5 μm. (B) Total fluorescence intensities of NS1. The error bars show the standard error of the mean. (C) Nuclear distribution of NS1 as a function of increasing distance from the NE in wt and NS2 mutants transfected cells (n = 29). (D) Numbers and (E) volumes of replication centers, identified by NS1 staining were analyzed in wt, splice donor and acceptor mutants transfected HeLa cells at 24 hpt (n = 29). The error bars show the standard error of the mean. Statistical significances were determined using Dunnett’s multiple comparison test. The significance values shown are denoted as ** (p<0.01), * (p<0.05) or ns (not significant).