Conceived and designed the experiments: TOI EN MVR. Performed the experiments: TOI EN JJ OP ND HS. Analyzed the data: TOI ND JT. Contributed reagents/materials/analysis tools: JL MVR. Wrote the paper: TOI EN JT MVR.
The authors have declared that no competing interests exist.
The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.
The nuclear replication strategies of DNA viruses and the virus-induced perturbations of host-cell nuclear structures differ considerably among virus families
The nucleus is a highly complex and dynamic organelle that hosts the chromosomes and a large number of proteinaceous nuclear bodies
The non-enveloped parvoviruses are among the smallest DNA viruses. Canine parvovirus (CPV) encapsidates its single-stranded negative-sense DNA genome of 5300 bases into an icosahedral capsid of ∼260 Å in diameter
An essential cellular replication protein, proliferating cell nuclear antigen (PCNA) encircles the dsDNA and enhances the DNA polymerase delta processivity in eukaryotic replication. PCNA has been shown to localize into the viral replication compartments in baculovirus
In this study, we use advanced confocal imaging techniques including photobleaching, fluorescence correlation spectroscopy and photoactivation to clarify the intranuclear processes and molecular interactions in parvovirus infection. The distributions of virus capsids and histone H2B, and dextrans of varying size, were determined so as to understand the size constraints of macromolecular dynamics within the viral replication bodies. Intranuclear diffusion of CPV virus like particles (VLPs) was studied with photoactivable VP2. Moreover, the dynamics and interactions of histone H2B, Enhanced Yellow Fluorescent Protein (EYFP), NS1 and PCNA in infected cells were assessed by quantitative fluorescence recovery after photobleaching (FRAP) and fluorescence fluctuation microscopy (FFM).
Norden laboratory feline kidney (NLFK) cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK) at 37°C in the presence of 5% CO2. HEK293, HeLa, T98G and TP366 cells were grown as described
The plasmids encoding fluorescent proteins, EYFP-PCNA, H2B-EYFP and H2B-ECFP, were generous gifts from Wim Vermeulen (Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands) and J. Langowski (German Cancer Research Center, Heidelberg, Germany). The pEYFP-N3 construct was purchased from Clontech Laboratories Inc. (Mountain View, CA). The plasmid construct, NS1-deYFP is a modification of NS1-EYFP
NS1-deYFP and PAGFP-VP2 transfections were performed with TransIT-LT1 reagent (Thermo Fisher Scientific Inc, Waltham, MA) according to the manufacturer's protocol. For studies of protein dynamics during infection, the cells were infected 20–24 h post transfection. NLFK cell lines stably expressing PCNA-EYFP, H2B-EYFP, or H2B-ECFP were established by transfection with an expression vector at 24 h after seeding. After 2 days the DMEM was replaced by DMEM containing 1 mg/ml of geneticin (Sigma Aldrich, St Louis, MO). The cells were then seeded at different intervals until a stable expression was observed by microscopy.
Incorporation of 5-bromo-2-deoxyuridine (BrdU, Sigma) was used to document the DNA synthesis in infected cells. The cells were incubated in DMEM containing 25 µM BrdU for 40 min at 24 h post infection (p.i). Cellular BrdU was detected with a mouse monoclonal Ab (MAb, Santa Cruz Biotechnology, Santa Cruz, CA) followed by Alexa-555-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR). BrdU and PCNA were labeled with anti-BrdU MAb and anti-PCNA (Abcam, Cambridge, UK) Ab, respectively, followed by Alexa-488-conjugated anti-mouse IgG and Alexa-633-conjugated anti-rabbit IgG. Labeling was performed either with or without denaturation of the DNA with 2 M HCl
CPV was immunostained either with Ab against VPs or with MAb against capsids
Microinjection of NLFK cells was carried out using a semiautomatic system consisting of a Transjector 5246 and a Micromanipulator 5171 (Eppendorf, Hamburg, Germany) on an Olympus IMT-2 inverted microscope. Needles were pulled from glass capillaries (Clark Electromedical Instruments, Reading, UK) using a P-97 needle puller (Sutter Instruments, Novato, CA). Cultures were grown to 80% confluency on 5 cm glass-bottom dishes. Cells were microinjected with 40 kDa FITC-dextran (2.5 mg/ml), 146 kDa TRITC-dextran (5 mg/ml), or 500 kDa FITC-dextran (2.5 mg/ml). Cells were infected 2 h prior to microinjection and imaged at 20–24 h p.i.
The imaging of chromatin marginalization and the change in nuclear volume was performed with Zeiss CellObserver HS widefield microscope (Zeiss, Göttingen, Germany). The microscope incubator was maintained at 37°C during the imaging process and the CO2 concentration was adjusted to 5%. The LD Plan-Neofluar 40× (NA = 0.6) objective was used. A 455 nm LED from a Colibri light source (Zeiss) was used for excitation of the H2B-ECFP and the emitted fluorescence was collected using a 458–502 nm band-pass filter and a Zeiss AxioCam MRm (chip pixel size 6.45 µm). A binning of 2×2 pixels was used to reduce the exposure time below 1 s. Imaging was carried out at 5 min intervals.
The images for deconvolution were acquired with a laser scanning confocal microscope LSM510 in Axiovert 100 M (Zeiss, Jena, Germany) using a Zeiss Plan-Neofluar 63× (NA = 1.25) oil immersion objective. For replication body imaging, live cells stably expressing H2B-ECFP were transfected with NS1-deYFP, infected and imaged 20–26 h p.i. The stage and the objective were warmed to 37°C before imaging. Image stacks of 30–45 slices were captured with a voxel size of 47 nm in the x and y, and 230 nm in the z (512×512 image, zoom factor 6) dimension. ECFP and EYFP were excited respectively with a 458 nm and a 514 nm laserlines. The ECFP fluorescence was collected using a 475–505 nm band-pass filter and a 530 nm long pass filter for EYFP. The pinhole was adjusted to 1 Airy unit. Capsids were immunolabeled with capsid MAb followed by Alexa-633-conjugated anti-mouse IgG, and their distribution was monitored in cells stably expressing H2B-ECFP. Capsids were detected with a HeNe 633 nm laser and a 650 nm long-pass filter. Imaging parameters used for ECFP were identical to those in replication body studies. The FITC-labeled dextran distribution was imaged using 488 nm excitation and 505–530 nm band-pass filter. The TRITC-labeled dextran was imaged with a 543 nm excitation and the fluorescence was detected with a 560 nm long-pass filter. The voxel size in the dextran imaging experiments was adjusted to 48 nm in the x and y, and to 154 nm in the z dimension. The pinhole was kept at 1 Airy unit. Stack were build-up from 30–55 slices of 512×512 pixel images (zoom factor 6). Multitracking was used to avoid crosstalk.
Imaging of BrdU labelled cells, DNaseI digested cells, and the nuclear volume was preformed with an Olympus FV1000 laser scanning confocal microscope attached to an IX-81 inverted microscope frame (Olympus Tokyo, Japan) with an UPLSAPO 60× (NA = 1.3) objective. For BrdU and DNaseI digestion assay imaging, single-section images were captured with an image size of 512×512 pixels. The pixel size was adjusted to 59 nm and 66 nm for BrdU and DNaseI, respectively. The nucleus size was imaged by capturing stacks of 20–30 images, with a pixel size of 92 nm in the x and y, and 500 nm in the z dimension. The pinhole was set to 1 Airy unit. DAPI and ECFP were excited with the 405 nm laserline, EYFP and Alexa-555 respectively with 515 nm and 543 nm laserlines. The fluorescence was collected respectively with 425–525 nm band-pass, 530–630 nm band-pass and 650 nm long-pass filters.
Various FRAP protocols were used to study the protein dynamics in live cells. Detailed descriptions of the methods employed in this study are provided in supporting information (
Fluorescence fluctuation microscopy was used to measure the diffusion of EYFP in the nuclei of NLFK cells and of EGFP in various cell lines. Comprehensive description of the method including data analysis is provided in supporting information (
Photoactivable (PA) GFP was fused to VP2 for a study of viral capsid protein dynamics in infected and non-infected cells. The Experimental protocol can be found in supporting information (
Various recovery models were used to obtain further information on protein recoveries, see supporting information (
Our previous studies of CPV infected cells have demonstrated the accumulation of fluorescent NS1 fusion protein into distinct nuclear foci followed by a thorough intranuclear distribution of NS1
Deconvoluted confocal microscopy images of CPV infected NLFK cells stably expressing H2B-ECFP studied 20–24 h post infection. (A) Live cell images of intranuclear histone H2B-ECFP (cyan) and NS1-deYFP (yellow). (B) Fixed cell images of intranuclear histone H2B-ECFP (cyan) and capsid Ab (magenta). Deconvoluted confocal microscopy images of living NLFK cells showing the distribtions of (C) 40 kDa, (D) 146 kDa and (E) 500 kDa dextrans in a pseudocolor scale. Scale bars, 5 µm.
To study the accessibility of the nuclear subcompartments, dextrans with a size of 40 kDa (the radius of gyration rg≈7 nm), 146 kDa (rg≈13 nm) and 500 kDa (rg≈24 nm) in size were microinjected to nuclei of infected and non-infected cells. Imaging with a confocal microscope at 20 h post injection showed a homogeneous intranuclear distribution the of the 40 kDa dextran in infected cells (
The dynamics of capsid protein VP2 was studied in cells expressing this protein fused to a photoactivable GFP (PAGFP). Western blot analyzis confirmed that the PAGFP-VP2 construct had the predicted molecular weight (92 kDa) and was recognized by both the VP antibody and the EGFP antibody (
Confocal microscopy images of PAGFP-VP2 photoactivation studies in (A) non-infected and (B) infected NLFK cell. The activation areas are marked with a white circles. (C) The normalized PAGFP (green) and PAGFP-VP2 (red) fluorescence intensity redistribution in the non-infected cells in addition to PAGFP (blue) and PAGFP-VP2 (black) Virtual Cell simulations of fluorescence redistribution. (D) The normalized fluorescence intensity of PAGFP-VP2 in infected NLFK cells (red) and the Virtual Cell simulation of its redistribution (black). Western-Blot strips of whole-cell lysates of CPV infected (V line) and PAGFP-VP2 (VP2 line) or EGFP (E line) transfected cells were analyzed for fusion protein expression using (E) anti-VP antibodies or (F) anti-EGFP antibodies. Error bars indicate the standard deviation. Scale bars, 5 µm.
In photoactivation studies the excitation of PAGFP at 488 nm was increased 10–20 fold by an activation laser pulse of 405 nm light. After photoactivation, PAGFP-VP2 diffused rapidly within the nucleus (
The localization of newly synthesized viral DNA was monitored by BrdU labelling with or without the denaturation step in infected cells stably expressing the chromatin marker H2B-ECFP (
Confocal microscopy images of CPV infected NLFK cells stably expressing H2B-ECFP or H2B-EYFP. Nuclei labelled with BrdU for 40 min at 24 h p.i. The BrdU (red) incorporation was examined (A) without and (B) with denaturation in comparison to H2B-ECFP (cyan). (C) Localization of endogenous DNA, labelled with BrdU prior to infections at 24 h p.i. BrdU (red) distribution in comparison to H2B-ECFP (cyan). (D) Qualitative FRAP analyzis of the H2B-EYFP recovery in the infected NLFK cell stably expressing H2B-EYFP. (E) FRAP recovery curves of H2B-EYFP infected (red) and non-infected (black) NLFK cells. Error bars indicate the standard deviation. Scale bars, 5 µm.
To explore the accessibility of DNA in the replication bodies, DNaseI was applied into detergent permeabilized cells (
Time-lapse analyzes were performed to analyze chromatin marginalization in the infected cells stably expressing H2B-ECFP. Imaging revealed a rapid enlargement of the ICD at 16–24 h p.i. (
FRAP experiments were performed on cells stably expressing H2B-EYFP to assess virus-infection-induced alterations in H2B binding. The fluorescence recovery of a bleached rectangular area (1 µm in width) was followed for 5 minutes. Interestingly, the recovery of H2B-EYFP fluorescence was found to be as slow in infected as in non-infected cells even though the corresponding distributions of H2B-EYFP were drastically different (
The above experiments showed that chromatin-depleted replication bodies were relatively homogeneous in structure, accessible to virus-size particles, and sensitive to DNaseI. Next, we measured the relative amount of DNA and the nuclear volume in infected cells. The DAPI labelling indicated that the DNA content was 2.5 times higher in infected than in non-infected cells (
Widefield microscopy images of NLFK cells. (A) Infected and non-infected G1/G2 and S phase cells labelled with anti-PCNA (red) antibody and DAPI (cyan). (B) DAPI fluorescence intensity measured from G phase, S phase and infected cells. (C) Timelapse imaging of infected H2B-ECFP expressing cells showing an increase in the nuclear size. (D) Nuclear volumes from fixed H2B-EYFP expressing cells. Error bars indicate the standard error of the mean. Confidence interval p<0.001 is marked with ***. Scale bars, 5 µm.
Timelapse imaging of the infected cells stably expressing H2B-ECFP showed an increase in the nuclear volume (
The above experiments indicated that in infected cells the nuclei are drastically reorganized as the replication bodies form. Next we studied if the intranuclear diffusion of proteins is affected by the virus infection. Subsequently, the general protein diffusion was analyzed by quantitative FRAP, FFM and Virtual Cell simulations of infected and non-infected cells expressing free EYFP. Confocal microscopy imaging revealed a homogeneous distribution of EYFP throughout the non-infected cells (
FRAP experiments and Virtual Cell Simulations of EYFP diffusion. (A) Non-infected cell with a homogeneous intranuclear distribution of EYFP. (B) Infected cell showing a uniform distribution of EYFP in the replication body, with a darker rim visible near the nuclear membrane. (C) FRAP experiments of infected cells performed with a high frame rate to capture the rapid fluorescence recovery. (D) Fluorescence recovery curves showing a faster recovery in the infected (black) than in the non-infected (red) cells. (E) Nuclear geometry in the simulated EYFP FRAP experiment. (F) Simulated FRAP recovery in non-infected cells. Measured recovery (black) in infected cell (G) and in non-infected cell nuclei (H) in comparison to the simulated experiment (red). (I) Summary of the results obtained with FRAP and FFM. Confidence interval p<0.05 is marked with *. Error bars indicate the standard deviation. Scale bars, 5 µm.
Next we analyzed protein dynamics in the replication structures. NS1-deYFP binding dynamics were studied by quantitative FRAP and mathematical modelling of the recovery data. A small circular area in the middle of the nucleus was bleached, and the fluorescence recovery was measured at 0.5 s intervals (
Infected NLFK cell expressing NS1-deYFP. (A) NS1-deYFP distribution shown in a pseudocolour scale. (B) NS1-deYFP fluorescence recovery (green) and a fit by the full model (blue). (C) Virtual Cell model result (black) for the NS1-deYFP recovery (red) with a mobile NS1-deYFP binding partner. (D) Virtual Cell model result (black) for the NS1-deYFP (red) recovery with two distinct binding sites with different affinities for NS1-deYFP. Error bars indicate the standard deviation. Scale bar, 5 µm.
The multiple-binding-site hypothesis was tested with the Virtual Cell software, assigning to NS1-deYFP two distinct and immobile binding sites with different affinities. We obtained again good fit results (
PCNA is an essential protein in parvovirus genome replication
BrdU (red) and PCNA (green) labels in NLFK cells with or without a denaturation step. (A) BrdU positive small foci in the PCNA labelled replication body of an infected cell observed without DNA denaturation. (B) Distributions of BrdU and PCNA in the S phase of a non-infected cell without DNA denaturation. (C) BrdU and PCNA labelled replication body in an infected cell with DNA denaturation (D) BrdU and PCNA in a non-infected cell with DNA denaturation. FRAP experiments were performed in cells stably expressing PCNA-EYFP. (E) A G phase cell with a homogeneous intranuclear distribution of PCNA-EYFP shown in a pseudocolour scale. (F) FRAP recovery of PCNA-EYFP (black), and EYFP (red) used as a control. (G) Recovery of PCNA-EYFP (green) fitted by the free diffusion model (blue). (H) An infected cell with PCNA-EYFP concentrated into the viral replication body. (I) Recovery of PCNA-EYFP in infected cells. (J) Recovery data (green) fitted by the full model (black). Error bars indicate the standard deviation. Scale bars, 5 µm.
The PCNA-EYFP dynamics in the replication bodies was then studied by FRAP. In non-infected cells the PCNA-EYFP distribution was homogeneous (
Exploitation of the cellular nuclear replication machinery by several DNA viruses is accompanied by alterations in the nuclear architecture and dynamics. Although the assembly steps of some DNA viruses are relatively well known, the intranuclear dynamics of replication structures are poorly understood. For parvoviruses the replication processes at the molecular level are relatively well established
Late atfter CPV infection, the fluorescent NS1-containing replication body filled the entire nucleus except the nucleolus, and chromatin was confined to the vicinity of the nuclear envelope. Also the viral capsids tended to accumulate close to the nuclear membrane. These results are consistent with observations made previously on herpesviruses and baculoviruses, showing that the RNA transcription, the DNA replication and the virion assembly take place in distinct locations
To examine whether it is the size constraint that explains the periferal localization of the capsids, we microinjected dextrans of various sizes to the nuclei of infected cells. We found that none of the dextrans were completely excluded from the replication compartment. It has been reported previously that the nucleoplasm is not fully accessible to dextrans; the dense chromatin regions exclude dextrans of >70 kDa
It is the radius of gyration of a particle that defines the size of the mesh pore through which it can enter. The dextran particles of 40 kDa, 146 kDa and 500 kDa have gyration radii of rg≈7 nm, rg≈13 nm and rg≈24 nm, respectively. For spherical proteins, the relation of particle radius (r) to the radius of gyration is
Observations on the structure of the replication body raised the question of capsid protein dynamics. The diffusion dynamics were examined using photoactivable GFP (PAGFP) fused N-terminally with the major capsid protein VP2. It is known that such a fusion allows for assembly of VLPs
The highly organized chromatin occupies a large proportion of the nucleus and controls the mobility of nuclear bodies
Within the nucleus, the histone H3 protein has been shown to associate with the DNA of herpes simplex virus after its release from the virion, but not with the newly replicated viral genome
The intranuclear dynamics of proteins and inert particles such as EGFP or fluorescent dextrans have previously been studied by FCS
The previous studies of nuclear diffusion have been carried out on non-infected interphase cells, whereas we examined the effects of virus infection on protein diffusion. FRAP and FFM studies revealed a two-component system for EYFP diffusion in non-infected NLFK cells, with the diffusion coefficient for faster-component of D = 50±5 µm2/s and D = 57±8 µm2/s. These data are in good agreement with those reported recently for EGFP inside the cell nuclei
The depletion attraction phenomenon has been suggested to be involved in vesicle clustering and in the formation of nuclear bodies
The proteins, viral DNA, and capsids accumulated into the replication body. The viral DNA has a loose conformation and does not hinder the diffusion of proteins. The replication body components continuously collide with the chromatin causing thereby an osmotic pressure (black arrows) leading to chromatin marginalization.
It has been suggested that the parvoviral NS1 protein shows non-specific DNA binding
PCNA is among the most important proteins in viral DNA replication, and has been found to accumulate in the parvoviral replication body
The binding times measured for PCNA-EYFP and NS1-deYFP were identical, even though the recoveries of these proteins were fitted by different models. These findings imply that both PCNA and NS1 stay bound to the viral genome during replication, thus supporting the parvoviral genome replication model as proposed in
Our results provide a comprehensive description of the parvovirus-infection-induced modifications in the nucleus. The parvoviral replication body is a complex structure that alters the binding properties of endogenous proteins, displaces the host DNA and modifies the nuclear microenvironment in a way that leads to increased protein mobility. The change in protein mobility can favour the viral replication by enhancing the rate of binding reactions and by reducing the likelihood of ssDNA hybridization.
Stably ECFP-H2B expressing cells were infected and imaged from 5 h p.i. to 24 h p.i. At 16–24 h p.i. clear increase in interchromosomal space was evident.
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Effect of DNaseI treatment on non-infected and infected NLFK cells. Fixed cell confocal microscopy images of dsDNA (DAPI label), chromatin (H2B-EYFP), NS1-deYFP and PCNA-EYFP after permeabilization and treatment with buffer or DNaseI. Unapparent nuclei are encircled. Scale bars, 5 µm.
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Intracellular localization of capsids and viral VP proteins in PAGFP-VP2 expressing cells. NLFK cells expressing PAGFP-VP2 were immunolabelled with antibodies that recognize intact capsids or VP proteins. Distribution of PAGFP-VP2 (green) together with (A) capsid MAb (red) or (B) VP Ab (red). Secondary antibodies used were Alexa-555-labled anti-mouse IgG and anti-rabbit IgG. Scale bars, 5 µm.
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Fluorescence in situ labelling of viral genome. Infected cells were labelled with CPV genome specific FISH probe at 24 h p.i. The FISH probe labelled the viral replication compartment inside the nucleus. Scale bar, 5 µm.
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Schematic presentation of NS1-deYFP Virtual Cell model. Schematic representation of the Virtual Cell simulation showing the molecular species (green) and reactions between them (yellow). In the model, fluorescent NS1-deYFP (NS1) reacts with the viral genome (CPV_Genome) and forms genome bound NS1 (Bound NS1). Similar reaction takes place between bleached, non-fluorescent NS1 (Bleached NS1) and the viral genome. This reaction forms non-fluorescent bound NS1 (Bleached Bound NS1). Bleaching laser induces the bleaching reaction, where fluorescent NS1 forms non-fluorescent bleached NS1 or where bound NS1 forms bleached, bound NS1. Imaging laser reacts with fluorescent forms of NS1 and simulates the bleaching reaction caused by the confocal imaging.
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Schematic presentation of PAGFP-VP2 Virtual Cell model. Schematic representation of the molecular species (green) and reactions between them (yellow) in PAGFP-VP2 activation study simulations. Activation laser reacts with dark, mobile and immobile capsids and leads to formation of bright capsids. Imaging laser simulates the bleaching caused by the confocal imaging.
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Intracellular distribution of EYFP and histone H2B in infected and non-infected cells. Confocal images show the distribution of EYFP and H2B-EYFP in (A) in infected and (B) non-infected cells at 24 h p.i. Line profile analysis revealed intensity profiles through the nuclear region. Scale bar, 5 µm.
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Free EYFP and EGFP diffusion in nuclei of living cells. Diffusion time of free EYFP was measured with FFM in the nucleoplasm of living NLFK cells at various positions. (A) Representative NLFK cell showing 3 measurement points and (B) the measured autocorrelation curves, respectively. (C) Summary of measured EYFP diffusion coefficients in NLKF cell nuclei and EGFP diffusion coefficients in HEK293, HeLa, T98G and TP366 nuclei. Scale bar, 5 µm.
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Supplementary Material and Methods
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We thank Colin Parrish for the infectious CPV clone and CPV antibodies, Jörg Langowski for the pEYFP-H2B and pECFP-H2B construct, Vim Vermeulen for the EYFP-PCNA construct and Jack Fransen for the PAGFP construct. We are grateful to Klaus Hedman for his comments on the manuscript. First-class experimental support by Milla Häkkinen, Jenni Reimari and Irene Helkala is gratefully acknowledged.