Parvovirus induced alterations in nuclear architecture and dynamics

Abstract

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, analysis 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.

Figure 1. Intranuclear distribution of viral proteins and dextrans.

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.

Figure 2. Intranuclear PAGFP-VP2 diffusion dynamics.

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.

Figure 3. Distribution of histone H2B in NLFK cells.

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.

Figure 4. DNA content and nuclear size in infected and 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.

Figure 5. EYFP diffusion in nuclei of infected and 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.

Figure 6. Intranuclear NS1-deYFP dynamics.

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.

Figure 7. PCNA-EYFP dynamics.

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.

Figure 8. Schematic representation of the replication body enlargement.

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.