Parvovirus minute virus of mice interacts with sites of cellular DNA damage to establish and amplify its lytic infection
- Christopher S. Bond Life Sciences Center, United States
- School of Medicine, University of Missouri-Columbia, United States
- MU Informatics Institute, University of Missouri-Columbia, United States
Figures
Figure 1
The replicating MVM genome associates with cellular sites undergoing DNA damage.
(A) Representative confocal images of Mock versus MVM infected murine A9 cells at 16 hpi, probing MVM-NS1 (red) and the DDR factors FANCD2 and γ-H2AX, and the irrelevant transcription factor NR5A2 (g…
Figure 2 with 2 supplements
The MVM genome associates with distinct sites on the cellular genome.
(A) Top Schematic of the V3C-seq assay showing how MVM- host cell genomic proximity is frozen by crosslinking, followed by digesting (with HindIII) and intramolecularly ligating to generate novel …
Figure 2—figure supplement 1
MVM replication during viral infection and correlation of V3C-seq interaction sites.
(A) MVM replication over the timecourse of viral infection in parasynchronized murine A9 cells. A9 cells were infected at an MOI of 5. Cells were harvested at the indicated timepoints and Southern …
Figure 2—figure supplement 2
Genome browser snapshots of MVM interaction sites on all chromosomes in the mouse genome.
12 hpi (red), 16 hpi (blue), 20 hpi (orange) and 24 hpi (black) timepoints are shown. The y-axis values are depicted on the right hand side. Since MVM interaction at 24 hpi did not show a …
Figure 3 with 2 supplements
The MVM genome initiated infection at sites of cellular DNA damage that in mock infected cells also exhibited DNA damage as the cells cycled through S-phase, and as infection progressed, localized to additional sites of induced damage.
(A) Representative quantile normalized ChIP-seq plots of γ-H2AX binding to the cellular genome on Chromosome 17 (top) and Chromosome (19). The tracks represent γ-H2AX ChIP-seq peaks in A9 cells that …
Figure 3—figure supplement 1
Genome browser snapshots of γ-H2AX occupancy over the entire mouse genome at different timepoints of MVM infection.
Occupancy in mock infected and HU treated cells is also shown. Chromosomes 17 and 19 are presented in Figure 3A. γ-H2AX binding at the different timepoints and treatments are compared with MVM …
Figure 3—figure supplement 2
MVM DNA damage interactions.
(A) Alkaline comet assays were performed in uninfected, doxorubicin treated (200 nM) or MVM infected murine A9 cells for 24 hr, and DNA fragmentation was visualized using immunofluorescence …
Figure 4 with 2 supplements
MVM NS1 colocalizes to sites of cellular DNA damage along with MVM genome.
(A) Representative quantile normalized ChIP-seq plots of MVM-NS1 (purple) and BRCA1 (pink) binding to the cellular genome on Chromosome 17 (top) and Chromosome 19 (bottom), with γ-H2AX and V3C at 16 …
Figure 4—figure supplement 1
Called peaks from DDR ChIP-seq in HU-treated primary mouse splenocytes were compared with DDR ChIP-seq in MVM infected murine A9 fibroblasts at 16 hpi.
The comparisons are presented in the form of heatmaps for (A) BRCA1 binding (green) and (B) γ-H2AX modification (blue). (C) The spatial interaction of MVM with common fragile sites were assayed in …
Figure 4—figure supplement 2
Comparisons of MVM-associated VAD sites with topological structure of the mouse nucleome.
Hi-C contact maps in murine CH12 cells were visualized in comparison with MVM-V3C in A9 cells at 16 hpi (grey tracks on top) using the Juicebox data visualization platform (presented as red …
Figure 5
FISH assays confirmed that MVM localized with cellular sites of DNA damage.
(A) Schematic of Chromosomes 19 and 6 showing where MVM associates with the mouse genome in A9 cells at 16 hr post infection, depicting the sites where FISH probes were designed. Representative …
Figure 6
MVM associates with artificially-engineered sites of DNA damage.
(A) Murine A9 fibroblasts were infected with MVM at an MOI of 10 for 18 hr. Cells were sensitized with Hoechst 33342 solution prior to irradiation in selected regions of interest with a 405 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus, Male) | A9 | ATCC, (Tattersall and Bratton, 1983) PMID: 6602222 | RRID:CVCL_3984 | Verified as mycoplasma- negative by PCR |
| Cell line (Mus musculus) | NIH-3T3 | ATCC | RRID:CVCL_0594 | Verified as mycoplasma- negative by PCR |
| Cell line (Homo sapiens) | NB-324K | (Tattersall and Bratton, 1983) PMID: 6602222 | RRID:CVCL_U409 | Verified as mycoplasma- negative by PCR |
| Cell line (Mus musculus) | EL4 | ATCC, (Tattersall and Bratton, 1983) PMID: 6602222 | RRID:CVCL_0255 | Verified as mycoplasma- negative by PCR |
| Cell line (Rattus norvegicus) | F111 | Fischer Rat Fibroblasts; (Freeman et al., 1975) | RRID:CVCL_6C52 | Verified as mycoplasma- negative by PCR |
| Antibody | NS1 | Salome and Pintel, unpublished | 2C9b | anti-mouse; Usage per sample: ChIP: 6 µg Immunofluorescence: 2 µg IB: 2 µg |
| Antibody | γ−Η2ΑX | EMD Millipore | EMD Millipore:05–636 | anti-mouse; Usage per sample: ChIP: 5 µg |
| Antibody | γ−Η2ΑX | Abcam:ab11174 | RRID:AB_297813 | anti-rabbit; Usage per sample: ChIP: 5 µg Immunofluorescence: 2 µg IB: 2 µg |
| Antibody | BRCA1 | Thermo Fisher Scientific:17F8 | RRID:AB_557804 | anti-mouse; Usage per sample: ChIP: 5 µg |
| Antibody | FANCD2 | Bethyl Laboratories: a302-174A | RRID:AB_1659803 | anti-rabbit; Usage per sample: Immunofluorescence: 2 µg |
| Antibody | NR5A2 | Abcam:ab189876 | RRID: AB_2732890 | anti-rabbit; Usage per sample: Immunofluorescence: 2 µg |
| Antibody | IgG | Cell Signaling | Cell Signaling:5415S | mouse; Usage per sample: ChIP: 5 µg |
| Antibody | AF 488 | Life Technologies: A11034 | RRID:AB_2576217 | anti-rabbit secondary; Usage per sample: Immunofluorescence: 1 µg |
| Antibody | AF 568 | Life Technologies: A11031 | RRID:AB_144696 | anti-mouse secondary; Usage per sample: Immunofluorescence: 1 µg |
| Antibody | AF 555 | Life Technologies: A27039 | RRID:AB_2536100 | anti-rabbit secondary; Usage per sample: Immunofluorescence: 1 µg |
| Antibody | AF 647 | Life Technologies: A32728 | RRID:AB_2633277 | anti-mouse secondary; Usage per sample: Immunofluorescence: 1 µg |
| Recombinant DNA reagent | Lenti-CRISPRv2 | Addgene; (Sanjana et al., 2014) PMID: 25075903 | Addgene plasmid 52961 | |
| Recombinant DNA reagent | pgRNA-humanized | Addgene; (Qi et al., 2013) PMID: 23452860 | Addgene plasmid 44248 | |
| Peptide, recombinant protein | HindIII | New England Biolabs | NEB:R0104 | |
| Peptide, recombinant protein | NlaIII | New England Biolabs | NEB:R0125 | |
| Peptide, recombinant protein | T4 DNA Ligase | New England Biolabs | NEB:M0202 | |
| Commercial assay or kit | FISH Tag DNA Multicolor Kit | Thermo Fisher Scientific | Thermo Fisher Scientific: F32951 | |
| Commercial assay or kit | QIAquick PCR purification kit | Qiagen | Qiagen:28106 | |
| Commercial assay or kit | NEBNext Ultra II Library Prep Kit for Illumina | New England Biolabs | NEB:E7645 | |
| Commercial assay or kit | Trevigen Comet Assay Kit | Trevigen | Trevigen: 4250–050 K | |
| Commercial assay or kit | StemCell | EasySep Human CD4+ T Cell Enrichment Kit | StemCell:19052 | |
| Chemical compound, drug | Hydroxyurea | Sigma Aldrich | Sigma Aldrich:H8627 | |
| Chemical compound, drug | Doxorubicin | Sigma Aldrich | Sigma Aldrich:D1515 | |
| Chemical compound, drug | Bovine Serum Albumin | Sigma Aldrich | Sigma Aldrich:A2153 | |
| Chemical compound, drug | ProLong Diamond Antifade Mountant with DAPI | Thermo Fisher Scientific | Thermo Fisher Scientific: P36966 | |
| Chemical compound, drug | Hoechst 33342 | Thermo Fisher Scientific | Thermo Fisher Scientific: 62249 | |
| Software, algorithm | Bowtie2 | (Langmead and Salzberg, 2012) PMID: 22388286 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml | |
| Software, algorithm | Samtools | (Li et al., 2009) PMID: 19505943 | RRID:SCR_006646 | http://samtools.sourceforge.net/ |
| Software, algorithm | Bedtools | (Quinlan and Hall, 2010) PMID: 20110278 | RRID:SCR_006646 | http://bedtools.readthedocs.io/en/latest/ |
| Software, algorithm | Deeptools | (Ramírez et al., 2016) PMID: 27079975 | RRID:SCR_016366 | https://deeptools.readthedocs.io/en/develop/ |
| Software, algorithm | UCSC Genome Browser | (Kent et al., 2002) PMID: 12045153 | RRID:SCR_005780 | https://genome.ucsc.edu/ |
| Software, algorithm | PreprocessCore | (Bolstad, 2013) | https://www.bioconductor.org/packages/release/bioc/html/preprocessCore.html | |
| Software, algorithm | Biostrings | (Pagès et al., 2017) | https://bioconductor.org/packages/release/bioc/html/Biostrings.html | |
| Software, algorithm | EPIC | (Xu et al., 2014) PMID: 24743992 | https://github.com/biocore-ntnu/epic | |
| Software, algorithm | Galaxy | (Afgan et al., 2016) PMID: 27137889 | RRID:SCR_006281 | https://usegalaxy.org/ |
| Software, algorithm | ImageJ | (Schneider et al., 2012) PMID: 22930834 | RRID:SCR_003070 | https://imagej.net/Welcome |
| Software, algorithm | Huygens Professional | Huygens professional version 17.10 (Scientific volume imaging, The Netherlands) | https://svi.nl/Huygens-Professional |
Table 1
Bioinformatic codes used.
https://doi.org/10.7554/eLife.37750.015| Program | Function | Code used |
|---|---|---|
| V3C-seq analysis | ||
| Bowtie2 | Alignment | bowtie2 --trim5 50 --very-sensitive -x/storage/htc/ biocompute/ircf/dbase/genomes/M_musculus/bowtie2/ index/mm10 -S 24hpi_1.sam 24hpi_1.fastq |
| Samtools | Sam to Bam | samtools view -b -S -o aligned_24hpi_1.bam 24hpi_1.sam |
| Sort | samtools sort -o aligned_sorted_24hpi_1.bam aligned_24hpi_1.bam | |
| BEDtools | Compute histogram | genomeCoverageBed -ibam aligned_sorted_24hpi_1.bam -bg - trackline -split -g. ..>24hpi_1.bedgraph |
| ChIP-seq analysis | ||
| Bowtie2 | Alignment | bowtie2 -x/storage/htc/biocompute/ircf/dbase/genomes/M_musculus/ bowtie2/index/mm10 -U 16hpi_gh2ax_1.fastq -S 16hpi_gh2ax_1.sam |
| Samtools | Sam to Bam | samtools view -b -S -o aligned_16hpi_gh2ax_1.bam 16hpi_gh2ax_1.sam |
| Sort | samtools sort -o aligned_sorted_16hpi_gh2ax_1. bam aligned_16hpi_gh2ax_1.bam | |
| BEDtools | Bam to BED conversion | bedtools bamtobed -i 16hpi_gh2ax_1.bam>16hpi_gh2ax_1.bed |
| EPIC | Peak Calling | epic -t 16hpi_gh2ax_1.bed -c 16hpi_ip.bed -gn mm10 -b BED -o epic_12hpi_gh2ax_1 |
| BEDtools | Intersection | bedtools intersect –a 16hpi_gh2ax_1.bed –b 16hpi_gh2ax_2. bed>16hpi_gh2ax_1_2.bed |
| Jaccard analysis | bedtools jaccard -a mock_gh2ax.bed -b 16hpi_gh2ax.bed | |
Table 2
Antibody table.
https://doi.org/10.7554/eLife.37750.016| Antibody | Concentration used |
|---|---|
| NS1 (see Key Resources Table) | ChIP: 6 μg Immunofluorescence: 2 μg IB: 2 μg |
| γ-H2AX (anti-mouse); EMD Millipore | ChIP: 5 μg |
| γ-H2AX (anti-rabbit); Abcam | ChIP: 5 μg Immunofluorescence: 2 μg IB: 2 μg |
| BRCA1 (anti-mouse); Thermo Fisher Scientific | ChIP: 5 μg |
| FANCD2 (anti-rabbit); Bethyl Laboratories | Immunofluorescence: 2 μg |
| NR5A2 (anti-rabbit); Abcam | Immunofluorescence: 2 μg |
| IgG (mouse); Cell Signaling | ChIP: 5 μg |
| AF 488; anti-rabbit secondary, Life Technologies | Immunofluorescence: 1 μg |
| AF 568; anti-mouse secondary, Life Technologies | Immunofluorescence: 1 μg |
| AF 555; anti-rabbit secondary, Life Technologies | Immunofluorescence: 1 μg |
| AF 647; anti-mouse secondary, Life Technologies | Immunofluorescence: 1 μg |
Additional files
-
Supplementary file 1
contains a table with the sequences of PCR primers and Taqman probes used in this study.
- https://doi.org/10.7554/eLife.37750.017
-
Transparent reporting form
- https://doi.org/10.7554/eLife.37750.018
