A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen University, China
Figures
Figure 1 with 1 supplement
The CRISPR-nuPin system.
(A) Schematics of the CRISPR-nuPin system. (B) Subcellular localization of transiently expressed Flag-dCas9-emerin and Flag-emerin in HEK293T cells. (C–D) dCas9-emerin cells were treated with doxycycline (+DOX), and the intracellular distribution of dCas9-emerin protein was examined by immunofluorescence staining with anti-Flag and anti-NUP98 (NUP) antibodies. Representative images scanned by confocal microscopy and 3D reconstructed are shown in D. (E–G) BAC-HSV-1 plasmids containing dCas9-emerin cells (+DOX) were electroporated with sgRNA targeting HSV-1 or ctrl sgRNA (E, F), or dCas9-emerin cells (+DOX) only were electroporated with telomere-targeting sgRNA or ctrl sgRNA (G, H). At the indicated time points post sgRNA electroporation, cells were fixed, and the intranuclear BAC or telomeres were stained by FISH and scanned under a confocal microscope. Representative images (upper panel) and their 3D reconstructions (bottom panel) are shown in (E and G). The percentage of the BACs or telomeres at the nuclear edge vs total intranuclear stains at each time point was calculated and plotted in F (BAC) and H (telomere), respectively. The total number of counted dots of each sampling group is shown as n at the bottom of (F and H). In panel (F) an average of approximately 30–50 nuclei were counted. In panel (H) approximately 27–30 nuclei were counted to generate the respective datasets. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05, ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, ‘***’ represents p≤0.001, and ‘****’ represents p≤0.0001.
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Figure 1—source data 1
Source files for western blot, statistic data and images in Figure 1.
This zip archive contains source data for western blot in panel B, original data collected for statistical analysis in panel F and H, and processed images (TIF files) in panel C, D, E and G. Original fluorescence images and confocal images in panel C, D, E, and G are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data1-v3.pdf
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Figure 1—source data 2
Uncropped western blot with labels in Figure 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data2-v3.pdf
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Figure 1—source data 3
Source files for processed images in Figure 1C.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data3-v3.zip
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Figure 1—source data 4
Source files for processed images in Figure 1D.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data4-v3.zip
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Figure 1—source data 5
Source files for processed images in Figure 1E.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data5-v3.zip
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Figure 1—source data 6
Source files for original data collected for statistical analysis in Figure 1F, H.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data6-v3.xlsx
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Figure 1—source data 7
Source files for processed images in Figure 1F.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-data7-v3.zip
Figure 1—figure supplement 1
Characterization of the CRISPR-nuPin system.
(A) Schematics of the DOX-induced CRISPR-nuPin expression cassette in dCas9-emerin cells. (B) dCas9 protein expression in transiently transfected HEp-2 cells was confirmed by immunofluorescence staining (left panel) and immunoblotting with an anti-dCas9 antibody (right panel). (C) dCas9-emerin cells were treated with DOX at the indicated concentration for 48 hr, and the expression of dCas9-emerin was examined by immunoblotting. (D–F) DOX-treated dCas9-emerin cells were stained with antibodies against calnexin and emerin and/or Flag (green) and NUP98 (NUP). (G) HEp-2 and dCas9-emerin cells seeded onto 96-well plates were treated with DOX (+DOX) or DMSO (-DOX). Cell viability was tested and plotted, data shown are mean ± SD, n = 3. (H–I) dCas9-emerin cells (+DOX) transfected with a 5 kb plasmid were electroporated with the plasmid-specific sgRNA. At the indicated time points post electroporation, cells were fixed, and the 5 kb plasmids were stained by FISH and imaged under a confocal microscope. Representative images (upper panel) and their 3D reconstructions (bottom panel) are shown (H). (I) The percentage of the nuclear edge-located plasmid stains vs total intranuclear stains at each time point was calculated and plotted. The total number of counted dots of each sample group is shown as n at the bottom of (I) , About 30–50 nuclei were counted per sample group. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘****’ represents p≤0.0001. (J) The cross-section of an image from Figure 1E HSV-1 sgRNA electroporated group is shown on the left panel. In the right panel, Fiji image software was used for the linescans of the relative fluorescence intensity of DAPI (blue) and BAC-HSV (red) along the white dotted line. (K) The cross-section of an image from Figure 1G Telomere targeting sgRNA electroporated group is shown on the left panel. In the right panel, a similar analysis as J is performed.
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Figure 1—figure supplement 1—source data 1
Source files for western blot, statiscic data and images in Figure 1—figure supplement 1.
This zip archive contains source data for western blot in panel B and C, original data collected for statistical analysis in panel G and I, and processed images (TIF files) in panel B, D, E, F, H, J, and K. Original fluorescence images and confocal images are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data1-v3.zip
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Figure 1—figure supplement 1—source data 2
Source files for uncropped western blot in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data2-v3.pdf
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Figure 1—figure supplement 1—source data 3
Source files for uncropped western blot with labels in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data3-v3.pdf
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Figure 1—figure supplement 1—source data 4
Source files for uncropped western blot in Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data4-v3.pdf
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Figure 1—figure supplement 1—source data 5
Source files for uncropped western blot with labels in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data5-v3.pdf
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Figure 1—figure supplement 1—source data 6
Source files for processed images in Figure 1—figure supplement 1D.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data6-v3.zip
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Figure 1—figure supplement 1—source data 7
Source files for processed images in Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data7-v3.zip
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Figure 1—figure supplement 1—source data 8
Source files for processed images in Figure 1-figure supplement 1 F.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data8-v3.zip
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Figure 1—figure supplement 1—source data 9
Source files for original data collected for statistical analysis in Figure 1-figure supplement 1 G and I.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data9-v3.xlsx
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Figure 1—figure supplement 1—source data 10
Source files for processed images in Figure 1—figure supplement 1H.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data10-v3.zip
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Figure 1—figure supplement 1—source data 11
Source files for processed images in Figure 1—figure supplement 1J.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data11-v3.zip
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Figure 1—figure supplement 1—source data 12
Source files for processed images in Figure 1—figure supplement 1K.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig1-figsupp1-data12-v3.zip
Figure 2 with 1 supplement
Transcription from the nuclear edge-localized HSV-1 genomes upon entry was strongly inhibited.
(A–F) dCas9-emerin cells transfected with HSV-1 sgRNA or ctrl sgRNA for 24 hr were infected with HSV-1 at an MOI of 1 (2 hr on ice) (A, F, G, H), an MOI of 5 (B, D, E), or the indicated MOIs (C). (A) Single-cycle growth kinetics of HSV-1 titrated by plaque assay, data shown are mean ± SD, n = 3. (B) Cells were fixed at 3 hpi and stained for HSV-1 genomes by FISH. The 3D reconstruction of the image is shown at the bottom. (C) Cell-associated HSV-1 viruses were titrated at 12 hpi, data shown are mean ± SD, n = 3. (D) Total RNA was extracted at 0, 0.17, 0.5, 1, 2, 3, 5 and 9 hpi, and the mRNA levels of ICP27, TK, ICP0 and VP16 were measured by qPCR (0.17 and 0.5 hpi labels were omitted), data shown are mean ± SD, n = 3. (E) The protein levels of ICP0, ICP8, TK, and VP16 at the indicated time points post infection were examined by immunoblotting. β-actin served as a loading control. (F) ChIP assays of differentially modified histones on the promoters of ICP27, ICP8 and VP16 of HSV-1 in HSV-1 sgRNA- or ctrl sgRNA-expressing dCas9-emerin cells at 0.5 hpi were performed using antibodies specific for H3K4me3, H3K9ac, H4K20me3, H3K9me3 and H3K27me3. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05, ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, ‘***’ represents ≤0.001, and ‘****’ represents p≤0.0001. (G) ChIP assays as in F were performed at the indicated time points post infection using an antibody against H3K9me3. For (F and G) the results were expressed as enrichment (fold) by comparing the fraction of viral promoters immunoprecipitated by the indicated antibodies to the fraction of GAPDH immunoprecipitated in the same reaction. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, ‘***’ represents ≤0.001, and ‘****’ represents p≤0.0001. (H) HSV-1 sgRNA- or ctrl sgRNA-expressing dCas9-emerin cells were treated with sodium butyrate (NaB) for 5 hr before infection (red lines) or not treated (black lines). The mRNA level of ICP27 at the indicated time points was measured by qPCR, data is shwon as mean ± SD, n = 3.
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Figure 2—source data 1
Source files for western blot, statiscic data and images in Figure 2.
This zip archive contains source data for western blot in panel E, original viral titers in panel A and C, qPCR data in panel D, F, G, and H, and processed images (TIF files) in panel B. Original confocal images in panel B are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-data1-v3.xlsx
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Figure 2—source data 2
Source files for processed images in Figure 2B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-data2-v3.zip
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Figure 2—source data 3
Source files for uncropped western blot in Figure 2E.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-data3-v3.pdf
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Figure 2—source data 4
Source files for uncropped western blot with relevant labels in Figure 2E.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-data4-v3.pdf
Figure 2—figure supplement 1
Nuclear edging of the viral genomes upon entry suppressed HSV-1 infection.
(A) HEp-2 cells were infected with HSV-1 at an MOI of 5. Cells were fixed at 0.5, 1, 1.5, and 2 hpi, and the intranuclear HSV-1 genomes were stained by FISH and scanned under a confocal microscope. Representative image of the 2 hpi sample and its 3D reconstruction are shown in the left panel. The percentage of the nuclear edge-located HSV-1 genomes vs total intranuclear stains at each time point was calculated, plotted and shown in the right panel. The total number of counted dots is shown as n on the top of the right panel. (B) The cross-section of a representative image from A is shown in the left panel. The while line measures the diameter of an HSV-1 FISH spot. The yellow line measures the center of an HSV-1 FISH spot to the edge of DAPI. The red arrow points to a nuclear edge-localized HSV-1 FISH spot. Scale bars, 2 μm. In the right panel, the distance of more than 100 nuclear edge-HSV-1 spots to the margin of the DAPI-stained area was measured and plotted. (C) Sequence, position, and number of targeting sites of 5 HSV-1 sgRNAs in the viral genome (GenBank accession number GU734771). Note that the figure is not drawn to scale as the sgRNA targeting site’s sequence is relatively short compared to the entire HSV-1 genome. (D) dCas9-emerin cells expressing a ctrl sgRNA or each of the 5 HSV-1 sgRNAs were infected with HSV-1 at an MOI of 1. Virus yield at 24 hpi was titrated by plaque assay. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘***’ represents ≤0.001 and ‘****’ represents p≤0.0001. (E) GFP-expressing HEp-2 cells (GFP), dCas9-expressing cells or dCas9-emerin cells were transfected with plasmids expressing HSV-1 sgRNA2 or ctrl sgRNA for 24 hr and infected with HSV-1 at an MOI of 1. Cell-associated viruses at 24 hpi were titrated by plaque assay. Data is shown as mean ± SD, n = 3. (F) dCas9 and dCas9-emerin cells were transfected with plasmids expressing HSV-1 sgRNA1 or sgRNA2 for 24 hr and infected with HSV-1 at an MOI of 1. Cell-associated viruses at 24 hpi were titrated by plaque assay. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘****’ represents p≤0.0001. (G) dCas9 and dCas9-emerin cells were transfected with plasmids expressing HSV-1 sgRNA2 and infected with R8515 (a GFP expressing recombinant HSV-1) at an MOI of 1. Cells were imaged at 24 hpi. (H) dCas9-emerin cells transfected with plasmids expressing ctrl sgRNA or HSV-1 sgRNA2 for 24 hr were infected with HSV-1 (2 hr on ice) at different MOIs. The mRNA level of ICP27 at the indicated time points was quantified by qPCR. Data is shown as mean ± SD, n = 3. (I) dCas9-emerin cells were transfected with plasmids expressing HSV-1 sgRNA2 or ctrl sgRNA for 24 hr and infected (2 hr on ice) with HSV-1 at an MOI of 1. Cells were collected at the indicated times and separated into cytosol (C), membranous organelles (M) and nuclear compartments (N). Successful separation of the subcellular compartments was confirmed by immunoblotting with marker antibodies (I upper panel). The viral DNA content in the nuclear compartment was quantified by qPCR targeting the ICP27 region of the HSV-1 genome and normalized to total cellular viral DNA load. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05, ‘*’ represents p≤0.05 and ‘**’ represents p≤0.01. Two independent experimental results are shown at the bottom panel of I. (J–K) dCas9-emerin cells with (+DOX) or without DOX (-DOX) were transfected with plasmids expressing HSV-1 sgRNA or ctrl sgRNA for 24 hr and then infected with HSV-1 at an MOI of 5. At the indicated time point post infection, cells were fixed and stained with anti-ICP8 (green) and anti-Flag (red) antibodies. Two representative images at each time point are shown in J. White arrows indicate nuclei with diffuse ICP8 (inefficient replication compartment formation), and red arrows indicate nuclei with aggregated/condensed ICP8 (replication compartment formation). (K) More than 200 nuclei in each sampling group were counted, and the HSV-1 replication center formation efficiency (nuclei of aggregated/condensed ICP8 vs total ICP8-positive nuclei) was quantified and plotted. Data is shown as mean ± SD, n = 3. (L) The fraction of viral DNA (targeting the ICP27 region; top panel) or host genomic DNA (targeting GAPDH) (bottom panel) immunoprecipitated with the indicated antibodies was quantified by qPCR and compared to their respective levels immunoprecipitated by a nonspecific antibody (ab) (IgG) and plotted. Data is shown as mean ± SD, n = 3. (M) ChIP assay examining the histone packaging level on the promoters of ICP27, ICP8 and VP16 of HSV-1 in HSV-1 sgRNA- or ctrl sgRNA-expressing dCas9-emerin cells at the indicated time points post infection (2 hr on ice) was performed using anti-H3 antibody. The results were displayed as enrichment (fold) by comparing the fraction of viral promoters immunoprecipitated by anti-H3 to the fraction of GAPDH immunoprecipitated in the same reaction. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05 and ‘**’ represents p≤0.01. (N) HSV-1 sgRNA- or ctrl sgRNA-expressing dCas9-emerin cells were treated with DMSO or different HDACi: valproic acid (VPA), trichostatin A (TSA), romidepsin (FK 228) or sodium butyrate (NaB) and infected with HSV-1 (2 hr on ice) at an MOI of 1 (drug treatment started 5 hr before the infection). The mRNA level of ICP27 at 3 hpi was quantified by qPCR, and the ICP27 level in ctrl sgRNA-expressing cells was set as 100%. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘***’ represents ≤0.001 and ‘****’ represents p≤0.0001.
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Figure 2—figure supplement 1—source data 1
Source files for western blot, statiscic data and images in Figure 2—figure supplement 1.
This zip archive contains source data for western blot in panel I, original data collected for statistical analysis in panel A, B, and K, qPCR data in panel H, I, L, M and N, virus titers in panel D, E and F, and processed images (TIF files) in panel B, G and J. Original fluorescence images are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data1-v3.xlsx
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Figure 2—figure supplement 1—source data 2
Source files for processed images in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data2-v3.zip
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Figure 2—figure supplement 1—source data 3
Source files for processed images in Figure 2—figure supplement 1G.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data3-v3.zip
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Figure 2—figure supplement 1—source data 4
Source files for uncropped western blot in Figure 2—figure supplement 1L.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data4-v3.pdf
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Figure 2—figure supplement 1—source data 5
Source files for uncropped western blot with labels in Figure 2—figure supplement 1I.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data5-v3.pdf
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Figure 2—figure supplement 1—source data 6
Source files for processed images in Figure 2—figure supplement 1J.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig2-figsupp1-data6-v3.zip
Figure 3 with 1 supplement
Dislodging HSV-1 genomes to the nuclear edge at 1 hpi promoted virus infection, termed ‘Escaping’.
(A) dCas9 cells (+DOX) mock-infected or infected with HSV-1 were fixed at 3 hpi, and the intracellular distribution of dCas9-emerin and NUP98 was stained with anti-Flag (green) and anti-NUP98 (NUP) (red) antibodies. (B) Schematics of the following experiments. dCas9-emerin cells were infected (2 hr on ice) with HSV-1 at an MOI of 1 and electroporated with HSV-1 sgRNA or ctrl sgRNA at 0 (immediately post virus inoculation), 0.5, 1 and 2 hpi. (C–D) At 5 min post sgRNA electroporation at the indicated time, the cells were fixed, and the intranuclear HSV-1 genomes were stained by FISH (red). Representative confocal images (upper panel) and their 3D reconstructions (3D reconstruction shows only the surface of the DAPI-stained area) are shown in C. More than 1000 total FISH-stained dots from approximately 155–250 nuclei were counted in each sampling group (n represents the total number of counted dots in each experimental group) The percentage of marginalized HSV-1 genomes vs total intranuclear counts is plotted in D. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘****’ represents p≤0.0001. (E). At 12 hpi, cell-associated viruses were titrated. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘*’ represents p≤0.05, ‘**’ represents p≤0.01. (F) Cell-associated virus yields during the first 12 hr post infection were examined. dCas9-emerin cells electroporated with ctrl sgRNA (empty rectangle) or HSV-1 sgRNA (filled rectangle) at 0 hpi served as a reference in the three charts in F. The blue arrow indicates the time of sgRNA electroporation. The red filled cycle represents the virus yield in cells that received HSV-1 sgRNA, and the green empty cycle represents the virus yield in cells that received ctrl sgRNA. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05, ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, and ‘***’ represents ≤0.001. (G) At 6 hpi, cells were fixed and stained with anti-ICP8 (green). Representative images of all stages of replication center formation are shown.
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Figure 3—source data 1
Source files for western blot, statiscic data and images in Figure 3.
This zip archive contains source data for original data collected for statistical analysis in panel D, virus titers in panels E and F, and processed images (TIF files) in panels A, C, and G. Original fluorescence images and confocal images in panels A, C, and G are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-data1-v3.zip
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Figure 3—source data 2
Source files for processed images in Figure 3C.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-data2-v3.zip
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Figure 3—source data 3
Source files for original data collected for statistical analysis and for virus titers in Figure 3D–F.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-data3-v3.xlsx
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Figure 3—source data 4
Source files for processed images in Figure 3G.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-data4-v3.zip
Figure 3—figure supplement 1
Localization of emerin during HSV-1 infection.
dCas9-emerin cells were infected with HSV-1 at an MOI of 5, collected at the indicated time and stained with antibodies against ICP8 (green) and emerin (red) (A, B) or antibodies against Flag (green) and NUP98 (NUP) (red) (C). Representative images are shown. In A and , cells were imaged under confocal microscopy (upper panel), and their 3D reconstruction is shown in the bottom panel. (D) dCas9 cells infected (2 hr on ice) with HSV-1 at an MOI of 1 were electroporated with HSV-1 sgRNA or ctrl sgRNA at the indicated time points post infection. At 12 hpi, cell-associated viruses were titrated by plaque assay. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant.
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Figure 3—figure supplement 1—source data 1
Source files for western blot, statiscic data and images in Figure 3—figure supplement 1.
This zip archive contains source data for original virus titers in panel D, and processed images (TIF files) in panels A, B, and C. Original confocal images are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-figsupp1-data1-v3.zip
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Figure 3—figure supplement 1—source data 2
Source files for processed images in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-figsupp1-data2-v3.zip
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Figure 3—figure supplement 1—source data 3
Source files for processed images in Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-figsupp1-data3-v3.zip
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Figure 3—figure supplement 1—source data 4
Source files for original virus titers in Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig3-figsupp1-data4-v3.xlsx
Figure 4 with 1 supplement
ICP0 is a sufficient but not necessary condition of the ‘Escaping’.
(A–E) dCas9-emerin cells infected with HSV-1 (2 hr on ice) at an MOI of 1 were electroporated with HSV-1 sgRNA or ctrl sgRNA at 1 hpi. (A) The mRNA levels of ICP27, TK, ICP0 and VP16 at the indicated time points were measured by qPCR. (B) The protein levels of ICP0, ICP8, TK and VP16 at the indicated time points were measured by immunoblotting. Data is shown as mean ± SD, n = 3. (C) dCas9-emerin cells were pretreated with PAA (500 µg/mL) or DMSO for 2 hr before infection and throughout the experiment and electroporated with HSV-1 sgRNA or ctrl sgRNA at 1 hpi. The mRNA levels of TK and VP16 at the indicated time points were measured by qPCR. Data is shown as mean ± SD, n = 3. (D) Five-fold more protein than B was loaded to detect early expression of viral genes by immunoblotting. (E) Cells were fixed at 1.5 hpi and stained with an anti-ICP0 antibody. (F) dCas9-emerin cells overexpressing ICP0 were infected with HSV-1 (2 hr on ice) at an MOI of 1 and electroporated with HSV-1 sgRNA or ctrl sgRNA at the indicated time points. The virus yields at 24 hpi were titrated. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. n.s. represents not significant, p>0.05, ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, ‘***’ represents ≤0.001, and ‘****’ represents p≤0.0001. (G) dCas9-emerin cells overexpressing either ICP0 or GFP were processed as in A. The fold change of virus titer induced by E1hpi in ICP0 overexpressing cells versus that in GFP overexpressing cells from three independent experiments were calculated by normalizing the virus titer in cells electroporated with HSV-1 sgRNA at 1 hpi to that in cells electroporated with control sgRNA at 1 hpi and plotted (same shape of symbols represents the same batch of experiments). Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘**’ represents p≤0.01. Expression of ICP0 was confirmed by immunoblotting. (H) Cells infected with ΔICP0 virus were processed similarly to F. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘*’ represents p≤0.05 and ‘***’ represents ≤0.001. (I) Cells infected with ΔICP0 virus were processed similarly to (A) and the mRNA levels of TK and VP16 at the indicated time points were measured by qPCR. Data is shown as mean ± SD, n = 3.
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Figure 4—source data 1
Source files for western blot, statiscic data and images in Figure 4.
This zip archive contains source data for western blot in panels B and D, qPCR data in panels A, C, and I, virus titers in panels F, G, and H, and processed images (TIF files) in panel E. Original images in panel E are deposited on DRYAD (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data1-v3.xlsx
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Figure 4—source data 2
Source files for uncropped western blot in Figure 4B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data2-v3.pdf
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Figure 4—source data 3
Source files for uncropped western blot with labels in Figure 4B.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data3-v3.pdf
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Figure 4—source data 4
Source files for uncropped western blot in Figure 4D.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data4-v3.pdf
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Figure 4—source data 5
Source files for uncropped western blot with labels in Figure 4D.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data5-v3.pdf
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Figure 4—source data 6
Source files for processed images in Figure 4E.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data6-v3.zip
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Figure 4—source data 7
Source files for uncropped western blot in Figure 4G.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data7-v3.pdf
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Figure 4—source data 8
Source files for uncropped western blot with labels in Figure 4G.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-data8-v3.pdf
Figure 4—figure supplement 1
Characterization of the growth of ΔICP0 in dCas9-emerin cells with viral genomes inserted into the nucleus upon their entry.
(A) HEp-2 cells were pretreated with PAA (500 µg/mL) or DMSO for 2 hr and infected (2 hr on ice) with HSV-1 at an MOI of 0.1 (PAA and DMSO were added all the time). Cells were collected at the indicated time points post infection, and the relative HSV-1 genome level (normalized to GAPDH) was quantified by qPCR. Data shown are mean ± SD, n = 3. (B) Three independent experimental results of Figure 4G. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘*’ represents p≤0.05, ‘**’ represents p≤0.01, and ‘****’ represents p≤0.0001. (C–F) dCas9-emerin cells transfected with HSV-1 sgRNA or ctrl sgRNA for 24 hr were infected with ΔICP0 virus at the indicated MOI (C) or an MOI of 1 (D–F). (C) Cell-associated ΔICP0 was titrated at 24 hpi. Data is shown as mean ± SD, n = 3. (D) Cell-associated ΔICP0 was titrated at 24 hpi. Data is shown as mean ± SD, n = 3. (E) The mRNA levels of ICP27, TK and VP16 were quantified by qPCR. (F) The protein levels of ICP27, ICP0, TK and VP16 were examined by immunoblotting. Data is shown as mean ± SD, n = 3. (G) dCas9-emerin cells infected with ΔICP0 virus at an MOI of 1 were electroporated with HSV-1 sgRNA or ctrl sgRNA at 1 hpi. (F) At 12 hpi, cell-associated viruses were titrated by plaque assay. (G) dCas9-emerin cells overexpressing either ICP0 or GFP were infected with ΔICP0 virus at an MOI of 1 and electroporated with HSV sgRNA or ctrl sgRNA at 1 hpi. Cell-associated viruses at 24 hpi were titrated. Data is shown as mean ± SD, n = 3, and P values are calculated using the Student's t-test. ‘*’ represents p≤0.05 and ‘****’ represents p≤0.0001.
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Figure 4—figure supplement 1—source data 1
Source files for western blot, statiscic data and images in Figure 4—figure supplement 1.
This zip archive contains source data for western blot in panel F, original virus titers in panels B, C, D, and G, qPCR data in panels A and E (https://doi.org/10.5061/dryad.vmcvdncxd).
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-figsupp1-data1-v3.xlsx
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Figure 4—figure supplement 1—source data 2
Source files for uncropped western blot in Figure 4—figure supplement 1F.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-figsupp1-data2-v3.pdf
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Figure 4—figure supplement 1—source data 3
Source files for uncropped western blot with labels in Figure 4—figure supplement 1F.
- https://cdn.elifesciences.org/articles/85412/elife-85412-fig4-figsupp1-data3-v3.pdf
Figure 5
Proposed stages of the early intranuclear life of HSV-1.
The schematic depicts the effects of intranuclear repositioning of viral genomes on HSV-1 infection within the initial hours of entering eukaryotic cells.
Additional files
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Supplementary file 1
Repeat numbers of key experiments.
The contents of this Excel file provide information about the repeat numbers of crucial experiments conducted during the research.
- https://cdn.elifesciences.org/articles/85412/elife-85412-supp1-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85412/elife-85412-mdarchecklist1-v3.pdf
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A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1
eLife 12:e85412.
https://doi.org/10.7554/eLife.85412
