(a–e) HFF were infected with HCMV strain AD169, IE1-deleted AD169 (HCMVΔIE1) or AD169 encoding IE1 mutant L174P (HCMV/IE1-L174P) at a MOI of 5 IEU/cell. Cells were harvested at 72 hpi for …
Numerical data that are represented as a graph in Figure 1b.
(a) Recruitment of nuclear body proteins to PML cages. HFF were infected with HCMV/IE1-L174P, based on strain AD169, at a MOI of 5 IEU/cell and harvested at 72 hpi for immunofluorescence staining of …
(a–d) MOI-dependent induction of PML cages. HFF were infected with HCMVΔIE1, based on strain AD169, at MOIs ranging from 0.1 to 10 IEU/cell or not infected (mock) and were harvested after 72 hr. …
Numerical data that are represented as graphs in Figure 3b, c and f.
Uncropped western blot images for Figure 3d.
(a–c) Reduced formation of PML cages in infected cells upon inhibition of the IFN pathway. HFF were infected with HCMV/IE1-L174P, based on strain AD169, at a MOI of 5 IEU/ml or were not infected …
Uncropped western blot images for Figure 4a and b.
Numerical data that are represented as graphs in Figure 4c, f, h, j, k and l.
(a) PML structures forming upon induction of DNA damage in HFF cells. HFF cells were seeded at low density (30,000 cells / well in 12-well plates) and were left untreated or treated with IFN (IFN-β, …
HFF were infected with EdC-labeled (HCMVΔIE1EdC) and unlabeled HCMVΔIE1 or respective wild-type viruses (HCMVEdC, HCMV), which are all based on strain AD169. At different times after infection, …
Numerical data that are represented as graphs in Figure 5a, d, f and h.
HFF cells were infected HCMV strain AD169 (MOI of 1) and, 44 hr later, were either left untreated (0 µM) or were treated with different concentrations (0.1 µM, 1 µM, or 10 µM) of EdC (a), EdU (b) or …
(a) IE1-deleted HCMV (strain AD169) was grown on HFF cells that stably express IE1 either in absence of nucleoside-analogs (mock) or in the presence of EdC or EdU (0.1 µM or 1 µM). Viral …
Numerical data that are represented as graphs in Figure 5—figure supplement 2a, b and d.
(a) Recruitment of heterochromatin protein HP1α to PML cages. HFF were non-infected or infected at a MOI of 5 IEU/cell with HCMV or HCMVΔIE1, based on strain AD169. After 72 hr, cells were fixed and …
Numerical data that are represented as graphs in Figure 6d and e.
(a) Localization of major capsid protein MCP to PML cages. HFF were infected with HCMV (MOI of 0.5) or HCMVΔIE1 (MOI of 5), based on strain AD169, and harvested at 3 or 6 days after infection, …
HFF expressing mCherry-tagged PML, isoform VI, were cultured in complete medium containing 10% FCS and were either mock treated (upper panel) of treated with 0.5 µg/ml doxycycline (lower panel). …
For CLEM analysis, HFF expressing mCherry-PML were seeded on carbon-patterned sapphire discs and, 1 day later, were infected with recombinant HCMV encoding IE2-eYFP, based von strain TB40E (MOI of …
(a) For CLEM analysis, HFF cells on carbon-patterned sapphire discs were infected with a TB40E-based recombinant HCMV lacking IE1 and encoding IE2-eYFP at a high MOI (MOI >30). 6 days after …
3D reconstruction of a cell nucleus after infection with HCMV-IE1-L174P and immunofluorescence staining as described in Figure 1a. The cell nucleus is shown in blue, PML cages in red, and viral …
FIB-SEM tomography was applied to analyze three volumes within a mCherry-PML expressing cell that was infected with HCMVΔIE1-IE2eYFP as described in Figure 7d. All regions had the same dimensions (5 …
3D reconstruction of a PML cage within a cell nucleus infected with HCMVΔIE1-IE2eYFP (see Figure 7d and e, panel c). FIB-SEM images containing the PML cage were assembled and cropped using ImageJ, …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Human cytomegalovirus) | AD169 | Hobom et al., J. Virol. 2000PMID:10933677 | based on BAC HB15 | |
Strain, strain background (Human cytomegalovirus) | TB40E | Sinzger et al., J. Gen. Virol. 2008PMID:18198366 | based on TB40E-Bac4 | |
Genetic reagent (Human cytomegalovirus) | AD169ΔIE1 | Scherer et al., J Virol. 2016PMID:26559840 | based on BAC HB15 | |
Genetic reagent (Human cytomegalovirus) | AD169/IE1-L174P | Scherer et al., J Virol. 2016PMID:26559840 | based on BAC HB15 | |
Genetic reagent (Human cytomegalovirus) | TB40E-IE2-EYFP | Wagenknecht et al., 2015PMID:26057166 | based on TB40E-Bac4 | |
Genetic reagent (Human cytomegalovirus) | TB40E-ΔIE1-IE2-EYFP | this paper | Deletion of IE1 from TB40E-IE2-EYFP | |
Cell line (Homo-sapiens) | U2OS | ATCC | HTB-96 | |
Cell line (Homo sapiens) | HEK293T | DMSZ | ACC 635 | |
Antibody | mAb-PML G8 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-377340 | IF (1:500) |
Antibody | pAb-PML #4 (Rabbit polyclonal) | Peter Hemmerich, Jena, Germany | IF (1:500) | |
Antibody | pAb-PML A301-167A (Rabbit polyclonal) | Bethyl Laboratories | Cat# A301-167ARRID: AB_873108 | IF (1:1000),WB (1:5000) |
Antibody | pAb-PML A301-168A (Rabbit polyclonal) | Bethyl Laboratories | Cat# A301-168ARRID: AB_2284081 | IF (1:1000),WB (1:5000) |
Antibody | pAb-Sp100 B01 (Mouse polyclonal) | Abnova | Cat# H00006672-B01, RRID:AB_982933 | IF (1:1000) |
Antibody | pAb-Sp100 GH3 (Rabbit polyclonal) | Hans Will, Hamburg, Germany | WB (1:10000) | |
Antibody | mAb-Daxx MCA2143 (Mouse monoclonal) | Bio-Rad | Cat# MCA2143, RRID:AB_2088900 | IF (1:400) |
Antibody | pAb-ATRX H300 (Rabbit polyclonal) | Santa Cruz Biotechnology | sc-15408 (discontinued) | IF (1:500) |
Antibody | Ab-HP1α 2,616 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2,616 | IF (1:200) |
Antibody | pAb-SUMO2/3 ab22654 (Rabbit polyclonal) | Abcam | Cat# ab22654, RRID:AB_2198415 | IF (1:500) |
Antibody | mAb-SUMO1 (Mouse monoclonal) | Gerrit Praefcke, Langen, Germany | IF (1:100) | |
Antibody | pAb-phospho-Histone H2A.X 20E3 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 9718, RRID:AB_2118009 | IF (1:500) |
Antibody | mAB-TRF1 TRF78 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-56807, RRID:AB_793407 | IF (1:250) |
Antibody | mAb-IE1 p63-27 (Mouse monoclonal) | Andreoni et al., J. Virol. Methods 1989PMID:2542350 | WB (1:100), IF (1:5) | |
Antibody | mAb-UL44 BS 510 (Mouse monoclonal) | Plachter et al., Virus Res. 1992PMID:1329369 | WB (1:5000), IF(1:1000) | |
Antibody | mAb-MCP 28–4 (Mouse monoclonal) | Waldo et al., Lancet 1989PMID:2463443 | WB (1:2), IF (1:2) | |
Antibody | pAb-IE2 pHM178 (Rabbit polyclonal) | Hofmann et al., J Virol 2000PMID:10684265 | WB (1:5000),IF (1:1500) | |
Antibody | pAb-UL84 (Rabbit polyclonal) | Hofmann et al., J Virol 2000PMID:10684265 | WB (1:5000),IF (1:1500) | |
Antibody | anti-FLAG (Mouse monoclonal) | Sigma-Aldrich | Cat# F1804, RRID:AB_262044 | IF (1:1000) |
Antibody | anti-Myc (Mouse monoclonal) | produced in hybridoma cells, ATCC | MYC 1-9E10.2 [9E10] | WB (1:10), IF (1:2) |
Antibody | anti-beta-Actin (Mouse monoclonal) | Sigma-Aldrich | Cat# A5441, RRID:AB_476744 | WB (1:10000) |
Antibody | anti-phospho-STAT2 (Tyr689) | Millipore | Cat# 07–224 | WB (1: 500) |
Antibody | anti-Interferon-alpha/beta Receptor Chain 2 (Mouse monoclonal) | Millipore | Cat# MAB1155, RRID:AB_2122758 | (5 µg/ml) |
Recombinant DNA reagent | pLVX-shRNA1 (plasmid) | Clontech | lentiviral construct for stable knockdown | |
Recombinant DNA reagent | pLVX-shPML (plasmid) | Wagenknecht et al., 2015PMID:26057166 | lentiviral construct for stable knockdown | |
Recombinant DNA reagent | pLVX-shSp100 (plasmid) | Wagenknecht et al., 2015PMID:26057166 | lentiviral construct for stable knockdown | |
Recombinant DNA reagent | pLVX-shDaxx (plasmid) | Wagenknecht et al., 2015PMID:26057166 | lentiviral construct for stable knockdown | |
Recombinant DNA reagent | pLVX-shATRX (plasmid) | this paper | cloning of shRNA directed against ATRX into pLVX-shRNA1 | |
Recombinant DNA reagent | pLKO-FLAG-PML I to VI (plasmid) | Cuchet et al., J Cell Sci 2011PMID:21172801 | lentiviral constructs for stable expression | |
Recombinant DNA reagent | pInducer20-CRSmut (plasmid) | Schilling et al., PLoS Pathog. 2021PMID:33770148 | lentiviral construct for inducible expression, mutated cis-repression signal | |
Recombinant DNA reagent | pInducer20-CRSmut-mCherry-PML VI (plasmid) | this paper | lentiviral construct for inducible expression, mutated cis-repression signal | |
Recombinant DNA reagent | pLKO-Myc-IE1 (plasmid) | Scherer et al., PLoS Pathog 2014PMID:25412268 | lentiviral construct for stable expression | |
Recombinant DNA reagent | pLKO-Myc-IE1 1–382 (plasmid) | Scherer et al., PLoS Pathog 2011PMID:26559840 | lentiviral construct for stable expression | |
Chemical compound, drug | Camptothecin; CPT | Santa Cruz Biotechnology | Cat# sc-200871 | |
Chemical compound, drug | Doxorubicin hydrochloride; Doxo | Cayman Chemical | Cat# Cay15007 | |
Chemical compound, drug | ATM inhibitor KU 55933; ATMi | Tocris Bioscience | Cat# 3,544 | |
Chemical compound, drug | 5-Ethynyl-2′-deoxycytidine; EdC | Sigma-Aldrich | Cat# T511307 | |
Chemical compound, drug | 5-Ethynyl-2′-deoxyuridine; EdU | Sigma-Aldrich | Cat# 900,584 | |
Chemical compound, drug | (2′S)–2′-Deoxy-2′-fluoro-5-ethynyluridine; F-ara-EdU | Sigma-Aldrich | Cat# T511293 |
Sequences of oligonucleotides used in this study.