Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies

  1. Myriam Scherer
  2. Clarissa Read
  3. Gregor Neusser
  4. Christine Kranz
  5. Anna K Kuderna
  6. Regina Müller
  7. Florian Full
  8. Sonja Wörz
  9. Anna Reichel
  10. Eva-Maria Schilling
  11. Paul Walther
  12. Thomas Stamminger  Is a corresponding author
  1. Institute of Virology, Ulm University Medical Center, Germany
  2. Central Facility for Electron Microscopy, Ulm University, Germany
  3. Institute of Analytical and Bioanalytical Chemistry, Ulm University, Germany
  4. Institute of Clinical and Molecular Virology, Friedrich Alexander Universität Erlangen-Nürnberg, Germany
8 figures, 3 videos, 1 table and 2 additional files

Figures

Formation of PML cages during infection with IE1-deficient HCMV.

(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 …

Protein composition of PML cages.

(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 …

Impact of virus dose and viral DNA replication on formation of PML cages.

(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. …

Figure 4 with 1 supplement
Role of IFN and DNA damage signaling for induction of PML cages.

(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 …

Figure 4—figure supplement 1
Effect of IFN and DNA damage on PML localization.

(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, …

Figure 5 with 2 supplements
Entrapment of HCMV input genomes by PML cages.

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, …

Figure 5—figure supplement 1
Detection of HCMV replication centers using alkyne-modified nucleosides.

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 …

Figure 5—figure supplement 2
Effect of alkyne-modified nucleosides on HCMV growth and IE gene expression.

(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 …

Abrogation of HCMV genome repression by disruption of PML cages.

(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 …

Figure 7 with 3 supplements
Entrapment of HCMV nuclear capsids by PML cages.

(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, …

Figure 7—figure supplement 1
Characterization of HFF with doxycycline-inducible expression of mCherry-PML.

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). …

Figure 7—figure supplement 2
Even distribution of viral capsids in HCMV-IE2eYFP-infected cell nuclei.

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 …

Figure 7—figure supplement 3
Clustering of viral capsids in HCMV∆IE1-infected cell nuclei.

(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 …

Author response image 1

Videos

Video 1
Formation of PML cages after infection with HCMV-IE1-L174P.

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 …

Video 2
FIB-SEM tomography showing HCMV capsid entrapment by PML cages.

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 …

Video 3
3D reconstruction of a PML cage containing HCMV capsids.

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, …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Human cytomegalovirus)AD169Hobom et al., J. Virol. 2000PMID:10933677based on BAC HB15
Strain, strain background (Human cytomegalovirus)TB40ESinzger et al., J. Gen. Virol. 2008PMID:18198366based on TB40E-Bac4
Genetic reagent (Human cytomegalovirus)AD169ΔIE1Scherer et al., J Virol. 2016PMID:26559840based on BAC HB15
Genetic reagent (Human cytomegalovirus)AD169/IE1-L174PScherer et al., J Virol. 2016PMID:26559840based on BAC HB15
Genetic reagent (Human cytomegalovirus)TB40E-IE2-EYFPWagenknecht et al., 2015PMID:26057166based on TB40E-Bac4
Genetic reagent (Human cytomegalovirus)TB40E-ΔIE1-IE2-EYFPthis paperDeletion of IE1 from TB40E-IE2-EYFP
Cell line (Homo-sapiens)U2OSATCCHTB-96
Cell line (Homo sapiens)HEK293TDMSZACC 635
AntibodymAb-PML G8 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-377340IF (1:500)
AntibodypAb-PML #4 (Rabbit polyclonal)Peter Hemmerich, Jena, GermanyIF (1:500)
AntibodypAb-PML A301-167A (Rabbit polyclonal)Bethyl LaboratoriesCat# A301-167ARRID: AB_873108IF (1:1000),WB (1:5000)
AntibodypAb-PML A301-168A (Rabbit polyclonal)Bethyl LaboratoriesCat# A301-168ARRID: AB_2284081IF (1:1000),WB (1:5000)
AntibodypAb-Sp100 B01 (Mouse polyclonal)AbnovaCat# H00006672-B01, RRID:AB_982933IF (1:1000)
AntibodypAb-Sp100 GH3 (Rabbit polyclonal)Hans Will, Hamburg, GermanyWB (1:10000)
AntibodymAb-Daxx MCA2143 (Mouse monoclonal)Bio-RadCat# MCA2143, RRID:AB_2088900IF (1:400)
AntibodypAb-ATRX H300 (Rabbit polyclonal)Santa Cruz Biotechnologysc-15408 (discontinued)IF (1:500)
AntibodyAb-HP1α 2,616 (Rabbit polyclonal)Cell Signaling TechnologyCat# 2,616IF (1:200)
AntibodypAb-SUMO2/3 ab22654 (Rabbit polyclonal)AbcamCat# ab22654, RRID:AB_2198415IF (1:500)
AntibodymAb-SUMO1 (Mouse monoclonal)Gerrit Praefcke, Langen, GermanyIF (1:100)
AntibodypAb-phospho-Histone H2A.X 20E3 (Rabbit monoclonal)Cell Signaling TechnologyCat# 9718, RRID:AB_2118009IF (1:500)
AntibodymAB-TRF1 TRF78 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-56807, RRID:AB_793407IF (1:250)
AntibodymAb-IE1 p63-27 (Mouse monoclonal)Andreoni et al., J. Virol. Methods 1989PMID:2542350WB (1:100), IF (1:5)
AntibodymAb-UL44 BS 510 (Mouse monoclonal)Plachter et al., Virus Res. 1992PMID:1329369WB (1:5000), IF(1:1000)
AntibodymAb-MCP 28–4 (Mouse monoclonal)Waldo et al., Lancet 1989PMID:2463443WB (1:2), IF (1:2)
AntibodypAb-IE2 pHM178 (Rabbit polyclonal)Hofmann et al., J Virol 2000PMID:10684265WB (1:5000),IF (1:1500)
AntibodypAb-UL84 (Rabbit polyclonal)Hofmann et al., J Virol 2000PMID:10684265WB (1:5000),IF (1:1500)
Antibodyanti-FLAG (Mouse monoclonal)Sigma-AldrichCat# F1804, RRID:AB_262044IF (1:1000)
Antibodyanti-Myc (Mouse monoclonal)produced in hybridoma cells, ATCCMYC 1-9E10.2 [9E10]WB (1:10), IF (1:2)
Antibodyanti-beta-Actin (Mouse monoclonal)Sigma-AldrichCat# A5441, RRID:AB_476744WB (1:10000)
Antibodyanti-phospho-STAT2 (Tyr689)MilliporeCat# 07–224WB (1: 500)
Antibodyanti-Interferon-alpha/beta Receptor Chain 2 (Mouse monoclonal)MilliporeCat# MAB1155, RRID:AB_2122758(5 µg/ml)
Recombinant DNA reagentpLVX-shRNA1 (plasmid)Clontechlentiviral construct for stable knockdown
Recombinant DNA reagentpLVX-shPML (plasmid)Wagenknecht et al., 2015PMID:26057166lentiviral construct for stable knockdown
Recombinant DNA reagentpLVX-shSp100 (plasmid)Wagenknecht et al., 2015PMID:26057166lentiviral construct for stable knockdown
Recombinant DNA reagentpLVX-shDaxx (plasmid)Wagenknecht et al., 2015PMID:26057166lentiviral construct for stable knockdown
Recombinant DNA reagentpLVX-shATRX (plasmid)this papercloning of shRNA directed against ATRX into pLVX-shRNA1
Recombinant DNA reagentpLKO-FLAG-PML I to VI (plasmid)Cuchet et al., J Cell Sci 2011PMID:21172801lentiviral constructs for stable expression
Recombinant DNA reagentpInducer20-CRSmut (plasmid)Schilling et al., PLoS Pathog. 2021PMID:33770148lentiviral construct for inducible expression, mutated cis-repression signal
Recombinant DNA reagentpInducer20-CRSmut-mCherry-PML VI (plasmid)this paperlentiviral construct for inducible expression, mutated cis-repression signal
Recombinant DNA reagentpLKO-Myc-IE1 (plasmid)Scherer et al., PLoS Pathog 2014PMID:25412268lentiviral construct for stable expression
Recombinant DNA reagentpLKO-Myc-IE1 1–382 (plasmid)Scherer et al., PLoS Pathog 2011PMID:26559840lentiviral construct for stable expression
Chemical compound, drugCamptothecin; CPTSanta Cruz BiotechnologyCat# sc-200871
Chemical compound, drugDoxorubicin hydrochloride; DoxoCayman ChemicalCat# Cay15007
Chemical compound, drugATM inhibitor KU 55933; ATMiTocris BioscienceCat# 3,544
Chemical compound, drug5-Ethynyl-2′-deoxycytidine; EdCSigma-AldrichCat# T511307
Chemical compound, drug5-Ethynyl-2′-deoxyuridine; EdUSigma-AldrichCat# 900,584
Chemical compound, drug(2′S)–2′-Deoxy-2′-fluoro-5-ethynyluridine; F-ara-EdUSigma-AldrichCat# T511293

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