Figure 5. Entrapment of HCMV input genomes by PML cages.

HFF were infected with EdC-labeled (HCMVΔIE1_EdC) and unlabeled HCMVΔIE1 or respective wild-type viruses (HCMV_EdC, HCMV), which are all based on strain AD169. At different times after infection, cells were fixed for antibody staining to detect IE2 and/or PML in combination with click chemistry to visualize HCMV input genomes (vDNA). (a) HFF were infected with HCMVΔIE1_EdC or unlabeled HCMVΔIE1 at a MOI of 0.1 IEU/cell and stained for PML and vDNA at 8 hpi. According to the number of genomes detected in the nucleus, cells were divided in three groups. Colocalization of PML and vDNA (entrapment) within these groups was determined in ≥160 cells derived from three independent experiments. Genomes encased by PML are marked by arrows, not entrapped genomes by arrowheads. Cell nuclei were visualized by DAPI staining. Asterisks indicate significant differences as compared to the group of cells with 1 viral genome/nucleus (**, p-value < 0.01; ***, p-value < 0.001). (b) 3D reconstruction of a confocal image stack showing PML-NBs in red and HCMV genomes in green. Scale bar, 5 µm. (c) HFF were infected with HCMVΔIE1_EdC or HCMVΔIE1 at a MOI of 1 IEU/cell for 8 hr, before they were fixed for immunofluorescence analysis of PML and IE2 combined with click chemistry. (d) HFF were infected, stained, and grouped as described in (a). IE2-positive cells were determined by immunofluorescence staining in ≥200 cells derived from three independent experiments and are shown as percentages of each group. Asterisks indicate significant differences as compared to the group of cells with 1 viral genome / nucleus (***, p-value < 0.001) (e) Infection of HFF was performed with HCMV_EdC at a MOI of 0.1 or 1 IEU/cell as indicated. PML and vDNA were stained at 8 hpi. (f) HFF were infected with HCMVΔIE1_EdC at a MOI of 0.1 or 1 IEU/cell as indicated and 72 hpi, cells were fixed for co-staining of PML, IE2, and vDNA. vDNA entrapment after infection with MOI 0.1 was determined in >190 cells derived from three independent experiments. (g, h) Association of entrapped vDNA with DNA damage sites. HFF were infected with HCMVΔIE1_EdC at a MOI of 0.1 IEU/cell and were fixed at 8 hpi or 72 hpi for staining of vDNA, γH2AX, and PML as indicated (g). Entrapped HCMV genomes were analyzed for an association with γH2AX in >160 cells derived from three independent experiments and the percentages of γH2AX-positive structures are presented as mean values ± SD (h). See also Figure 5—source data 1.

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 F-ara-EdU (c). At 72 hr post-infection, cells were fixed and subjected to click chemistry to visualize newly synthesized HCMV DNA. Cell nuclei were stained with DAPI. EdC, ethynyl-deoxycytidine; EdU, ethynyl-deoxyuridine; F-ara-Edu, deoxy-fluoro-ethynyluridine.

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 supernatants were subjected to protease K treatment followed by HCMV gB-specific real-time PCR to determine HCMV genome numbers. Values are derived from triplicate samples and represent mean values ± SD. (b) IE1-deleted HCMV, strain AD169, was grown on HFF-IE1 in the absence (HCMV∆IE1) or presence (HCMV∆IE1_EdC) of 0.1 µM EdC. After purification of viral supernatants by centrifugation, viral genome copies were quantified by protease K treatment and HCMV gB-specific real-time PCR (genomes in viral supernatant). To determine intracellular viral genome copies, HFFs were infected with HCMV∆IE1 or HCMV∆IE1_EdC followed by extraction of total DNA at 16 hpi and HCMV gB-specific real-time PCR (genomes in infected cells). To measure the influence of EdC labeling on IE gene expression, HFF were infected with dilutions of HCMV∆IE1 or HCMV∆IE1_EdC. 24 hr after infection, cells were stained for IE2 and the number of IE2-positive cells was determined to calculate viral titers (immediate-early units). All values were extrapolated to 1 ml of input virus. Unlabeled and labeled viral supernatants showed similar ratios of extra- and intracellular genome equivalents (110–120 x) as well as similar ratios of intracellular genome equivalents and immediate-early units (^~ 55x). (c, d) Efficiency of viral DNA labeling. HFF cells were infected with HCMV∆IE1_EdC or HCMV∆IE1_EdU at an MOI of 0.1 for 6 hr, followed by staining of the HCMV tegument protein pp150 and visualization of viral DNA (vDNA) by click chemistry. A representative image of HCMV∆IE1_EdC-infected cells is shown. Calculation of the number of vDNA signals per nucleus resulted in a mean value of 1.2 viral genomes/nucleus (c). Pp150 signals at the cell nuclei as well as signals of viral genomes that were released into the nuclei were assessed in three independent experiments (EdC labeling: > 500 pp150 signals, EdU labeling: > 350 p150 signals) to determine the vDNA labeling efficiencies (vDNA/pp150 ratio), which are shown as mean values ± SD. See also Figure 5—figure supplement 2—source data 1.