Direct visualization of bio-orthogonally labelled input HSV-1 genomes at low MOI

Microscopy studies have played pivotal roles in the identification of host factors and signalling pathways that contribute to the intracellular regulation of intrinsic and innate immunity during herpesvirus infection (reviewed in [1, 4, 7, 32, 39]). However, the detection of viral genomes under low MOI conditions, which do not saturate intrinsic host defences, remains a significant technical challenge. To date, viral genome localization studies have relied on the indirect detection of vDNA through immunolabelling or fluorescent tagging of vDNA binding proteins, for example the viral immediate early (IE) transcription factor ICP4 or the early (E) single-stranded vDNA binding protein ICP8 [8–10, 13, 14, 40]. The use of vDNA binding proteins limits temporal resolution of host factor recruitment to infecting viral genomes, as genome detection requires the successful expression of viral gene products that may compete with, or displace, host factors bound to vDNA. Consequently, this strategy is suboptimal for the examination of early intrinsic host immune defences that influence the cellular restriction of viral gene expression. While direct vDNA labelling strategies have been employed, most notably fluorescent in situ hybridization (FISH; [8, 10]), such approaches require harsh denaturing conditions which impair host antigen detection ([41], personal communication J. Brown), and have not been widely adopted. Recent advances in direct bio-orthogonal nucleic acid labelling, using Ethynyl-tagged deoxynucleotides in combination with fluorescent labelling by click chemistry techniques, have enabled the direct visualization of vDNA during both Adeno- and Herpesvirus infection ([42–46]). We sought to apply this technique by purifying either EdU or EdC labelled HSV-1 virions (HSV-1^EdU or HSV-1^EdC, respectively) and infecting cells at low MOI (≤ 3 PFU/cell) to examine the temporal recruitment of intrinsic and innate immune regulators to input viral genomes following nuclear entry.

Successful labelling of vDNA and the purification of high titre WT or ICP0-null mutant HSV-1^EdU or HSV-1^EdC stocks was achieved by infecting Retinal Pigmented Epithelial (RPE) cells (see Materials and methods; S1A–S1C Fig). Notably, many laboratory cell lines were unable to support efficient viral propagation at nucleotide concentrations exceeding 1 μM in an Ethynyl-tag dependent manner (S1 Table, S1D–S1F Fig). As RPE cells were restrictive to HSV-1 ICP0-null mutant replication (see below) and sensitive to Ethynyl-tagged deoxynucleotide labelling in the absence of ICP0 (S1G–S1I Fig), we selected the lowest dose of 0.5 μM EdU or EdC for genome labelling to enable comparative recruitment studies to input viral genomes in the presence or absence of ICP0. In vitro genome release assays demonstrated that ≥ 60% of virions contained EdU or EdC labelled viral genomes detectable by click chemistry following partial denaturation of the HSV-1 capsid by 2M guanidine hydrochloride (GuHCl, Fig 1A–1C, S2 Fig; [47]). Particle to plaque forming unit (PFU) analysis of virus preparations grown in the presence of EdU demonstrated that HSV-1^EdU labelled virions had a roughly equivalent ratio to that of unlabelled control virus preparations (within 3-fold; S2 Table). These data demonstrate that EdU labelling of vDNA was not significantly detrimental to virion production or infectivity under these labelling conditions.

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Fig 1. Direct visualization of bio-orthogonally labelled HSV-1 genomes.

Quantitation of bio-orthogonally labelled vDNA in HSV-1 virions. HSV-1^EdU or HSV-1^EdC virions were subjected to partial denaturation with 2M GuHCl at 4°C for 60 mins to release vDNA [47]. vDNA (red) was detected by click chemistry and capsid (green) by indirect immunofluorescence staining. (A) Representative confocal images showing vDNA release and expansion from partially denatured HSV-1^EdU virions following GuHCl treatment. (B,C) Stack plots showing the relative population of EdU or EdC positive vDNA labelled HSV-1 virions. n ≥ 3000 particles per population derived from 3 independent experiments. Minimum estimates for viral genome labelling efficiency are shown (%). (D,E) HFt cells were mock or HSV-1 infected with either unlabelled or EdU labelled WT (HSV-1^EdU) or ICP0-null mutant HSV-1 (ΔICP0^EdU) at an MOI of 3 PFU/cell. Cells were fixed and permeabilized at the indicated times minutes post-infection (mpi; post-addition of virus). vDNA was detected by click chemistry (white arrows) and nuclei stained with DAPI (blue). Representative confocal images showing the nuclear accumulation of HSV-1^EdU and ΔICP0^EdU genomes over time (30–120 mpi; as indicated), with genome detection specific to input EdU labelled virus. (F) Quantitation of HSV-1^EdU or ΔICP0^EdU genome positive nuclei over time (as in D). Boxes: 25^th to 75^th percentile range; black line: median; whiskers: 5^th to 95^th percentile range. n ≥ 300 cells per sample population derived from a minimum of 3 independent experiments. (G) Number of genomes detected within infected nuclei at 120 mpi. Boxes: 25^th to 75^th percentile range; black line: median; whiskers: minimum and maximum range of sample. n ≥ 200 cells per sample population derived from a minimum of 3 independent experiments. (H) Nuclear (Nuc.) and cytoplasmic (Cyto.) distribution of genome foci detected at 120 mpi. n ≥ 300 cells per sample population derived from a minimum of 3 independent experiments. Percentage of total genomes detected within the nucleus is shown (%).

https://doi.org/10.1371/journal.ppat.1006769.g001

A time course of infection of human foreskin fibroblast (HFt) cells with WT (HSV-1^EdU) or ICP0-null (ΔICP0^EdU) mutant HSV-1 demonstrated that vDNA could be readily detected within the nuclei of infected cells as early as 30 minutes post-infection (mpi; post-addition of virus), with > 70% of nuclei containing at least 1 (median average of 2) HSV-1^EdU genome foci by 120 mpi (Fig 1D, 1F and 1G). Signal detection was dependent on both HSV-1 infection and EdU vDNA labelling, demonstrating that fluorescent click signal(s) were specific to input EdU labelled vDNA (Fig 1E), with the majority of genome signals (≥ 70%) observed within the nucleus (Fig 1H). Notably, qPCR analysis (S2 Table) revealed that the majority of particles (> 50%) had yet to release their genomes by 120 mpi (post-addition of virus). As vDNA is undetectable within native capsids (S2 Fig), these data suggest that the process of nuclear infection is still ongoing at 120 mpi. We note that under equivalent MOI conditions, ΔICP0^EdU infected cells had a reduced number of vDNA positive nuclei at each time point (Fig 1F–1H), a phenotype that likely reflects the efficiency of ICP0-null mutant EdU vDNA labelling in restrictive RPE cells (S1G–S1I Fig).

PML-NBs entrap WT HSV-1 genomes upon nuclear entry

With the ability to detect input vDNA within the nuclei of infected cells as early as 30 mpi (post-addition of virus), we next assessed the utility of this approach to investigate the recruitment of intrinsic immune factors to infecting WT HSV-1^EdU genomes over a short time-course of infection (30 to 90 mpi; Fig 2). Using a combination of click chemistry to detect vDNA and immuno-labelling to detect PML-NB intrinsic host factors, PML (the main scaffolding protein of PML-NBs; [48]) and Daxx (a core constituent protein of PML-NBs; [48]) were observed to stably colocalize with vDNA over the time course of infection (30–90 mpi; Fig 2A). High-resolution Z-series imaging revealed input vDNA to be encased in PML following nuclear infection (Fig 2B). At 90 mpi, ICP0 could be observed to colocalize with PML-NBs prior to PML degradation and PML-NB disruption (Fig 2C and 2D; [22, 25–27]). ICP0 localization at multiple PML-NBs demonstrates that vDNA entrapment within any single PML-NB is not sufficient to target ICP0 to that specific body (Fig 2C). Western blotting of infected cell lysates demonstrated that EdU labelling of vDNA was not detrimental to the initiation of IE gene expression (ICP0, ICP4) or the degradation of PML (Fig 2D). Collectively, these data that demonstrate infecting WT HSV-1 genomes are rapidly encased by PML-NB intrinsic host factors from the outset of nuclear infection prior to the onset of lytic replication. Our data contrast with previous recruitment studies, which have reported PML-NB constituent proteins to localize to sites in close proximity to infecting viral genomes [8–10, 40]. However, we note that these studies have typically relied on higher MOI conditions (≥ 10 PFU/cell; [10]), the use of vDNA binding proteins to enable genome detection by proxy, or time points post-infection where vDNA replication proteins are readily detectable. We conclude that PML-NB host factors rapidly entrap viral genomes shortly after nuclear entry prior to the robust onset of viral gene expression (Fig 2D).

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Fig 2. PML-NBs stably entrap vDNA upon nuclear entry.

HFt cells were mock or HSV-1^EdU infected at an MOI of 3 PFU/cell. Cells were fixed and permeabilized at the indicated times (mpi; post-addition of virus). vDNA, or PML-NB host factors and ICP0, were detected by click chemistry and indirect immunofluorescence staining, respectively. (A) Localization of PML (green) and Daxx (cyan) to HSV-1 vDNA (red, white arrows) at 30–90 mpi (as indicated). Insets show magnified regions of interest (dashed boxes) highlighting PML-NB colocalization with vDNA. Cut mask (yellow) highlights regions of colocalization between PML, Daxx, and vDNA (as indicated). Weighted colocalization coefficients are shown. (B) 3D reconstruction of high-resolution confocal Z-series images showing PML (green) entrapment of vDNA (red, white arrow) at 90 mpi. Single channel maximum intensity projections (left-hand panels) and 3D rendered image (right-hand panel). Pearson coefficient for PML-vDNA colocalization is shown. Scale bar 2 μm. (C) Nuclear localization of ICP0 (green) to vDNA (red, white arrow) and PML (cyan) in HSV-1^EdU infected cells at 90 mpi. Cut mask (yellow) highlights regions of colocalization between ICP0, PML, and vDNA (as indicated). Weighted colocalization coefficients are shown. Nuclei stained with DAPI (blue). (D) EdU labelling of viral genomes does not affect the initiation of infection. HFt cells were mock or HSV-1 infected with unlabelled (HSV-1) or EdU labelled (HSV-1^EdU) HSV-1 at an MOI of 3 PFU/cell. Whole cell lysates were collected at the indicated times (hours post-infection; hpi) for western blot analysis to monitor the rate of PML degradation and the accumulation of the viral immediate early (IE) gene products (ICP0, ICP4). Actin is shown as a loading control. Molecular mass markers are shown, < denotes the detection of a non-specific background band.

https://doi.org/10.1371/journal.ppat.1006769.g002

Asynchronous plaque-edge recruitment studies have shown that PML-NB host factors are independently recruited to infecting viral genomes [49, 50] in an IFI16-dependent manner [11, 12, 14], where de novo PML-NB like foci are reformed [10]. We therefore assessed the composition of vDNA containing PML-NBs, as well as the localization of the PRR IFI16, to infecting WT HSV-1^EdU or HSV-1^EdC genomes (Fig 3, S3 Fig). High colocalization frequencies (weighted colocalization coefficients > 0.7) were observed for all PML-NB component proteins examined (PML, Daxx, Sp100, ATRX, and SUMO2/3) to infecting viral genomes irrespective of genome label, with equivalent paired colocalization frequencies observed for resident PML-NB proteins in mock-infected cells (Fig 3A–3D and 3F, S3 Fig). These data demonstrate that PML-NBs that contained vDNA were indistinguishable in composition from other PML-NBs within the same infected cell or in mock-infected cells at 90 mpi. Surprisingly, the colocalization frequency between IFI16 and input viral genomes was below coincident threshold levels (weighted colocalization coefficients < 0.2; solid line), demonstrating that IFI16 does not stably localize with viral genomes entrapped within PML-NBs at 90 mpi (Fig 3E and 3F, S3 Fig). These data again contrast with published asynchronous plaque-edge recruitment studies that have used vDNA binding proteins for genome detection, which have shown IFI16 and PML to both localize to infecting HSV-1 ICP0-null mutant genomes [11, 13, 14]. As ICP0 has been reported to promote the degradation of IFI16 [11, 28–31], we examined the recruitment of IFI16 to ΔICP0^EdU genomes to investigate the potential effect of ICP0 expression on IFI16 localization to vDNA. No stable localization of IFI16 could be observed to infecting ΔICP0^EdU genomes (S4 Fig), demonstrating that a lack of stable IFI16 recruitment to viral genomes was not due to low levels of ICP0 expression at 90 mpi (Fig 2C). We note that IFI16 localization to viral genomes has been reported to be highly dynamic [12, 14], which could potentially be inhibited by PML-NB entrapment of vDNA. We therefore investigated the influence of MOI (1, 10, and 50 PFU/cell) and time (15 or 30 mpi) on the recruitment of IFI16 and PML to nuclear infecting HSV-1^EdU genomes (S5 Fig). While vDNA-IFI16 colocalization could be observed under very high MOI conditions (50 PFU/cell; S5A Fig), quantitation (n ≥ 250 genomes) revealed the frequency of these colocalization events was not significantly altered by MOI or time (S5B–S5E Fig). In contrast, PML colocalization with vDNA was significantly reduced in a MOI dependent manner (1–50 PFU/cell), indicative of PML-NB saturation by viral genomes or disruption by ICP0 under these high MOI conditions. PML recruitment to vDNA also occurred in a time dependent manner, with a significant increase in colocalization frequency between 15 and 30 mpi (MOI of 10 PFU/cell). As bio-orthogonal nucleic acid labelling is incompatible with live-cell kinetic studies, we conclude that IFI16 does not form a stable association with input vDNA following genome entry into the nucleus.

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Fig 3. Core PML-NB proteins, but not IFI16, associate with infecting HSV-1 genomes.

HFt cells were mock or infected with either HSV-1^EdU or HSV-1^EdC at an MOI of 3 PFU/cell. Cells were fixed and permeabilized at the indicated times (mpi; post-addition of virus). vDNA and PML-NB host factors were detected by click chemistry and indirect immunofluorescence staining, respectively. (A-E) Localization of PML (green), and either Daxx, Sp100, ATRX, SUMO2/3 (SUMO), or IFI16 (cyan; as indicated) to infecting HSV-1^EdU vDNA (red, white arrows). Insets show magnified regions of interest (dashed boxes) highlighting host protein localization with vDNA. Cut mask (yellow) highlights regions of colocalization between host proteins and vDNA (as indicated). Weighted colocalization coefficients are shown. Individual channel images shown in S3 Fig. Nuclei were stained with DAPI (blue). (F) Quantitation of host protein recruitment to infecting viral genomes labelled with EdU (as shown in A-E) or EdC. Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). n ≥ 50 vDNA foci per sample population from a minimum of three independent infections.

https://doi.org/10.1371/journal.ppat.1006769.g003

IFI16 recruitment to input viral genomes is not enhanced in the absence of PML

To test if PML-NBs competitively exclude IFI16 from binding vDNA following nuclear entry, we investigated the recruitment of IFI16 and Daxx (as a positive control; [49]) to input HSV-1^EdU and ΔICP0^EdU genomes in cells depleted of PML (Fig 4, S6 Fig). HFt cells were stably transduced with lentiviral vectors expressing non-targeting control or PML-targeting short hairpin RNAs (shCtrl and shPML, respectively; [49]). qRT-PCR and western blotting confirmed PML depletion without influencing Daxx or IFI16 expression (Fig 4A and 4B). HSV-1^EdU or ΔICP0^EdU infection of shCtrl cells recapitulated observations made in parental HFt cells (Fig 3, S3 Fig, S4 Fig), demonstrating that lentiviral transduction, shRNA expression, or puromycin selection did not affect PML-NB entrapment of vDNA or alter IFI16 localization (Fig 4C–4E). PML depletion did not increase the frequency of IFI16 colocalization with vDNA during either HSV-1^EdU or ΔICP0^EdU infection (Fig 4D and 4E, S6 Fig), demonstrating that PML-NBs do not competitively exclude IFI16 from binding vDNA. In contrast, while Daxx colocalized with vDNA in a subset of PML depleted cells (Fig 4C, S6A Fig), quantitation (n ≥ 100 genomes) revealed that the frequency of this colocalization was significantly reduced compared to control cells irrespective of ICP0 expression (Fig 4E). These data contrast with asynchronous plaque-edge recruitment studies, where Daxx recruitment to infecting viral genomes occurs in a PML independent manner under high MOI conditions [49]. We conclude that under infection conditions that do not saturate or disrupt PML-NBs by 90 mpi (MOI < 10 PFU/cell; S5 Fig), Daxx colocalization with vDNA is stabilized by PML at PML-NBs where Daxx is a resident protein (Figs 2–4, S3 Fig; [48]).

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Fig 4. Depletion of PML does not enhance the recruitment of IFI16 to infecting viral genomes.

HFt cells were stably transduced with lentiviruses expressing PML-targeting (shPML) or non-targeting control (shCtrl) shRNAs. (A) qRT-PCR quantitation of PML or IFI16 mRNA levels in HFt shCtrl and shPML cells. Mean (RQ) and standard deviation (RQmin/max) shown and expressed relative to HFt shCtrl cells (1). (B) Western blot analysis of the expression levels of PML, Daxx and IFI16 in whole cell lysates derived from HFt shCtrl and shPML cells. Actin is shown as a loading control. Molecular mass markers are shown. (C, D) Localization of PML (green), and either Daxx or IFI16 (cyan; as indicated), to infecting HSV-1^EdU vDNA (red, white arrows) in HFt shCtrl and shPML cells at 90 mpi (post-addition of virus). Cells were infected with HSV-1^EdU or ΔICP0^EdU at an MOI of 3 PFU/cell. Insets show magnified regions of interest (dashed boxes) highlighting host protein localization with vDNA. Cut mask (yellow) highlights regions of colocalization between PML, IFI16, Daxx, and vDNA (as indicated). Weighted colocalization coefficients are shown. Nuclei were stained with DAPI (blue). Images for ΔICP0^EdU infected cells are shown in S6 Fig. (E) Quantitation of host protein recruitment to infecting viral genomes (as shown in C, D, S6). Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). n ≥ 50 vDNA foci per sample population from a minimum of four independent infections. *** P < 0.001, Mann-Whitney U-test.

https://doi.org/10.1371/journal.ppat.1006769.g004

Live cell microscopy studies and asynchronous plaque-edge recruitment assays have reported that IFI16 is required for PML and Daxx recruitment to infecting viral genomes [11, 12, 14], although no direct evidence of IFI16 colocalization with vDNA or deposition within PML-NBs was reported to support this hypothesis. We therefore investigated if IFI16 played a role in PML-NB entrapment of vDNA (Fig 5). HFt cells were stably transduced with lentiviral vectors expressing non-targeting control or IFI16-targeting short hairpin RNAs (shCtrl and shIFI16, respectively; [11]). qRT-PCR and western blotting confirmed IFI16 depletion without influencing PML or Daxx expression (Fig 5A and 5B). HSV-1^EdU infection of shCtrl or shIFI16 cells demonstrated that both PML and Daxx strongly colocalized with vDNA independently of IFI16 (Fig 5C–5E). We conclude that IFI16 does not play an essential role in the entrapment of vDNA within PML-NBs. As we failed to observe any significant recruitment of IFI16 to infecting HSV-1 genomes under a range of infection conditions, our data suggest that the stable recruitment of PML and IFI16 to infecting viral genomes occurs with temporally distinct kinetics.

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Fig 5. IFI16 does not influence the recruitment of PML or Daxx to infecting viral genomes.

HFt cells were stably transduced with lentiviruses expressing IFI16-targeting (shIFI16) or non-targeting control (shCtrl) shRNAs. (A) qRT-PCR quantitation of IFI16 or PML mRNA levels in HFt shCtrl and shIFI16 cells. Mean (RQ) and standard deviation (RQmin/max) shown and expressed relative to HFt shCtrl cells (1). (B) Western blot analysis of the expression levels of IFI16, PML, and Daxx in whole cell lysates from HFt shCtrl and shIFI16 cells. Actin is shown as a loading control. Molecular mass markers are shown. (C, D) Localization of PML (green), and either IFI16 or Daxx (cyan; as indicated) to infecting HSV-1^EdU vDNA (red, white arrows) in HFt shCtrl and shIFI16 cells at 90 mpi (post-addition of virus). Cells were infected with HSV-1^EdU at an MOI of 3 PFU/cell. Insets show magnified regions of interest (dashed boxes) highlighting host protein localization with vDNA. Cut mask (yellow) highlights regions of colocalization between IFI16, PML, Daxx, and vDNA (as indicated). Weighted colocalization coefficients are shown. Nuclei were stained with DAPI (blue). (E) Quantitation of host protein recruitment to infecting viral genomes (as shown in C, D). Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). n ≥ 50 vDNA foci per sample population from a minimum of four independent infections. ns (not significant), Mann-Whitney U-test.

https://doi.org/10.1371/journal.ppat.1006769.g005

vDNA entry into the nucleus is not sufficient to induce ISG expression

As asynchronous plaque-edge recruitment assays have played a key role in defining the recruitment of IFI16 to infecting HSV-1 genomes [11, 13, 14], we conducted analogous assays to assess the temporal recruitment of PML and IFI16 to both vDNA and ICP4, an IE vDNA binding protein commonly utilized as a proxy for vDNA in genome recruitment studies [9–11, 14, 40]. Viral DNA labelling was achieved by pulse labelling HSV-1 ICP0-null mutant infected cell monolayers at 24 hours post-infection (hpi) with 1 μM EdU for 6 h. Under these conditions, DNA replication compartments within the body of a developing plaque were clearly detected (S7 Fig). Cells on the periphery of the plaque-edge were readily observed to contain EdU positive vDNA foci asymmetrically distributed around the nuclear rim prior to ICP4 detection (Figs 6A, 6C top panels, S7), indicative of input EdU labelled viral genomes that have yet to initiate a productive gene expression programme. The recruitment of PML to infecting viral genomes occurred independently of ICP4 expression, with many genome foci observed to localise in close proximity to PML foci (Fig 6A and 6B). In contrast, IFI16 recruitment only occurred in cells that expressed ICP4 localized to vDNA at the nuclear rim (ICP4 NR; Fig 6C and 6D, S7 Fig). These data support previous asynchronous recruitment studies that have used ICP4 as a proxy for genome detection [11, 14] and demonstrate that PML and IFI16 are recruited to infecting viral genomes with temporally distinct kinetics, which in the case of IFI16 correlates with the expression and localization of viral gene products with vDNA (Fig 6D). Importantly, these data suggest that vDNA entry into the nucleus alone may not be sufficient to stimulate the induction of an IFI16-dependent innate immune response, but instead require the expression of specific viral gene products or the initiation of vDNA replication.

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Fig 6. Nuclear entry of vDNA is not sufficient to induce robust Mx1 ISG expression.

(A-D) Recruitment of PML, IFI16, and eYFP.ICP4 to ΔICP0 infecting viral genomes in an asynchronous plaque-edge recruitment assay (as described in [10]). HFt cells were infected with ΔICP0 expressing eYFP.ICP4 under conditions (MOI 2 PFU/cell) that enable plaque-formation to occur. Cells were pulse labelled at 24 hpi with 1 μM EdU for 6h to label vDNA. (A, C) Localization of eYFP.ICP4 (green), PML or IFI16 (cyan; as indicated) to vDNA (red) in newly infected cells on the periphery of a developing plaque-edge. Cut mask (yellow) highlights regions of colocalization between either PML or IFI16 and vDNA (as indicated). Weighted (w.) colocalization coefficients shown. (B, D) Quantitation of PML and IFI16 recruitment to infecting vDNA in the presence or absence of eYFP.ICP4 at the nuclear rim (ICP4 NR and ICP4 -ve, respectively; as shown in A and C). Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). n = 100 plaque-edge cells +ve for vDNA per PML or IFI16 sample population from 4 independent infections. *** P < 0.001, ns (not significant), Mann-Whitney U-test. (E-I) Mx1 expression is only induced under infection conditions that permit ΔICP0 plaque formation. (E) Number of plaques detected at 24 hpi following ΔICP0 infection of HFt cells at an input MOI of 0.1 or 1 PFU/cell (as indicated). n = 3, means and standard deviations shown. (F) Quantitation of ΔICP0^EdU nuclear genomes in HFt cells (as infected in E). Boxes: 25^th to 75^th percentile range; black line: median; whiskers: 5^th to 95^th percentile range. n ≥ 250 cells derived from 3 independent experiments per condition. (G) Frequency of ΔICP0^EdU genomes detected within infected cell nuclei (as described in E/F). Boxes: 25^th to 75^th percentile range; black line: median; whiskers: minimum and maximum range of sample. (H) HFt cells were mock treated, IFN-β stimulated (100 IU/ml), or infected with ΔICP0 at a MOI of 0.1 or 1 PFU/cell. Samples were fixed at 24 post-treatment and analysed by confocal microscopy for Mx1 (green) ISG expression. (I) Quantitation of Mx1 positive cells (as shown in H). n ≥ 250 cells derived from 3 independent experiments per condition. (J) PML-NB entrapment of vDNA is maintained under low MOI conditions (0.1 PFU/cell) that restrict ΔICP0^EdU replication and plaque formation at 24 hpi. 3D reconstruction of high-resolution Z-series confocal images showing PML entrapment of ΔICP0^EdU vDNA. Single channel maximum intensity projection images (left), 3D rendered images of the whole nucleus showing PML (green) and vDNA (red), with a single vDNA focus entrapped by PML (dashed box; right). Scale bar 2 μm. Enlargement of PML entrapped vDNA (centre-right). Scale bar 0.5 μm. Cut mask (yellow) highlights colocalization between PML and vDNA (far-right). Pearson coefficient for PML-vDNA colocalization shown. Nuclei were stained with DAPI (blue).

https://doi.org/10.1371/journal.ppat.1006769.g006

To test this hypothesis, we examined the induction of Mx1, a well-characterized ISG product [51], during HSV-1 ICP0-null mutant infection by confocal microscopy. HFt cells were mock treated, stimulated with IFN-β (as a positive control), or infected with ΔICP0^EdU at input levels which either restrict or permit the initiation of HSV-1 ICP0-null mutant replication and plaque formation at 24 hpi (MOI 0.1 and 1.0 PFU/cell, respectively; Fig 6E). vDNA was readily detectible within the nuclei of infected cells under restrictive MOI conditions (0.1 PFU/cell) with a frequency close its expected input genome ratio (Fig 6F) and copy number (1–2 genomes/infected cell; Fig 6G, S2 Table). Notably, the number of genomes per infected cell nuclei under permissive conditions (MOI 1 PFU/cell) was lower than expected based on our input qPCR analysis (~ 25 genomes/cell; Fig 6G, S2 Table). These data indicate that under MOI conditions that begin to saturate intrinsic host defences (~ 10–20 genome copies/nuclei), ΔICP0^EdU genome detection is lost following the onset of vDNA replication by 24 hpi.

As expected, IFN-β stimulation efficiently induced Mx1 expression 24 h post-treatment (Fig 6H and 6I). In contrast, infection with ICP0-null mutant HSV-1 only stimulated Mx1 expression at genome input levels sufficient to stimulate the onset of viral replication and plaque formation (MOI 1.0 PFU/cell; Fig 6E, 6H and 6I). High-resolution Z-series imaging revealed that viral genomes remained stably entrapped within PML-NBs under restrictive MOI conditions (0.1 PFU/cell) at 24 hpi (Fig 6J). These data demonstrate that under infection conditions that restrict the initiation of ICP0-null mutant HSV-1 replication, viral genome entry into the nucleus alone is not sufficient to stimulate the induction Mx1 ISG expression.

vDNA replication is required for the induction of innate immunity

As PML-NB entrapped HSV-1 ICP0-null mutant genomes failed to stimulate the induction of Mx1 expression, we next examined the kinetics of ISG induction during HSV-1 infection under MOI conditions that enabled the onset of viral replication (MOI 1 PFU/cell; Fig 7). As expected, HSV-1 ICP0-null mutant infection efficiently induced the transcription (by 8–9 hpi) and expression (by 16 hpi) of three independent ISG products (Mx1, ISG15, and ISG54), a host response that was significantly impaired during WT HSV-1 infection (Fig 7A–7C). Importantly, the induction of ISGs only occurred under infection conditions that enabled the onset of ICP0-null mutant HSV-1 replication and plaque-formation (≥ 1.0 PFU/cell; Figs 6E and 7D). Consistent with our microscopy observations (Fig 6H and 6I), these data demonstrate that saturation of intrinsic host defences is required for the robust induction of ISGs during HSV-1 ICP0-null mutant infection. As IFI16 binds to a range of DNA structures that may be produced during vDNA replication [52], we next investigated the role of vDNA replication in the induction of ISGs. ISG transcript levels were monitored in the presence of the vDNA replication inhibitors PAA (phosphonoacetic acid) and ACG (acycloguanosine), two well-characterized herpesvirus DNA replication inhibitors [53–57]. PAA efficiently inhibited the induction of both Mx1 and ISG15 transcript levels in a dose-dependent manner (Fig 7E), while ACG treatment had only a modest inhibitory effect at concentrations sufficient to restrict HSV-1 plaque formation (≥ 50 μM; Fig 7F, S1 Table). Inhibition of ISG induction by PAA was virus-specific, as ISG transcript levels were readily induced by IFN-β stimulation in the presence of PAA (Fig 7G). By way of contrast, JAK inhibition by Ruxolitinib (Ruxo) effectively blocked ISG induction following IFN-β stimulation (Fig 7G; [58, 59]), consistent with a key role for JAK in IFN-mediated innate immune signalling [60]. JAK inhibition also effectively blocked ISG induction during HSV-1 ICP0-null mutant infection (Fig 7H). Importantly, this inhibition occurred in the presence 50 μM ACG (Fig 7I), demonstrating that JAK activity is specifically required to induce ISG expression during the initiating cycle(s) of HSV-1 ICP0-null mutant vDNA replication by 9 hpi. Together with our microscopy observations (Fig 6), these data demonstrate that the onset of vDNA replication is required for the robust induction of ISG expression during HSV-1 ICP0-null mutant infection.

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Fig 7. Inhibition of vDNA replication impairs the induction of innate immunity during HSV-1 infection.

HFt cells were mock, WT, or ΔICP0 HSV-1 infected at an MOI of 1 PFU/cell (unless stated otherwise). Samples were collected at the indicated times (hours; h) post-treatment or infection. (A) Relative mRNA levels of Mx1, ISG15, and ISG54 during WT or ΔICP0 HSV-1 infection. n = 2, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to mock (1). (B) Western blot analysis of the expression levels of ISGs (ISG15, Mx1, ISG54), viral proteins (ICP0, ICP4, VP5), and actin (as a loading control), from whole cell lysates of mock, IFN-β stimulated (100 IU/ml), WT or ΔICP0 HSV-1 infected HFt cells at the indicated times. Molecular mass markers are shown. (C) Quantitation of ISG expression levels (as shown in B). n = 3, means and standard deviations shown expressed relative to mock (1). ** P < 0.01, *** P < 0.001, ns (not significant); two-tailed t-test. (D) Relative mRNA levels of Mx1 and ISG15 in mock or ΔICP0 infected HFt cells (MOI of 0.01 to 1 PFU/cell, as indicated). n = 3, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to ΔICP0 MOI 1 (1). (E, F) Relative mRNA levels of Mx1 and ISG15 in mock or ΔICP0 infected HFt cells in the presence of phosphonoacetic acid (PAA) or acycloguanosine (ACG) at the indicated concentrations. n = 3, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to ΔICP0 (1). * P < 0.05, ** P < 0.01, *** P < 0.001, ns (not significant); two-tailed t-test. (G) Relative mRNA levels of Mx1 and ISG15 in mock or IFN-β stimulated (100 IU/ml) HFt cells co-incubated in the presence Ruxolitinib (5 μM; Ruxo) or PAA (1.6 mg/ml), as indicated. n = 2, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to IFN-β treatment (1). (H, I) Relative Mx1 and ISG15 mRNA levels in mock or ΔICP0 infected HFt cells treated with Ruxo (2.5–10 μM, as indicated) or Ruxo (5 μM) and ACG (50 μM), as indicated. n = 2, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to ΔICP0 infection (1).

https://doi.org/10.1371/journal.ppat.1006769.g007

Dual roles for both PML and IFI16 in the regulation of intrinsic and innate immunity to HSV-1 infection

As the induction of innate immunity during HSV-1 ICP0-null mutant infection was inhibited by Ruxolitinib, we next examined the effect of JAK inhibition on WT and ICP0-null mutant HSV-1 replication (Fig 8). At a concentration sufficient to inhibit ISG induction (5 μM; Fig 7G–7I), Ruxolitinib treatment had no effect on the relative plaque formation efficiency (PFE) of either WT or ICP0-null mutant HSV-1 (Fig 8A). These data indicate that innate immune signalling and the induction of ISGs does not directly contribute to the cellular restriction and plaque-formation defect of an HSV-1 ICP0-null mutant observed in restrictive cell types (≥ 1000 fold; [33, 35, 61]). In contrast, virus yield assays demonstrated that Ruxolitinib treatment enhanced the levels of ICP0-null mutant, but not WT, HSV-1 propagation (Fig 8B). Thus, under infection conditions that saturate intrinsic host defences and enable the onset of HSV-1 ICP0-null mutant replication (MOI of ≥ 1 PFU/cell; Fig 6E), innate immune defences act to restrict virus propagation. By way of contrast, depletion of IFI16 or PML enhanced both the PFE and virus yield of an HSV-1 ICP0-null mutant (Fig 8C and 8D; [11, 49]). Importantly, no additional increase in virus yield was observed on Ruxolitinib treatment of IFI16 or PML depleted cells (Fig 8D), indicative of an impaired innate immune response in these cells during HSV-1 ICP0-null mutant infection. Correspondingly, qRT-PCR demonstrated that the induction of ISGs (Mx1, ISG15, ISG54) was significantly impaired in both IFI16 or PML depleted cells in response to HSV-1 ICP0-null mutant infection at 9 hpi (Fig 8E and 8F). These data identify a novel role for PML in the induction of ISGs and the regulation of innate immunity during HSV-1 infection. Collectively, our data demonstrate that PML plays dual roles in the temporal regulation of intrinsic and innate immune defences that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively.

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Fig 8. Dual roles for PML and IFI16 in the regulation of intrinsic and innate immunity to HSV-1 infection.

(A) HFt cells were DMSO or Ruxolitinib (5μM; Ruxo) treated and infected with serial dilutions of WT or ΔICP0 HSV-1 for 24 h prior to immuno-staining for plaque formation. Plaque counts expressed relative to control cell monolayers (# of plaques treated / # of plaques DMSO control) at equivalent serial dilutions of virus and presented as relative plaque formation efficiency (PFE). n ≥ 3, means and standard deviations shown. (B) HFt cells were DMSO or Ruxo treated and infected with WT (MOI 0.001 PFU/cell) or ΔICP0 (MOI 1 PFU/cell) HSV-1. Cell released virus (CRV) was harvested at the indicated times (hpi) and CRV titres determined on U2OS cells. n = 3, means and standard deviations shown. (C) Stably transduced HFt cells expressing non-targeting control (shCtrl), or targeting IFI16 (shIFI16) or PML (shPML) shRNAs, were infected with WT or ΔICP0 HSV-1 (as in A). Plaque counts expressed relative to infected shCtrl cell monolayer plaque counts (1) and presented as relative PFE. n = 3, means and standard deviations shown. (D) shCtrl, shIFI16, or shPML HFt cells were treated with DMSO or Ruxo and infected with either WT or ΔICP0 HSV-1 (as in B). CRV was collected at the indicated times (hpi) and titres determined on U2OS cells. n = 3, means and standard deviations shown. (E, F) Relative Mx1, ISG15, and ISG54 mRNA levels in HFt shCtrl, shIFI16, or shPML cells mock or ΔICP0 infected (MOI 1 PFU/cell) at 9 hpi. n = 3, means (RQ) and standard deviations (RQmin/max) shown and expressed relative to ΔICP0 infected shCtrl (1). *** P < 0.001; two-tailed t-test.

https://doi.org/10.1371/journal.ppat.1006769.g008

As intrinsic and innate immune defences to HSV-1 infection are known to be cell-type dependent [34, 35, 62], we next investigated if there was a correlation between cell line permissiveness to HSV-1 ICP0-null mutant replication and the entrapment of viral genomes by PML-NB host factors (Fig 9). Relative to permissive osteosarcoma cells (U2OS, SAOS), which do not require ICP0 to stimulate the onset of HSV-1 replication [34], RPE cells demonstrated equivalent levels of HSV-1 ICP0-null mutant restriction to HFt cells (≥ 1000-fold reduction in PFE, Fig 9A). Western blot analysis revealed that all these cell lines expressed similar levels of PML, Daxx, and IFI16 (Fig 9B). However, infection with HSV-1^EdU demonstrated a significant reduction in the colocalization frequency of PML and Daxx to infecting viral genomes between permissive (U2OS, SAOS) and restrictive (HFt, RPE) cell-types (Fig 9C and 9D). Importantly, in many instances neither PML nor Daxx was observed to localize with infecting viral genomes in permissive cell types (Fig 9C, bottom panels). Thus, we have identified a correlation between the stable entrapment of vDNA by PML-NBs and the requirement for ICP0 to stimulate the efficient onset of HSV-1 infection in restrictive (HFt, RPE), but not permissive (U2OS, SAOS), cell-types. As U2OS and SAOS cells do not express ATRX (Fig 9B; [63, 64]), a known core constituent protein of PML-NBs and an intrinsic antiviral regulator to HSV-1 infection [64–66], we investigated the requirement for ATRX to mediate PML-NB entrapment of vDNA. HFt cells were stably transduced with lentiviral vectors expressing non-targeting control or ATRX-targeting short hairpin RNAs (shCtrl and shATRX, respectively; [65]). qRT-PCR and western blotting confirmed ATRX depletion, which had a modest effect on PML mRNA transcript levels without influencing PML or Daxx expression levels (Fig 9E and 9F). HSV-1^EdU infection of shCtrl or shATRX cells demonstrated that depletion of ATRX led to a distinct population of viral genomes with a reduced colocalization frequency with PML (left-hand dotted box; Fig 9G). Notably, low levels of ATRX colocalization were still observed with vDNA in a significant proportion of ATRX depleted cells (right-hand dotted box; Fig 9G). Quantitation (n ≥ 200 genomes per condition) revealed that there was a significant difference in PML recruitment to vDNA in ATRX depleted cells (Fig 9H). We conclude that ATRX, either directly or indirectly, contributes to the entrapment of vDNA within PML-NBs following nuclear entry.

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Fig 9. PML-NB entrapment of infecting HSV-1 vDNA occurs in a cell type and ATRX dependent manner.

(A) U2OS, SAOS, RPE, and HFt cells were infected with serial dilutions of WT or ΔICP0 HSV-1 for 24 h. Plaque counts expressed relative to U2OS, a cell line permissive to ΔICP0 replication [34], (# of plaques / # of plaques U2OS control) at equivalent serial dilutions of virus and presented as relative plaque formation efficiency (PFE). n ≥ 3, means and standard deviations shown. (B) Western blot analysis of the relative expression levels of PML, Daxx, ATRX, and IFI16 in whole cell lysates derived from HFt, RPE, U2OS and SAOS cells. Actin is shown as a loading control. Molecular mass markers are indicated. (C) Localization of PML (green) and Daxx (cyan) to HSV-1^EdU vDNA (red, white arrows) in restrictive (HFt, RPE) and permissive (U2OS, SAOS) cell types at 30 mpi (post-addition of virus; MOI of 3 PFU/cell). Insets show magnified regions of interest (dashed boxes) highlighting host protein localization with vDNA. Cut mask (yellow) highlights regions of colocalization between PML, Daxx, and vDNA (as indicated). Weighted (w.) colocalization coefficients shown. (D) Quantitation of PML and Daxx recruitment to infecting vDNA in restrictive (HFt, RPE) and permissive (U2OS, SAOS) cell lines (as shown in C). Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). n ≥ 50 vDNA foci per sample population from 4 independent infections. (E-H) ATRX is required for efficient PML-NB entrapment of vDNA. HFt cells were stably transduced with lentiviruses expressing ATRX-targeting (shATRX) or non-targeting control (shCtrl) shRNAs. (E) qRT-PCR quantitation of ATRX or PML mRNA levels in HFt shCtrl and shATRX cells. Mean (RQ) and standard deviation (RQmin/max) shown and expressed relative to HFt shCtrl cells (1). (F) Western blot analysis of the relative expression levels of ATRX, PML, and Daxx in whole cell lysates from HFt shCtrl and shATRX cells. Actin is shown as a loading control. Molecular mass markers are shown. (G) Scatter plot showing paired w. colocalization coefficients of ATRX and PML to individual nuclear infecting viral genomes in shCtrl (blue) and shATRX (red) cells infected with HSV-1^EdU at an MOI of 3 PFU/cell at 90 mpi (post-addition of virus). n ≥ 200 genomes per sample population. Dotted boxes highlight genome populations identified to have altered distribution of colocalization frequency in comparison to infected shCtrl cells. (H) Quantitation of PML and ATRX recruitment to infecting viral genomes (as shown in G). Boxes: 25^th to 75^th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5^th to 95^th percentile range. Solid line indicates coincident threshold level (weighted colocalization coefficients < 0.2). * P < 0.05, *** P < 0.001; Mann-Whitney U-test.

https://doi.org/10.1371/journal.ppat.1006769.g009

Finally, we compared restrictive (HFt, RPE) and permissive (U2OS, SAOS; [62]) cell types to mount an innate immune response to HSV-1 ICP0-null mutant infection under infection conditions that permitted the onset of viral replication (MOI 1 PFU/cell). Surprisingly, qRT-PCR analysis demonstrated that only HFt cells induced ISG (Mx1, ISG15, ISG54) expression during HSV-1 ICP0-null mutant infection (Fig 10A, top panels; [62]). This was not due to a defect in IFN pathway signalling, as all four cell-types were responsive to exogenous IFN-β stimulation (Fig 10A, bottom panels). Correspondingly, only HFt cells showed enhanced levels of HSV-1 ICP0-null mutant propagation following JAK inhibition by Ruxolitinib (Fig 10B). We conclude that RPE cells, which are highly restrictive to HSV-1 ICP0-null mutant replication (Fig 9A), are defective in aspects relating to intracellular innate signalling in response to HSV-1 infection. These data support our microscopy observations (Fig 6) and inhibitor studies (Figs 7 and 8), and collectively demonstrate that intrinsic and innate host immune responses to HSV-1 infection are temporally distinct and functionally separable arms of host immunity.

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Fig 10. PML-NB entrapment of vDNA does not lead to the induction of innate immunity.

(A) Relative Mx1, ISG15, and ISG54 mRNA levels in cell lines restrictive (HFt, RPE) or permissive (U2OS, SAOS) to HSV-1 ICP0-null mutant (ΔICP0) infection. Cells were mock treated, IFN-β (100 IU/ml) stimulated, or infected with ΔICP0 at an MOI of 1 PFU/cell for 9 h. n = 3, means (RQ) and standard deviations (RQmin/max) are shown and expressed relative to mock for each cell line (1). (B) Restrictive (HFt, RPE) or permissive (U2OS, SAOS) cell lines were infected with ΔICP0 at an MOI of 1 PFU/cell and treated with either DMSO or Ruxolitinib (Ruxo, 5μM). CRV was harvested at 48 hpi and titres determined on U2OS cells. n = 3, means and standard deviations shown and expressed as relative fold-change to DMSO titres (1) for each cell line. * P < 0.05, *** P < 0.001, ns (not significant); two-tailed t-test.

https://doi.org/10.1371/journal.ppat.1006769.g010

In summary, we show that the temporal recruitment of host immune regulators to infecting viral genomes plays an important role in the sequential regulation of intrinsic and innate immunity during HSV-1 infection. We identify PML-NBs to entrap vDNA shortly after nuclear entry in an ATRX-dependent and IFI16-independent manner. We identify a novel role for PML in the induction of innate immunity in response to HSV-1 infection that correlates with the recruitment of IFI16 into vDNA complexes associated with ICP4 and the onset of vDNA replication. These intracellular host defences are counteracted by ICP0, which induces the degradation of PML from the outset of infection to release viral genomes entrapped within PML-NBs to stimulate the onset of HSV-1 lytic replication.

Cells and drugs

Primary human foreskin fibroblast cells (HFs) were obtained from Thomas Stamminger (Department of Urology, University of Erlangen; [49]) and immortalized (HFt) by retrovirus transduction to express the catalytic subunit of human telomerase, as previously described [18]. HFt, retinal pigmented epithelial (RPE-1; ATCC, CRL-4000), Human osteosarcoma (U2OS and SAOS; ECACC, 92022711 and 89050205), primary human foetal lung fibroblast (MRC-5; ATCC, CCL-171), and adult human keratinocyte (HaCat; a gift of F. Rixon, MRC-UoG CVR) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, 41966). HFt cells were cultured in the presence of 5 μg/ml of Hygromycin (Invitrogen, 10687–010) to maintain hTERT expression. Transduced HFt cells expressing shRNAs were cultured in the presence of Puromycin (Sigma-Aldrich, P8833; 1 μg/ml or 0.5 μg/ml for selection or maintenance, respectively). Primary human embryonic lung fibroblast (HEL 299; ECACC, 87042207) cells were maintained in Minimum Essential Medium Eagle (MEM; Sigma-Aldrich M5650) supplemented with 2 mM L-Glutamine (Life Technologies, 25030–024) and 1 mM Sodium Pyruvate (Life Technologies, 11360–039). Baby hamster kidney fibroblast (BHK-21 C13; a gift of R. Everett, MRC-UoG CVR) cells were grown in Glasgow Minimum Essential Medium (GMEM; Life Technologies, 21710–025) supplemented with 10% Tryptose Phosphate Broth (TPB; Life Technologies, 18050–039). Medium for all cell lines was supplemented with 10% foetal bovine serum (FBS; Life Technologies, 10270), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, 15140–122). All cell lines were maintained at 37°C in 5% CO₂. 5-Ethynyl-2’-deoxyuridine (EdU; Sigma-Aldrich, T511285), 5-Ethynyl-2’-deoxycytidine (EdC; Sigma-Aldrich, T511307), 2’-deoxyruridine (dU; Sigma-Aldrich, D5412), and Ruxolitinib (Ruxo; Sellechchem, S1378) were prepared in DMSO and used at the indicated concentrations. Acycloguanosine (ACG, Sigma-Aldrich, A4669), Phosphonoacetic acid (PAA, Sigma-Aldrich, 284270), and Interferon beta (IFN-β; Calbiochem, 407318) were prepared in Milli-Q H₂O and used at the indicated concentrations.

Viruses

Wild-type HSV-1 strain 17syn+ (HSV-1), its ICP0-null mutant derivative dl1403 (ΔICP0; [33]), and their respective variants that express eYFP.ICP4 [40] were propagated and titrated as described [35]. For EdU labelling of viral genomes, RPE cells were infected with either HSV-1 (MOI 0.001 PFU/cell) or ΔICP0 (MOI 0.5 PFU/cell). At 24 h post-infection (hpi), EdU or EdC was added at a final concentration of 0.5 μM, unless otherwise indicated. Fresh EdU/EdC was pulsed into infected cultures at 24 h intervals until extensive cytopathic effect was observed, typically 3 to 4 days post-infection. Supernatants containing labelled cell released virus (CRV) were clarified by centrifugation (423 xg for 10 min) and filtered through a 0.45 μm sterile filter and pelleted using a Beckman TLA100 Ultracentrifuge (33,800 xg for 3h at 4°C). Virion pellets were resuspended and pooled in 500 μl complete DMEM medium, and titrated in U2OS cells as described [35].

Plasmids and lentiviral transduction

Plasmids encoding short hairpin (sh) RNAs against a non-targeted control sequence (shCtrl; 5’-TTATCGCGCATATCACGCG-3’), PML (shPML; 5’-AGATGCAGCTGTATCCAAG-3’), ATRX (shATRX; 5’- CGACAGAAACTAACCCTGTAA-3’), or IFI16 (shIFI16; 5’-CCACAATCTACGAAATTCA-3’) were used to generate lentiviral supernatant stocks for transduction of HFt cells as described [11, 49, 65]. Pooled and stably transduced cells were used for experimentation.

Antibodies

The following antibodies were used for immunofluorescence or western blotting: Primary rabbit polyclonal: anti-actin (Sigma-Aldrich, A5060), anti-Daxx (Upstate, 07–471), anti-ATRX (Santa Cruz, H300), anti-PML (Bethyl Laboratories, A301-167A; Jena Biosciences, ABD-030), anti-Sp100 (GeneTex, GTX131569), anti-SUMO2/3 (Abcam, ab22654), anti-Mx1 (Santa Cruz, sc-50509;ProteinTech, 13750-1-AP), anti-ISG15 (ProteinTech, 15981-1-AP), and anti-ISG54 (IFIT2, proteinTech, 12604-1-AP). Primary mouse monoclonal: anti-ICP0 (11060, [92]), anti-ICP4 (58s, [93]), anti-VP5 (DM165, [94]), anti-SUMO2/3 (Abcam, ab81371), anti-PML (abcam, ab96051), anti-IFI16 (abcam, ab55328; Santa Cruz, sc-8023). Primary antibodies were detected using the following secondary antibodies: DyLight-680 or -800 conjugated anti-rabbit or -mouse (Thermo; 35568 and SA5-35571), Alexa -488, -555, or -647 conjugated anti-rabbit, or -mouse (Invitrogen; A21206, A21202, A31572, A31570, A31573, A31571), HRP conjugated anti-mouse (Sigma-Aldrich, A4416).

Plaque Forming Efficiency (PFE)

Unless otherwise stated, cells were infected with serial dilutions of HSV-1 or ΔICP0 and rocked every 10 min for 1 h prior to overlay with medium supplemented with 2% Human Serum (HS; MP Biomedicals, 2931149). 24 to 36 hpi, cells were washed twice in PBS (Sigma-Aldrich, D1408), simultaneously fixed and permeabilized in 1.8% formaldehyde (Sigma-Aldrich, F8775) and 0.5% NP40 (BDH, 56009) in PBS for 10 min, then washed twice in 0.1% Tween in PBS (PBST). Cells were blocked with 5% skimmed milk powder (SMP; Marvel) in PBST (blocking buffer) for 30 min before incubation with an anti-VP5 monoclonal antibody diluted in blocking buffer for 90 min. Cells were washed three times with PBST, incubated with HRP conjugated anti-mouse IgG diluted in blocking buffer for 60 min, then washed with PBST three times. Plaques were visualized with True Blue peroxidase developing solution (Insight, 50-78-02) according to the manufacturer’s instructions, and washed with Milli-Q H₂O prior to plaque counting or imaging using an Axio Observer Z.1 microscope (Zeiss) with differential interference contrast. For plaque formation efficiency (PFE) assays, plaque counts are expressed relative to the number of plaques on control HFt or U2OS infected cell monolayers (as indicated) at the equivalent dilution of input virus. Results presented as relative fold change (number of plaques sample/number of plaques sample control). Plaque diameters were measured using Zen blue (Zeiss) imaging software.

Viral yield assays

Cells were infected with either HSV-1 (MOI 0.001 PFU/cell), or ΔICP0 (MOI 1 or 2 PFU/cell, as indicated) and rocked every 10 min for 1 h prior to overlay with complete medium containing either 5 μM Ruxolitinib or DMSO as a carrier control. Supernatants containing cell released virus (CRV) were collected at the indicated times post-infection. Virus titres were calculated by titration on U2OS cells as described [35].

Particle counting

Equal volumes of virus suspension and polystyrene latex spheres (Agar Scientific, AGS130-02) at a known concentration per ml were mixed in 2 volumes of TNE buffer (20 mM Tris [pH 7.5], 0.5 M NaCl, and 1 mM EDTA). 5 μl of suspension was then added to a glow discharged EM grid (Agar Scientific, S162-4), allowed to rest for 1 min, washed three times in deionised water, and stained with Ammonium Molybdate (2% (w/v) pH 7.2). Dry grids were examined using a JEM2200 FS electron microscope (JEOL) and images captured using an Ultrascan 4000 charge-coupled-device (CCD) camera (Gatan). Multiple images (n ≥ 6) per sample were used for virus particle and latex bead enumeration and used to calculate the number of particles per ml of virus stock inoculum.

Western blotting

Treated or infected cells were washed twice with PBS. Whole cell lysates were collected in SDS-PAGE loading buffer containing 4 M Urea (Sigma-Aldrich, U0631) and 50 mM Dithiothreitol (DTT; Sigma-Aldrich, D0632). Proteins were resolved on NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen, NP0322BOX) in MES (Invitrogen; NP0002) or MOPS buffer (Invitrogen, NP0001) and transferred onto 0.2 μm nitrocellulose membrane (Amersham, 15249794) for 90 min at 30 volts in Novex transfer buffer (Invitrogen, NP0006-1) according to the manufacturer’s instructions. Membranes were blocked in PBS with 5% FBS (Block) for a minimum of 1 h at room temperature. Membranes were incubated in primary antibody diluted in Block for a minimum of 1 h, washed three times with PBST for 5 min each, then incubated in secondary antibody diluted in Block for 1 h. Following three 5 min washes in PBST, one 5 min wash in PBS, and one rinse in Milli-Q H₂O, membranes were imaged on an Odyssey Infrared Imager (LiCor). The intensity of protein bands was quantified with Odyssey Image Studio Software.

Immunofluorescence and confocal microscopy

Cells were seeded overnight on to 13 mm coverslips prior to treatment or infection at the indicated MOI and time points at 37°C. For click chemistry assays, cells were washed in serum free DMEM prior to overlay in complete medium or fixation. At indicated time points, cells were washed twice in CSK buffer (10 mM HEPES, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl₂, 5 mM EGTA), simultaneously fixed and permeabilized in 1.8% formaldehyde and 0.5% Triton-X100 (Sigma-Aldrich, T-9284) in CSK buffer for 10 min, and washed twice in CSK. Coverslips were then blocked with 2% HS in PBS for 30 min prior to click chemistry followed by immunostaining. Where applicable, EdU-labelled vDNA was detected using the Click-iT Plus EdU Alexa Fluor 555 Imaging Kit (ThermoFisher scientific, C10638) according to the manufacturer’s instructions. For host and viral protein labelling, cells were incubated with primary antibodies diluted in 2% HS in PBS for 60 min, then washed in PBS three times, before incubation with secondary antibodies and DAPI (Sigma-Aldrich, D9542) in 2% HS in PBS for 60 min. Coverslips were then washed in PBS three times, and twice in Milli-Q H₂O prior to mounting on Citiflour AF1 (Agar Scientific, R1320). Coverslips were examined using a Zeiss LSM 880 confocal microscope using the 63x Plan-Apochromat oil immersion lens (numerical aperture 1.4) using 405 nm, 488 nm, 543 nm, 594 nm, and 633 nm laser lines. Zen black software (Zeiss) was used for image capture, generating cut mask channels, and calculating weighted colocalization coefficients. High-resolution Z-series images were captured under LSM 880 Airy scan deconvolution settings using 1:1:1 capture conditions at 0.035 μm intervals. Images were processed using Imaris (Bitplane) software to produce rendered 3D image reconstructions and to calculate Pearson colocalization coefficients. Exported images were processed with minimal adjustment using Adobe Photoshop and assembled for presentation using Adobe Illustrator.

Virion genome release assay

In vitro virion DNA release assays were conducted as essentially described in [47]. Briefly, 1x10⁸ PFU of virus preparation was diluted in ice-cold TNE buffer in the presence or absence of GuHCl (2M final concentration; Sigma-Aldrich, G3272). Samples were incubated on ice for 60 mins prior to the addition of ice-cold Methanol (final concentration 60%). Samples were dried onto poly-D-lysine (Sigma-Aldrich, P7405) treated coverslips for 60–90 mins at 4°C prior to fixation in PBS containing 1.8% formaldehyde and 0.5% Triton-X100 for 10 mins at RT. Samples were washed twice in PBS and blocked in PBS containing 2% FBS for 10 mins at RT. Samples were processed for click chemistry to detect EdU or EdC labelled vDNA and immuno-stained for VP5 to detect viral capsids (as described above). High-resolution Z-series images were captured under LSM 880 Airy scan deconvolution settings at 0.2 μm intervals (as described above) and the number of capsids and EdU or EdC labelled viral genomes were quantified in Zen blue (Zeiss) from maximum intensity projection images.

Plaque-edge recruitment assays

HFt cells were infected with ΔICP0 EYFP.ICP4 at an MOI of 2 PFU/cell to enable the initiation of viral replication and plaque formation, as previously described [9]. At 24 hpi, infected cell monolayers were pulsed with 1 μM EdU for 6h prior to fixation and immunostaining, as described above.

Quantitative RT-PCR

Cells were mock, HSV-1, or ΔICP0-infected at the indicated MOI, and total RNA collected at 9 hrs post-infection unless otherwise stated. Where applicable, all drug treatments were added at the indicated concentration 1 h after inoculum adsorption. Total RNA was isolated using the RNAeasy Plus Kit (Qiagen, 74134) according to the manufacturer’s instructions. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). Relative mRNA levels were determined using the ΔΔCt method, normalized to GAPDH, and expressed relative to indicated treatments. Data presented is from a minimum of two independent biological replicates, each analysed in triplicate (RQ/RQmin/max). Means (RQ) and standard deviations (RQmin/max) are presented. For input viral genome quantitation, vDNA was extracted from infected HFt cells harvested at 90 mpi. Cells were trypsinised, pelleted by centrifugation (500 xg, 10 min), washed twice in PBS, and resuspended in PBS containing 1% SDS and 300mM Sodium acetate (pH 5.2). Total DNA was isolated by phenol chloroform extraction and ethanol precipitation, and resuspended in Tris buffer (10mM Tris-HCl pH 8.5). qPCR was performed using two virus specific (UL30 and UL36) primer-probe sets with distinct fluorophores (Sigma-Aldrich; S3 Table) in duplex reactions performed in triplicate per biological replicate. Quantitation was performed against standards of known concentration derived from a purified infectious HSV-1 17syn+ BAC clone (SR27 DNA, [95]; a kind gift from Andrew Davison MRC-UoG CVR).