Fig 1.
Successful differentiation of SH-SY5Y into neuron-like cells.
(A) Schematic representation of the differentiation process showing representative pictures of SH-SY5Y before treatment with collagenase (left) at day 10 post-differentiation, epithelial-like cells that remained in the well (middle) and neuron-like cells after seeding onto a new Matrigel-coated well. Scale bar: 100 µm. (B) Representative pictures of SH-SY5Y during the differentiation process. Scale bar: 100 µm. (C) Representative pictures showing expression of neuronal markers in dSH-SY5Y at 18 dpd. Scale bar: 10 µm. (D) Representative images showing KI67 expression in SH-SY5Y during the differentiation process. Scale bar: 20 µm. The results shown in this figure are representative from three independent differentiations. Abbreviations: dpd, days post-differentiation; MAP2, microtubule-associated protein 2; DBH, dopamine beta-hydroxylase; Nav1.7, voltage-gated sodium channel Nav1.7; TuJ1, beta-III-tubulin antibody.
Fig 2.
VZV productively infects dSH-SY5Y cells.
(A) Schematic representation of the recombinant v63R/11G showing the terminal repeats long and short (TRL and TRS, respectively) the unique long and short regions (UL and US, respectively) and the internal repeat long and short (IRL and IRS, respectively). ORF63 and ORF70 correspond to duplications of the same ORF, with ORF63 located in the IRS and ORF70 in the TRS. (B) Images showing spread of v63R/11G in dSH-SY5Y cells. (C-F) Graphs showing VZV genome copies n=3-9 (C) and relative expression of VZV genes, n=3-6 (D-F) at different times post-infection. The results in (C-F) are from 3 biological replicates. Values are presented as mean ± s.e.m. Statistical comparisons between infected cells at 0 dpi and rest of the conditions were performed using one-way ANOVA on log-transformed raw data to stabilize variances and improve normality. P > 0.05 (ns), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). Abbreviations: dpi, days post-infection.
Fig 3.
Incubation with ACV progressively represses VZV in dSH-SY5Y cells.
(A) Schematic representation of the experiment. (B) Graph showing the day post-infection when wells containing ORF63-RFP/ORF11-GFP positive dSH-SY5Y cells were detected following incubation with ACV for 2-6 days. Each symbol represents one well containing ORF63-RFP/ORF11-GFP positive cells. “n” refers to the number of wells infected with VZV for each condition. (C) Graph showing the number of wells containing ORF63-RFP/ORF11-GFP positive (orange) and negative (blue) dSH-SY5Y cells following incubation with ACV for 2-6 days. The percentage on top of each column indicates the percentage of wells with ORF63-RFP/ORF11-GFP positive dSH-SY5Y cells at the end of the experiment (30 dpi). The data corresponds to 4, 8 and 14 biological experiments (differentiation plus infection) for samples treated with ACV for 2-4 dpi, 5 dpi and 6 dpi, respectively. The “n” in (B) indicates the number of analyzed wells per condition. Statistical comparisons were performed using one-way ANOVA on log-transformed raw data to stabilize variances and improve normality. P > 0.05 (ns), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). Abbreviations: ACV, acyclovir; dpi, days post-infection.
Fig 4.
Incubation with ACV during 5 days results in a repressive phenotype that can be released.
(A) Schematic representation of the experiment. The arrows indicate days when cells were collected for further analysis. (B) Representative images showing dSH-SY5Y cells infected with v63R/11G in the absence (acute) or presence of ACV for 5 dpi and lacking or containing ORF63-RFP/ORF11-GFP positive dSH-SY5Y cells. The dSH-SY5Y cells were labelled with an anti-TuJ1 antibody and the nuclei were stained with DAPI. Scale bar: 100 µm. (C) Western blot detecting VZV gE (top blot) and actin (bottom blot) in dSH-SY5Y cell lysates obtained from mock- or v63R/11G-infected cells without ACV (acute) or with 5 days incubation with ACV. (D-F) Relative gene expression of VZV genes, n=3-9 (D,E) and quantification of VZV genomes, n=3-9 (F) in mock- or v63R/11G-infected dSH-SY5Y cells in the absence (acute) or presence of ACV for 5 dpi. Statistical comparisons between acute infection at 5 dpi and other infection conditions were performed using one-way ANOVA on log-transformed raw data to stabilize variances and improve normality. P > 0.05 (ns), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). (G) Detection of VZV genomes by in situ hybridization in dSH-SY5Y infected with v63R/11G in the absence (acute) or presence of ACV for 5 days in wells where no ORF63-RFP/ORF11-GFP positive cells were detected. DAPI was used to stain nuclei. The white arrows point to the VZV genomes. The data corresponds to 41 and 14 fields of view acquired randomly in for 12 and 20 dpi, respectively, obtained from three biological experiments. Scale bar: 10 µm. (H) Detection of ORF63-RFP/ORF11-GFP in ARPE19 cells incubated with dSH-SY5Y cells (infected in the presence of ACV for 5 days) from wells lacking (NR) or containing (R) ORF63-RFP/ORF11-GFP-positive cells. DAPI was used to stain nuclei. In all panels NR refers to “non-replicating” VZV, while R refers to “replicating” VZV, determined by the expression of ORF63-RFP/ORF11-GFP. Abbreviations: ACV, acyclovir; dpi, days post-infection. The results in (D-F) are from 3 biological replicates. The images in (G) are representative from more than six samples. Values are presented as mean ± s.e.m.
Fig 5.
A small percentage of dSH-SY5Y cells incubated with ACV for six days maintain non-replicating viral genomes for up to 20 days.
(A) Schematic representation of the experiment. The arrows indicate days when cells were collected for further analysis. (B) Representative images showing dSH-SY5Y cells infected with v63R/11G in the absence (acute) or presence of ACV for 6 dpi and lacking or containing ORF63-RFP/ORF11-GFP positive dSH-SY5Y cells. The dSH-SY5Y cells were labelled with an anti-TuJ1 antibody and the nuclei were stained with DAPI. Scale bar: 100 µm. (C) Western blot detecting VZV gE and IE4 (top blot) and actin (bottom blot) in dSH-SY5Y cell lysates obtained from mock- or v63R/11G-infected cells without ACV (acute) or with 6 days incubation with ACV. (D-G) Relative gene expression of VZV genes, n=3-6 (D-F) and quantification of VZV genomes, n=3-6 (G) in mock- or v63R/11G-infected dSH-SY5Y cells in the absence (acute) or presence of ACV for 6 dpi. Statistical comparisons between acute infection at 6 dpi and other infection conditions were performed using one-way ANOVA on log-transformed raw data to stabilize variances and improve normality. P > 0.05 (ns), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). (H) Detection of VZV genomes (grey dots) by in situ hybridization in dSH-SY5Y cells infected with v63R/11G in the absence (acute) and presence of ACV for 6 days in wells where no ORF63-RFP/ORF11-GFP positive cells were detected. DAPI was used to stain nuclei. The white arrows point to the VZV genome. The data was obtained from 41 and 16 random images for 12 and 20 dpi, respectively, from three biological experiments. Scale bar: 10 µm. The graph on the right shows the percentage of cells containing the VZV genome following infection in the presence of ACV during 6 days and subjected to in situ hybridization at 12 and 20 dpi Abbreviations: dpi, days post-infection. The results in (D-G) are from 3 biological replicates. Values are presented as mean ± s.e.m.
Fig 6.
Exogenous expression of pVLT-ORF63 results in production of infectious VZV from cells exposed to ACV during 6 days.
(A) Schematic representation of the experiment. The arrows indicate days when cells were analyzed. (B) Relative VZV gene expression in dSH-SY5Y cells infected with v63R/11G in the presence of ACV for 6 days and incubated with LY294002 or SAHA at 8 dpi, n=3-4. The results are from 3 biological replicates. Values are presented as mean ± s.e.m. (C) Representative images of dSH-SY5Y infected cells in the absence (acute) or presence of ACV during 6 days and incubated or nor with LY294002 or SAHA at 8 dpi during 4 days. The pictures were taken at 6 dpi for acute infected cells and at 14 dpi for the samples treated with ACV. (D) Representative images of dSH-SY5Y cells infected with v63R/11G in the presence of ACV for 6 days and transduced with a lentivirus expressing VLT-ORF63 (top) or eGFP (bottom). The left and middle panels show direct fluorescent expression of ORF63-RFP, ORF11-GFP, RFP and GFP, while the right panels show the merge of the fluorescent channels with phase-contrast images. Scale bar: 100 µm. (E) Graph showing the percentage and number of wells containing ORF63-RFP/ORF11-GFP positive dSH-SY5Y cells (representing replicating virus), following transduction with lentivirus expressing VLT-ORF63 or eGFP. Abbreviations: ACV, acyclovir; dpi, days post-infection.
Fig 7.
Incubation with ACV leads to low level genome-wide VZV transcription in dSH-SY5Y cells.
(A,B) Genome-wide transcription profiles of dSH-SY5Y cells infected with v63R/11G in the absence (acute, violet) and presence of ACV for 6 dpi (orange, A; blue, B) or for 3, 4, 5 and 6 dpi (orange, B). Bulk RNA-Seq was performed at different times post-infection, as labelled. Transcription from both DNA strands is shown with the depth of coverage labelled on the y-axis. A representation of the VZV genome and all encoded transcripts is shown. The samples labelled as 12, 16, 20, 30 dpi correspond to transcriptomic analyses of dSH-SY5Y cells infected with VZV in the presence of ACV during the first 6 days of infection. Total RNA was extracted on days 12, 16, 20 and 30 post-infection and subjected to RNA-sequencing. The samples labelled as 5, 6, 7 and 8 dpi correspond to cells that were infected in the presence of ACV during 3, 4, 5 and 6 days and were processed for RNA extraction two days after removal of ACV. (C) Detection by in situ hybridization of VZV mRNA (grey dots) with a probe that detects VLT and VLT-ORF63 transcripts in dSH-SY5Y infected in the absence (acute) or presence of ACV for 6 days in wells where no ORF63-RFP/ORF11-GFP positive cells were detected. DAPI was used to stain nuclei. The white arrows point to the transcripts. Scale bar: 10 µm. The graph on the right shows the percentage of cells expressing VLT or VLT-ORF63 transcripts following infection in the presence of ACV during 6 days and subjected to in situ hybridization at 12 and 20 dpi. Abbreviations: ACV, acyclovir; dpi, days post-infection.
Fig 8.
ChIP-seq and ATAC-seq analysis of differentiated SH-SY5Y cells quiescently infected with VZV at 13 days post-infection.
(A) Read density coverage tracks of histone marks and ATAC-seq signal on two host loci and VZV, determined by ChIP-seq. Coverage track of VZV shows data from dSH-SY5Y cells latently infected with VZV pOKA WT as a representative of the two independent virus strains used in ChIP experiments. (B) Input-normalized quantification of ChIP-seq signals in a 10 kb sliding window across the VZV genome (VZV, right panel), relative to the 200 most significantly enriched host regions (hg38 positives, left panel) and an equal number of size matched, randomly selected host control loci (hg38 negatives, center). Signals observed in host control regions were set to 1 (10E0). Experiments were independently performed with BAC-derived, wild-type VZV (WT; VZV pOKA WT) and a BAC-derived VZV carrying fluorescent markers (v63R/11G; VZV pOKA Bac ORF11-GFP/ORF63-RFP). (P > 0.05 (n.s), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***) Wilcoxon-Mann-Whitney-Test of indicated sample compared to corresponding hg38 negatives). (C) Fragment size distribution of ATAC-seq data. Size distribution of human reads (hg38) are shown for all aligned reads (top panel) and randomly subsampled to match numbers of aligned reads to VZV (middle panel). Lower panel shows fragment size disruption of reads aligned to VZV (VZV pOKA WT). Peaks indicative of nucleosome free regions (NFR), mononucleosomes (mono), dinucleosomes (di) and trinucleosomes (tri) are indicated within the upper graph.
Table 1.
Sequences of primers used to quantify gene expression.
Table 2.
Sequences of primers used to determine genome copy number.