RNA expression in productively and quiescently-infected hESC-derived neurons.

<p>A) The upper two histograms show the counts of reads from RNASeq analysis of quiescently-infected neurons and the lower two those of productively infected neurons aligned to the annotated vOKA genome. Note the difference in the Y-axis scale between the sets of histograms depicting productive and quiescently infected cultures. In the annotated genome at the bottom of the Fig, ORFs depicted in red (increased) and green (reduced) are those displaying statically significant differences in enrichment between quiescent and productively infected neurons. B) Fold-changes between the relative expression of transcripts of VZV ORFs between quiescent and productively infected neurons. The duplicated genes of the short repeats region of the genome are enriched in quiescently-infected neurons. ORFs for which significant differences were detected are denoted by asterisks.</p

DNA fluorescent <i>in situ</i> hybridization confirms the presence of VZV genomes in nuclei of hESC-derived neurons quiescently infected with VZV.

<p>Neurons were infected with VZV-ORF66-GFP and <i>in situ</i> hybridization performed on isolated nuclei as described in the methods. (A) shows FISH of nuclei from productively and (B) from quiescently infected neurons. Almost all quiescently, infected FISH+ neurons contained only one puncta in their nuclei. (C) FISH for VZV genomes in quiescently infected neurons receiving the PI3K inhibitor LY as a reactivation stimulus. After LY treatment there was a slight decrease in the percentage of FISH+ nuclei in the preparations, but 25% of labeled nuclei contained 2 or more puncta, suggesting additional sites of VZV genomes had appeared. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004885#ppat.1004885.t001" target="_blank">Table 1</a> for quantification of the puncta. Scale Bar = 5 μm.</p

Reactivation stimuli increase the number of VZV genomes and transcripts in quiescently-infected human neurons.

<p>Wells of quiescently infected neurons were induced to reactivate VZV using growth factor withdrawal (GF n = 2 for each time point) or treatment with PI3K inhibitor LY294002 (LY) 2, 4, or 7 weeks (n = 5 for each time point) after infection. DNA and RNA were extracted from the wells and VZV genomes (A) and transcripts (B) of ORF63 and ORF31 were quantified. Both treatments increased the levels of both viral genomes and transcripts to varying degrees at all time points tested, indicating at least a partial reactivation of VZV. In all experiments reactivating VZV with LY, changes in nucleic acid levels measured were statistically significant.</p

Reactivation of VZV in quiescently infected hESC-derived neurons by PI3K inhibition at 34°C results in a productive, spreading infection.

<p>(A-F) A microscopic field showing neurons expressing GFP after reactivation at 34°C for 2 (A,B), 6 (C,D) and 14 (E,F) days after initiation of treatment. GFP expression is first observed in individual neurons, and spreads over time in the same initial foci of expression. (G-H) Another experiment showing the results of LY-induced reactivation at 34°C. The area depicted by the box in G is presented at higher magnification in H, showing the diffuse filling of neurons with GFP at 34<b>°</b>C. (I-N) In another reactivation experiment at 34°C, a focus of GFP expression (I&L) initially spreads over 3 days (J&M), but then contracts over a period of 4 days (K&N). Scale bars = 100μm.</p

Reactivation of quiescent VZV in hESC-derived neurons induced by growth factor withdrawal.

<p>(A&C) hESC-derived neurons were incubated with low MOI VZV in the presence of ACV. Two weeks after exposure to virus and one week after removal of ACV, no GFP expression was detected in 50% of neuron-containing wells. At this time point, growth factors (GF) were withdrawn from the medium in wells that were GFP negative. By 4 days after GF withdrawal, massive loss of neurites was observed (B), eventually resulting in death of the cells in the wells by day 5 after treatment. However 30% of the initially GFP negative wells receiving GF-withdrawal treatment contained single and small foci of ORF66 protein-expressing neurons. (D). A and B are phase micrographs, C and D fluorescence micrographs of the same microscopic fields. Scale bar = 100μm.</p

VZV DNA and transcripts are present in hESC-derived neurons productively or quiescently infected with VZV.

<p>Neurons were infected with high or low MOI VZV-ORF66-GFP in the presence of acyclovir, and ACV removed after 6 days incubation. (A) DNA and RNA were extracted from the GFP negative wells 2, 4 or 7 weeks after infection. Levels of VZV genomes and transcripts for ORF63 and ORF31 (gB) were quantified using Taqman probes and digital qPCR and normalized to GAPDH. (B) Transcripts levels detected from both ORFs in quiescently and productively-infected neurons, showing much higher levels in productively-infected neurons.</p

Percentage of neuronal nuclei containing fluorescent puncta following DNA FISH to detect VZV genomes.

<p>Percentage of neuronal nuclei containing fluorescent puncta following DNA FISH to detect VZV genomes.</p

Quiescent infection and reactivation of VZV in hESC-derived neurons without the use of ACV.

<p>hESC-derived neurons were infected via their axons in compartmentalized microfluidic chambers as detailed in the methods. A) Quantification of VZV genomes and transcripts from ORF63 and ORF31 were quantified by digital qPCR from nuclei acids extracted from the cell-body compartment two weeks after infection. B) & C) Reactivation of VZV in axonally-infected neurons. (B) Reactivation of VZV in hESC-derived neurons by LY 2 weeks after quiescent axonal infection. Neurons infected as in A were treated for 4 days with LY at 37°C and DNA and RNA extracted. qPCR revealed an increase in VZV genomes and transcripts from ORF62 and ORF31. No GFP-positive neurons were observed under these reactivation conditions. (C) Reactivation of neurons axonally-infected by VZV at 34°C. A photomicrograph of a microfluidic chamber where the cell body compartment (CB) containing the somata of axonally infected neurons was treated for 4 days with LY at 34 degrees is shown. A cluster of neurons (upper box) expressing ORF66GFP as a result of the treatment (higher magnification image in upper inset). The axons in the axonal compartment (Ax) are coated by the GFP-fluorescent debris (lower box, higher magnification image in the lower inset) used to infect the axons. Ch = microfluidic channels connecting the cell body and axonal compartments. Scale Bars = 100μm.</p

GrB activates PAR-1 receptor.

<p>(A) Human neuronal cells were treated with or without GrB for 30 min and then lysed with RIPA buffer. Cell lysates were incubated with GrB protein-bound protein G beads overnight at 4°C. After washing, immunoprecipitates were resolved by SDS-PAGE and subjected to Western blot analysis using a PAR-1 specific antibody. A representative blot of three independent experiments is shown. (C) Human neurons were treated with GrB cell lysates were collected for PAR-1 detection using Western-blot analysis. A representative blot of three independent experiments is shown. GrB treatment diminished PAR-1 protein as early as 5 min following exposure, indicating that GrB may cleave the PAR-1 receptor, a classical way for PAR-1 activation. (D) Intracellular cAMP level was detected 15 min after GrB treatment in the presence and absence of SCH, a PAR-1 inhibitor. Results represent average ± SEM from six independent experiments. (E) Effect of PAR-1 activation on GrB-induced neurotoxicity was studied by quantifying neurite length. Human fetal neurons on coverslips in 24-well plates were treated with GrB (4 nM) with or without SCH (50 nM) pretreatment. After 24 hr, neurons were immunostained for beta-III-tubulin. Average neurite lengths were measured as described in the Methods section. Results represent average ± SEM from three independent experiments.</p

GrB activates Kv1.3 channel in neurons.

<p>(A) Human fetal neurons were treated with GrB (4 nM) for 24 hr. Cells were then fixed and immunostained for Kv1.3 and beta-III tubulin and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (B) Human fetal neurons were pretreated with cycloheximide (CHX, 100 µg/ml) or actinomycin D (Act D, 10 µM) for 30 min prior to GrB (4 nM) treatment. 24 hr later, cells were fixed and immunostained for Kv1.3 and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (C) Primary human neuronal cultures were first transfected with siRNA specific to Kv1.3 (KvSi). After 48 hr, GrB (4 nM) was used to treat the cells. Cells were fixed after 24 hr and immunostained for beta-III-tubulin. Neurite lengths were measured as detailed in Methods. Results represent average ± SEM from three independent experiments. (D) Human neuronal cells were transfected with PAR-1 specific siRNA (PARsi) or a nonspecific control siRNA (Nsi) 48 hr prior to GrB treatment and Western-blot analysis was used to detect Kv1.3 expression after 24 hr of GrB treatment. Representative blot is shown (Lane 1: control; lane 2: PARsi; lane 3: Nsi; lane 4: GrB; lane 5: GrB/PARsi: Lane 6: GrB/Nsi) and results are presented as average ± SEM from three independent experiments. (E) Primary human neuronal cultures were pretreated with corresponding inhibitors 30 min prior to GrB treatment (4 nM). Cell viability was determined using Cytoquantiblue assay 24 hr later. Results represent mean ± SEM. (F) Cells were incubated with a K free solution containing 5 uM PBFI AM for 2 hours. After washing, the cells were treated with GrB (10 nM) with/without MgTX (10 nM) pretreatment. Intracellular K+ concentration was determined by measuring the florescence at Ex 340 nM and Em 500 nM. Data represents mean ± SEM from five replicates.</p