16 research outputs found
Loss of GFP expression over time in rKSHV.219-infected primary MM cell cultures.
<p>Expression of GFP among MM cells infected with rKSHV.219 was evaluated over time post-infection by traditional flow cytometry. (A) The percentage of cells expressing GFP is shown over the first 3 weeks of culture when MM cells were infected at passage 7 (black circles), 36 (gray triangles), or 113 (gray squares). (B) Percentages of MM7.219 (black circles) and MM36.219 (gray triangles) cells expressing GFP after one month of continuous unselected culture.</p
Imaging flow cytometry analysis of LANA expression in rKSHV.219-infected MM cell cultures.
<p>Infected MM cells were stained for LANA and assayed by MIFC for the presence of punctate nuclear LANA dots. (A) Representative panel of MM7.219 cells at 21 days post-infection, showing examples of cells by bright-field (BF), GFP, DAPI, and LANA. Percentages of GFP + cells (green circles) are shown over time as compared to the percentage of cells which contain one or more nuclear LANA dots (black squares), for MM7.219 (B), MM36.219 (C), and MM113.219 (D). (E) The specificity of GFP for detecting LANA-dot positive cells is shown across time points and cultures as a percentage of GFP + cells that were also positive for 1 or more nuclear LANA dot. (F) Sensitivity of GFP for detection of infected cells is plotted for the same samples, with percentage of LANA-dot + cells that are also GFP + shown.</p
Chromatin Immunoprecipitation comparison of divergent MM7.219 and MM36.219.
<p>(A) Enrichment of the activating H3K4me3 histone mark across seven regions of the rKSHV.219 episome at 75 days post infection is plotted as a percentage of ChIP input for MM7.219 (dark gray bars) and MM36.219 (light gray bars). (B) Enrichment of the H3K4me3 mark at the viral terminal repeat for the two cell populations, graphed separately due to scale. (C) Enrichment of the repressive H3K27me3 histone mark across eight positions on the rKSHV.219 episome. Values represent the mean +/- S.D. of 4 to 5 PCR replicates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111502#s2" target="_blank">Methods</a>).</p
Reversal of MM7.219 GFP silencing by valproic acid treatment.
<p>At 79 days post-infection, MM7.219 (dark gray bars) and MM36.219 cultures (light gray bars) were exposed to valproic acid (VPA) at the listed concentrations for 54 hours. The percent cells that were GFP + are shown for the listed doses of VPA (A). The percentage of cells scoring positive for RFP at these doses is also shown (B). In a separate experiment performed 32 days post infection, silenced MM7.219 cultures were treated with 5 mM VPA for 2 days. Enrichment of the active H3K4me3 histone mark across four positions on the rKSHV.219 episome are shown as % of input DNA (C). Similarly, repressive H3K27me3 association is shown for the same four regions (D). Values represent the mean +/- S.D. of 4 to 5 PCR replicates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111502#s2" target="_blank">Methods</a>).</p
Rapamycin treatment significantly decreases induction of lytic KSHV proteins.
<p>(A) BCBL-1 cells were pre-treated for 2 hours with rapamycin (12 nM), then induced, in the presence of rapamycin with either CoCl<sub>2</sub> or TPA. (A) BCBL-1 cells were pre-treated for 2 hours with rapamycin 120 nM or DMSO vehicle control two hours prior to induction with CoCl<sub>2</sub> (200 mM), TPA (20 ng/mL) or VPA (600 µM). Nuclear extracts were analyzed by immunoblot for HIF1α expression 24 hours (left), or vIRF3 expression (middle) or LANA expression (right) 48 hours post-treatment. (B) Uninduced or VPA-induced BCBL-1 cells were harvested 48 h post-rapamycin treatment (12 nM), then fixed and stained with antibodies against ORF59, K8.1 and LANA. Leftmost set of dot plots show ORF59 staining on representative samples from uninduced (left panels) and induced (right panels) BCBL-1 cells treated with either vehicle (top panels) or rapamycin (bottom panels). Similar data was collected for both K8.1 and LANA expression. Graphs depict results from multiple replicates of these experiments and indicate the percent of live-gated cells positive for indicated protein stain; horizontal lines depict the mean of replicates (individual circles). Numbers above graph depict p values of comparisons.</p
Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.
<p>BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA<sup>+</sup> cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl<sub>2</sub> (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA<sup>+</sup> cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.</p
RTA regulation by rapamycin is mediated at both the mRNA and protein level.
<p>Messenger ORF50 RNA and RTA protein levels were assessed in BCBL-1 pre-treated with rapamycin for 2 hours, and then induced to lytic reactivation using VPA, TPA, or CoCl<sub>2</sub>. Samples were collected at 0, 6, 24, and 48 hours post-treatment. (A) Top panel, nuclear extracts were used to determine RTA protein levels at each time point using non-enzymatic, immunoblot quantification and normalization to Ran. Graphs show mean ± s.e.m. of triplicate experiments for DMSO (solid line) and rapamycin-treated (dashed line) samples. Individual experiments were normalized to the maximal protein expression levels in DMSO sample at 48 h post-induction. Bottom panel, total mRNA was also collected for quantitative RT-PCR analysis of ORF50 mRNA from parallel whole lysate samples. Graph shows pooled data from 3 experiments with DMSO (solid line) and rapamycin-treated (dashed line) cultures shown. Means ± s.e.m. (B) BCBL-1 cells treated with rapamycin (dashed lines) or DMSO (solid lines) were induced with either TPA (left panels) or CoCl<sub>2</sub> (right panels). RTA protein levels (top) and mRNA levels (bottom) are shown from representative experiments. (C) For each timepoint and induction, viability was determined for both DMSO (black bar) and rapamycin-treated (gray bar) samples by staining an aliquot from each culture with a live/dead exclusion dye and assessing for dye uptake (cell death) by flow cytometry. Graphs show % viable, mean ± s.e.m. of triplicates.</p
Virion production is significantly decreased by rapamycin treatment.
<p>BCBL-1 +/− rapamycin 12 nM were induced with CoCl<sub>2</sub>, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA<sup>+</sup> HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.</p
Progressive Accumulation of Activated ERK2 within Highly Stable ORF45-Containing Nuclear Complexes Promotes Lytic Gammaherpesvirus Infection
<div><p>De novo infection with the gammaherpesvirus Rhesus monkey rhadinovirus (RRV), a close homolog of the human oncogenic pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV), led to persistent activation of the MEK/ERK pathway and increasing nuclear accumulation of pERK2 complexed with the RRV protein, ORF45 (R45) and cellular RSK. We have previously shown that both lytic gene expression and virion production are dependent on the activation of ERK <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004066#ppat.1004066-Woodson1" target="_blank">[1]</a>. Using confocal microscopy, sequential pull-down assays and FRET analyses, we have demonstrated that pERK2-R45-RSK2 complexes were restricted to the nucleus but that the activated ERK retained its ability to phosphorylate nuclear substrates throughout infection. Furthermore, even with pharmacologic inhibition of MEK beginning at 48 h p.i., pERK2 but not pERK1, remained elevated for at least 10 h, showing first order decay and a half-life of nearly 3 hours. Transfection of rhesus fibroblasts with R45 alone also led to the accumulation of nuclear pERK2 and addition of exogenous RSK augmented this effect. However, knock down of RSK during bona fide RRV infection had little to no effect on pERK2 accumulation or virion production. The cytoplasmic pools of pERK showed no co-localization with either RSK or R45 but activation of pERK downstream targets in this compartment was evident throughout infection. Together, these observations suggest a model in which R45 interacts with pERK2 to promote its nuclear accumulation, thereby promoting lytic viral gene expression while also preserving persistent and robust activation of both nuclear and cytoplasmic ERK targets.</p></div