19 research outputs found
NLRX1 negatively modulates type I IFN to facilitate KSHV reactivation from latency
<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV) is a herpesvirus that is linked to Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent latent infection in the human host. KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle. During KSHV reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that NLRX1, a negative regulator of the type I interferon response, is important for optimal KSHV reactivation from latency. Depletion of NLRX1 in either iSLK.219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group. Concomitantly, fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells. Further analysis revealed that upon NLRX1 depletion, higher IFNβ transcription levels were observed, which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines. To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation, we treated control and NLRX1 depleted cells with a TBK1 inhibitor (BX795) or TBK1 siRNA to block IFNβ production. Upon BX795 or TBK1 siRNA treatment, NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production, suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses. Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation, which might serve as a potential target for restricting KSHV replication and transmission.</p></div
NLRX1 depletion results in enhanced interferon responses upon KSHV reactivation.
<p>iSLK.219 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with Dox for various time points. (A) IFNβ mRNA levels were monitored by qRT-PCR. (B-D) RNA was extracted from duplicate samples and various JAK/STAT related mRNA levels were analyzed using a JAK/STAT real-time qPCR-based gene array (details are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006350#ppat.1006350.s008" target="_blank">S1 Table</a>). mRNA levels of genes were normalized to the mRNA levels of human β-actin to yield dCT as a measure or relative expression without clustering. Scatter plot comparison of relative mRNA level at 0 (B), 24 (C) and 48 (D) hours between NLRX1 siRNA treated cells and NS siRNA treated cells. (E-H) Scatter plot of relative mRNA level at 24 and 48 hours. NS siRNA group at 0 hours were set as normalization control. (I) Heat map of the JAK/STAT microarray. Relative Z-scores of each gene level was calculated by subtracting mean values for each individual gene, and then dividing by each gene standard deviation. Higher Z-scores are indicated by red, lower levels by blue as shown in the key. Data are presented as mean ± s.d. from at least three independent experiments. *indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
NLRX1 is required for optimal KSHV reactivation in BCBL-1 cells.
<p>BCBL-1 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with TPA and sodium butyrate for various time points. (A) KSHV genome copy number in reactivated BCBL-1 cells. (B) KSHV genome copy number in the supernatants of reactivated BCBL-1 cells. (C) qRT-PCR of NLRX1 in reactivated BCBL-1 cells. (D) qRT-PCR of ORF57 in reactivated BCBL-1 cells. (E) qRT-PCR of K8.1 in reactivated BCBL-1 cells. (F) qRT-PCR of ORF36 in reactivated BCBL-1 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
NLRX1 knock down results in enhanced interferon responses upon KSHV reactivation in BCBL-1 cells.
<p>BCBL-1 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with TPA and sodium butyrate for various time points. (A) IFNβ mRNA in BCBL-1 cells was monitored by qRT-PCR. (B-D) RNA was extracted from duplicate samples and various JAK/STAT related mRNA levels were analyzed using a JAK/STAT real-time qPCR-based gene array. mRNA levels of viral genes were normalized to the mRNA levels of human β-actin to yield dCT as a measure or relative expression without clustering. Scatter plot comparison of relative mRNA level at 0 (B), 24 (C) and 48 (D) hours between NLRX1 siRNA treated cells and NS siRNA treated cells. (E-H) Scatter plot of relative mRNA level at 24 and 48 hours. NS siRNA group at 0 hours were set as the normalization control. (I) Heat map of JAK/STAT microarray. Relative Z-scores of each gene level was calculated by subtracting mean values for each individual gene, and then dividing by each gene standard deviation. Higher Z-scores are indicated by red, lower levels by blue as shown in the key. Data are presented as mean ± s.d. from at least three independent experiments. *indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
NLRX1 is required for optimal KSHV reactivation in iSLK.219 cells.
<p>(A) iSLK.219 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with Dox for various time points. A representative image of a field of cells expressing GFP and RFP are shown at 0, 24, 48 and 72 hours post-Dox treatment. (B-C) Whole well GFP/RFP intensities were monitored and quantitated by a Clariostar plate reader. (D) KSHV genome copy numbers in the supernatants of reactivated iSLK.219 cells. (E) KSHV genome copy numbers in reactivated iSLK.219 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
TBK1 siRNA rescues KSHV reactivation.
<p>iSLK.219 cells were transfected with NS, NLRX1 siRNA or NLRX1+TBK1 siRNAs for 48 hours. iSLK.219 were then reactivated with Dox for 0, 24 and 48 hours. (A) A representative image of a field of cells expressing GFP and RFP are shown at 0, 24, and 48 and hours post-Dox treatment. (B-C) Whole well GFP/RFP intensities were monitored and quantitated by a Clariostar plate reader. (D) NLRX1 knockdown efficiency was monitored by qRT-PCR. (E-G) qRT-PCR of ORF57, K8.1 and vIRF1 in reactivated iSLK.219 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
NLRX1 negatively regulates MAVS-dependent type I interferon response.
<p>(A) iSLK.219 cells were transfected with NS or NLRX1 siRNA for 72 hours. Cells were then transfected with poly I:C at 2 μg/μl for 4 hours before being harvested. RNA was subjected to IFNβ qRT-PCR analysis. (B) NLRX1 knockdown efficiency was monitored by qRT-PCR. (C) iSLK.219 cells were transfected with NS, NLRX1 siRNA for 72 hours. 1 μM BX795 was then added to one set of NLRX1 siRNA transfected samples 6 hours before the cells were reactivated with Dox for 0, 24 and 48 hours. IFNβ mRNA was monitored by qRT-PCR. (D) NLRX1 knockdown efficiency was monitored by qRT-PCR.</p
Block of TBK1 by BX795 partially rescues KSHV reactivation.
<p>iSLK.219 cells were transfected with NS, or NLRX1 siRNA for 72 hours. 1 μM BX795 was then added to one set of NLRX1 siRNA treated samples 6 hours before the cells were reactivated by Dox for 0, 24 and 48 hours. (A) A representative image of a field of cells expressing GFP and RFP are shown at 0, 24, and 48 hours post-Dox treatment. (B-C) Whole well GFP/RFP intensities were monitored and quantitated by Clariostar plate reader. (D) KSHV genome copy number in reactivated iSLK.219 cells. (E) qRT-PCR of ORF57 in reactivated iSLK.219 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
Loss of NLRX1 attenuates global KSHV gene expression upon reactivation.
<p>iSLK.219 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with Dox for various time points. (A) qRT-PCR of NLRX1 in reactivated iSLK.219 cells. (B) qRT-PCR of K8.1 in reactivated iSLK.219 cells. (C) qRT-PCR of ORF57 in reactivated iSLK.219 cells. (D) Cells were harvested at the indicated time points for Western blot analysis of NLRX1, ORF45, and K8alpha. Actin was monitored as loading control. (E) iSLK.219 cells were treated as described in the text. RNA was extracted from duplicate samples and KSHV viral transcript levels were analyzed using a KSHV real-time qPCR-based whole genome array. mRNA levels of viral genes were normalized to the mRNA levels of multiple cellular housekeeping genes to yield dCT as a measure of relative expression. These were then subjected to unsupervised clustering. A heat map and dendrogram depicted by the brackets is shown. Higher transcript expression levels are indicated by red and lower expression levels by blue as shown in the key. Data are presented as mean ± s.d. from at least three independent experiments. *indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p
pUL135 and pUL138 impact phosphorylation of EGFR.
<p>Fibroblasts were infected with WT, <i>UL135</i><sub>STOP</sub>, and <i>UL138</i><sub><i>STOP</i></sub> virus at an MOI of 1. At 48 hpi, infected cells were pulsed with 10nM EGF for 15min and then lysed. (A) EGFR was immunoprecipitated with ms α-EGFR and both IP and lysate samples were separated by SDS-PAGE. Blots were stained with rb α-EGFR, rb α-EGFR phosphotyrosine 1068, ms α-phosphotyrosine, and ms α-IE1/2. (B) The quantification of phosphorylation over three experiments is shown. To control for the variation in EGFR levels in different infections, we normalized signals associated with pY1068 or pY to total EGFR. Statistical significance relative to WT was calculated by student t-test (* p-value ≤ 0.05). (C) Serum-starved or fed fibroblasts expressing EGFR<sub>3XFLAG</sub> were infected with WT CMV at 20 hours post transduction. Cells were stained with ms α-EGFR, rb α-EGFR pY1068 at 48 hpi. A merge of all three images in shown to the right. (D) Serum-starved, infected fibroblasts were pulsed with Alexa Fluor 647-conjugated EGF ligand on ice, fixed 20 min after a shift to 37C and stained with rb α-EGFR. Cells were imaged by deconvolution microscopy. For C and D, nuclei are stained with DAPI.</p