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. 2016 Apr;97(4):941-954.
doi: 10.1099/jgv.0.000407. Epub 2016 Jan 20.

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter

Xue-Feng Liu  1 , Chunfa Jie  1 , Zheng Zhang  1 , Shixian Yan  1 , Jiao-Jing Wang  1 , Xueqiong Wang  1 , Sunil Kurian  2 , Daniel R Salomon  2 , Michael Abecassis  3   1 , Mary Hummel  3   1
Affiliations

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter

Xue-Feng Liu et al. J Gen Virol. 2016 Apr.

Abstract

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1β, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.

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Figures

Fig. 1.
Fig. 1.
Transcription factor activation induced by allogeneic transplantation. Kidneys from latently infected BALB/c mice were transplanted into allogeneic C57BL/6 recipients and harvested 3, 24 or 48 h post-transplant (G), as indicated on the x-axis. The contralateral latent donor kidney (C) was harvested at the time of the transplant as a matching Day 0 control. Nuclear extracts were prepared and analysed by TransAm assays, n = 5/time point. Results are expressed as the mean plus standard error. **P < 0.01; *P < 0.05.
Fig. 2.
Fig. 2.
Expression of transcription factor RNAs in control and kidney allografts. RNA expression was analysed in control (C) kidneys and in grafts harvested 3 (G 3 h), 24 (G 24 h) or 48 h (G 48 h) after transplant and normalized to hypoxanthine phosphoribosyltransferase (HPRT) RNA. Results are expressed as the mean plus standard error. P values were determined by comparison of expression in the graft to pooled controls. ***P < 0.001; **P < 0.01; *P < 0.05; n = 4.
Fig. 3.
Fig. 3.
Reactivation of IE gene expression correlates with recruitment of transcription factors and remodelling of the MIEP. (a) Expression of IE-3 in matching control (C) and transplanted (G) kidneys 48 h after transplant. MCMV IE-3 RNA expression was normalized to expression of cellular Eef2; n = 5. Error bars show the mean plus standard error. *P < 0.05. (b) ChIP analysis of p65/RelA and p50 NF-κB subunits bound to the MCMV MIEP (left panel) and to the promoters of the cellular Ccl2 and Hes1 genes (right panel) in pooled chromatin from control (C) or 48 h allografts (G). (c) Binding of junD to the MIEP (left panel) and to the promoters of the IL-6 and Ant4 genes (right panel). (d) Binding of actin to the MIEP. The data shown are representative of at least two independent assays, except NF-κB p50 (n = 1). Error bars show sd of technical replicates analysed in triplicate.
Fig. 4.
Fig. 4.
Allogeneic transplantation induces release of inflammatory proteins into the plasma. (a) Heat map of differentially regulated plasma proteins in control (C) and recipient mice at 3 and 24 h after transplant, n = 5/group. (b) Graphical representation of the plasma protein values of selected cytokines. Results are expressed as the mean plus sd. ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 5.
Fig. 5.
Dynamic changes in inflammatory signalling pathways are induced by allogeneic transplantation. (a) Ingenuity Pathway Analysis of selected signalling pathways upregulated by allogeneic transplantation at 3, 24 and 48 h. Pathways with –log P value >1.3 (dashed line) are statistically significant. (b) Heat map of RNA expression of genes within these pathways determined by microarray analysis.
Fig. 5.
Fig. 5.
Dynamic changes in inflammatory signalling pathways are induced by allogeneic transplantation. (a) Ingenuity Pathway Analysis of selected signalling pathways upregulated by allogeneic transplantation at 3, 24 and 48 h. Pathways with –log P value >1.3 (dashed line) are statistically significant. (b) Heat map of RNA expression of genes within these pathways determined by microarray analysis.
Fig. 6.
Fig. 6.
Expression of TNF and IL-6 is induced in kidney allografts. RNA expression was analysed in control (C) kidneys and in grafts harvested 3 (G 3 h), 24 (G 24 h) or 48 h (G 48 h) after transplant and normalized to HPRT RNA. Results are expressed as the mean plus standard error. P values were determined by comparison of expression in the graft to pooled controls. ***P < 0.001; **P < 0.01; *P < 0.05; n = 4.
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