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. 2001 May;75(10):4814-22.
doi: 10.1128/JVI.75.10.4814-4822.2001.

Allogeneic transplantation induces expression of cytomegalovirus immediate-early genes in vivo: a model for reactivation from latency

M Hummel  1 , Z Zhang, S Yan, I DePlaen, P Golia, T Varghese, G Thomas, M I Abecassis
Affiliations

Allogeneic transplantation induces expression of cytomegalovirus immediate-early genes in vivo: a model for reactivation from latency

M Hummel et al. J Virol. 2001 May.

Abstract

Reactivation of cytomegalovirus (CMV) from latency is a frequent complication of organ transplantation, and the molecular mechanism by which this occurs is unknown. Previous studies have shown that allogeneic stimulation induces reactivation of human CMV (HCMV) in vitro (64). We find that transplantation of vascularized allogeneic kidneys induces murine CMV (MCMV) and HCMV immediate-early (ie) gene expression. This induction is accompanied by increased expression of transcripts encoding inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-2, and gamma interferon, and by activation of NF-kappaB. TNF alone can substitute for allogeneic transplantation in inducing HCMV and MCMV ie gene expression in some tissues. Our studies suggest that reactivation is a multistep process which is initiated by factors that induce ie gene expression, including TNF and NF-kappaB. Allogeneic transplantation combined with immunosuppression may be required to achieve complete reactivation in vivo.

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Figures

FIG. 1
FIG. 1
Effect of transplantation on expression of MCMV IE1 RNA. Kidneys from latently infected mice were transplanted into allogeneic or syngeneic recipients and were removed 1, 2, or 5 days after transplant (lanes 5, 7, and 9, respectively). RNAs from transplanted kidneys (T) or contralateral control (C) kidneys or from productively infected murine embryo fibroblasts (lanes 1 and 2) were analyzed for MCMV IE1 RNA expression by RT-PCR. Transplanted and contralateral control kidneys are indicated by brackets. Lane 3, RNA from lung tissue of a latently infected mouse injected with TNF. β-Actin RNA was analyzed as a positive control.
FIG. 2
FIG. 2
Putative transcription factor binding sites in the MCMV and HCMV ie1 promoter/enhancer regions. Binding sites in the MCMV promoter/enhancer are based on analysis of the nucleotide sequence (20). Binding sites in the HCMV promoter/enhancer are based on previous analyses (41).
FIG. 3
FIG. 3
Effect of transplantation on activation of NF-κB and AP-1. Nuclear extracts from transplanted (T) and contralateral control kidneys (C) were analyzed for activation of NF-κB and AP-1 by electrophoretic mobility shift assay. Kidneys were transplanted into allogeneic (lanes 4 and 8) or syngeneic (lanes 2 and 6) recipients and were removed 1 day (lanes 2 and 4) or 2 days (lanes 6 and 8) after transplant. Results shown are representative of two sets of experiments. S, syngeneic transplant; A, allogeneic transplant.
FIG. 4
FIG. 4
Effect of transplantation on expression of inflammatory cytokines. Kidneys were transplanted (lanes T) into allogeneic or syngeneic mice and were removed 1, 2, and 5 days after transplant. The contralateral kidney was removed at the time of transplant and analyzed as a control (lanes C). RNAs were analyzed for expression of TNF, IL-2, IFN-γ, or IL-1 by RT-PCR. PCR products were detected by agarose gel electrophoresis (D) or by Southern blot hybridization (A to C). Induction of TNF expression was observed in six of six allogeneic and zero of six syngeneic transplants.
FIG. 5
FIG. 5
Effect of TNF on MCMV ie1 gene expression and transcription factor activation in lung and kidney tissue of latently infected mice. (A) RT-PCR analysis of IE1 RNA expression in lungs of latently infected mice at 8, 16, 24, and 48 h after injection with TNF. RNA from productively infected murine embryo fibroblasts (Acute) was analyzed as a positive control. NT, no template. (B and C) Electrophoretic mobility shift assay analysis of activation of NF-κB and AP-1 in lung (L) and kidney (K) extracts 2 h after injection with TNF (+) or with phosphate-buffered saline (−). Results shown are representative of two sets of experiments. Competition assays (lanes 8 to 10) were performed by incubating extracts with excess unlabeled oligonucleotides containing mutant (lane 8) or wild-type NF-κB binding sites (lane 9) or no competitor (lane 10) with kidney extracts from mice injected with TNF. wt, wild type; mut, mutant.
FIG. 6
FIG. 6
Effect of TNF and transplantation on HCMV ie1 gene expression in vivo. (A) Histochemical analysis of expression of the β-galactosidase transgene under the control of the HCMV major ie promoter in the kidneys of control (left) and TNF-injected (right) MIEP-lacZ mice. (B) Quantitative chemiluminescence analysis of β-galactosidase activity in kidneys of control mice and mice injected with TNF, in kidneys transplanted into allogeneic recipients, and in contralateral control kidneys. Values shown are average light units per nanogram of protein plus standard deviation. TNF (P = 0.01, two-tailed t test) and allogeneic transplantation (P = 0.001, paired t test) induce a statistically significant increase in β-galactosidase activity over that of controls. allo tx, allogeneic transplant.
FIG. 7
FIG. 7
Model for reactivation of CMV from latency. See text for explanation.
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