Advances in immunosuppressive therapy, which have led to the application of transplantation for a wide spectrum of diseases, have been limited in part by the development of infectious complications. Despite the development of effective antiviral therapy, CMV represents the most common viral infection following transplantation resulting in loss of life and graft, as well as significant cost. In addition, the emergence of viral mutants has resulted in increasing resistance to antiviral therapy, underscoring the importance of preventive strategies. Reactivation of CMV from latency requires the virus to become transcriptionally active, and culminates in replication of infectious virus resulting in active infection. A crucial first step is expression of the Immediate Early (IE) genes to activate expression of other viral genes. The investigators hypothesize that reactivation of latent MCMV occurs in latently infected cells as a result of induction of IE gene expression. They further hypothesize that inflammatory cytokines such as TNF-a, released as a result of ischemia/reperfusion injury and/or allogeneic stimulation, activate the transcription factor NF-kB which in turn induces IE gene expression, an essential first step in MCMV reactivation. The investigators will induce transcription of MCMV genes in latently infected organs by intraperitoneal injection of TNF-a, ischemia/reperfusion injury, and allogeneic stimulation. They anticipate that this will be accompanied by activation of NF-kB, AP-1, and ATF. Immunophenotyping in combination with in situ amplification of RNA will be used to identify the cell types in which induction of viral transcripts occurs. The mechanism of induction of IE transcription will be investigated using transgenic mice that express beta -gal under the control of the IE promoter. The investigators propose to show that these mice are a valid model for viral reactivation by demonstrating that treatments which induce IE gene expression in latently infected mice also induce beta -gal expression in the IE-beta -gal transgenic mice. They will investigate the role of TNF in induction of IE gene expression by breeding IE-B-gal mice to mice deficient in TNF receptors. The requirement for NF-kB in activation of IE gene expression will be confirmed by using inhibitors that prevent activation of NF-kB. By defining the specific cytokine/transcription factor interactions that govern upregulation of relevant CMV gene expression, future strategies which modulate gene expression may be designed that prevent reactivation of CMV from latency, therefore eliminating the direct and indirect sequelae of CMV infection.
