Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients. The majority of HCMV- associated morbidity and mortality is the result of the spread of persistent virus. The long term goal of our laboratory is to understand at the host, cellular, and molecular level the mechanisms by which HCMV persists, to develop novel strategies for blocking HCMV persistence, and ultimately reduce the incidence of HCMV disease in transplantation. A critical means by which HCMV establishes a persistent infection is to evade host innate and adaptive immune responses. Recently, we have discovered that one of these key immunoevasion strategies is the blockade of interferon (IFN) stimulated responses in HCMV infected fibroblasts and endothelial cells. HCMV blocks IFN-gamma and IFN-alpha/beta stimulated signal transduction, IFN-stimulated transcription factor activation, and IFN- stimulated gene expression in infected cells. Analyses of the JAK/STAT signal transduction pathway reveals that protein levels of JAK1, a tyrosine kinase required for activation of IFN-gamma and IFN- alpha/beta signal transduction, are decreased in HCMV infected cells. The focus of this proposal is to determine the molecular mechanism for the HCMV-mediated decrease in JAK1 expression, identify the HCMV gene that decreases JAK1 expression and disrupts IFN signal transduction, and investigate the significance of IFN-blocking activity in mutant HCMV strains lacking this capability.
In Specific Aim I, we will test the hypothesis that JAK1 protein levels are decreased by a proteasome-dependent process in HCMV infected cells. We will analyze the mechanism for the degradation of JAK1 proteins in infected cells and the upstream events prior to JAK1 degradation including phosphorylation, ubiquitination, and IFN-receptor binding. Moreover, the role of suppressors of cytokine signaling (SOCS) proteins in blocking the kinase activity of JAK1 and mediating JAK1 degradation will be investigated.
In Aim II, we test the hypothesis that HCMV encodes a gene product that mediates the decrease of JAK1 levels thereby blocking IFN signal transduction in infected cells. 2C4 cells, which are stably transfected with an IFN-responsive promoter linked to the cell surface CD2 glycoprotein, will be utilized to expression clone the HCMV gene with IFN signal transduction blocking activity.
In Aim III, we will test the hypothesis that HCMV-mediated IFN-blocking activity is critical for HCMV gene expression, replication, and reducing MHC-based antigen presentation. To test this hypothesis, we will generate a mutant HCMV strain lacking IFN-blocking activity and investigate HCMV gene expression, replication, and MHC based antigen presentation in the presence of IFNs.
| Raghavan, Bindu; Cook, Charles H; Trgovcich, Joanne (2014) The carboxy terminal region of the human cytomegalovirus immediate early 1 (IE1) protein disrupts type II inteferon signaling. Viruses 6:1502-24 |
| Machesky, Nicholas J; Zhang, Guojuan; Raghavan, Bindu et al. (2008) Human cytomegalovirus regulates bioactive sphingolipids. J Biol Chem 283:26148-60 |
| Zhang, Guojuan; Raghavan, Bindu; Kotur, Mark et al. (2007) Antisense transcription in the human cytomegalovirus transcriptome. J Virol 81:11267-81 |
