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Title: Characterising the role of interferon regulatory factor binding elements within the promoter of murine gammaherpesvirus-68 ORF50 gene in virus lytic infection
Author: Manso, Bruno Manuel Tomas
ISNI:       0000 0004 2733 8343
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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In y2-herpesviruses the balance between lytic infection and reactivation from latency depends entirely on expression of an immediate-early viral gene (ORFSO) encoding the Replication and Transcriptional Activator (Rta) protein. Although studies have elucidated how Rta can activate viral genome leading to a cascade of events necessary for the viral lytic infection, much less is understood about the regulation of Rta expression itself. Nonetheless, extensive work has produced a body of evidence describing several interactions between cellular factors signaling pathways and physiological stimuli, which are able to induce or repress Rta gene expression and hence influence lytic infection cycle as well as reactivation from latency. In this study, the murine model, MHV-68, was used to characterize transcription activity of different domains within the Rta promoter sequence. Initial observations demonstrated that Rta promoter activity is both cell-type dependent and domain-specific. Overall, Rta promoter had a greater basal activity in immunocompetent cells than cells which were unable to respond to type I IFN signaling. Specifically, the transcription activity of a region between 1019 and 391 bp upstream of ORFSO ORF was down regulated when cells are pre-treated with IFN-a/B. Detailed bioinformatics analysis of this domain revealed a series of putative binding sites most closely associated with IFN response elements, including interferon regulatory factors 3 and 7 (IRF3 and IRF7). Site-specific mutation of these viral sequences within the Rta promoter resulted in cell type dependent modulation of Rta promoter. Interestingly, data obtained from different cell types strongly suggested that endogenous IRF7 was involved in upregulation of the promoter in murine B Iymphocytes which was different from non-B cells. Paradoxically, exogenous expression of IRF7 reduced activation of the Rta promoter in fibroblasts with an abrogated type I IFN pathway and in a promoter sequence-specific manner. Although, physical interaction between IRF7 and Rta promoter using EMSA could not be demonstrated further work is required to show how IRF7 may affect Rta promoter and under different cell conditions. Taken together, these findings show for the first time the domain- and cell-type-specific activities of the Rta promoter and the complexities of Rta promoter regulation by type I IFN mediated responses. These findings also give credence to previous work which demonstrated that y- herpesviruses have evolved strategies alongside type I IFN-dependent cellular pathways which modulate their lytic as well as latent (persistent) stages of infection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral