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Understanding KSHV vIRF-2-cell interactions

Mutocheluh, Mohamed (2011)
Ph.D. thesis, University of Birmingham.

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Abstract

Kaposi’s sarcoma-associated herpes virus (KSHV) encodes genes with immunomodulatory potential, one of which is vIRF-2 that shares homology to cellular interferon regulatory factors. The innate antiviral mechanism mediating the type I interferons is an essential host cell defence mechanism limiting viral replication. The aim of this study was to determine the range and type of cellular gene sets and associated biological pathways whose expression is deregulated by vIRF-2. HEK 293-derived cell clones were engineered to express doxycycline-inducible vIRF-2. Interferon (IFN) responses were induced with recombinant (r) IFN-α and measured by an IFN stimulated response elements (ISRE) luciferase reporter gene assay. The effects of vIRF-2 on cell transcriptome profile in response to rIFN-α were determined by DNA microarray analysis and confirmed by immunoblot assay. vIRF-2 protein inhibited activation of ISRE-luc by over 50% and significantly (p<0.05) down-regulated the expression of 57/78 (73%) of rIFN-α regulated genes. The DAVID and GSEA software packages revealed vIRF-2 down-regulates the RIG-I-like receptor, JAK-STAT and Ubiquitin ligase pathways and many gene sets involved in antiviral response, transcriptional regulation and apoptosis. Immunoblot assays demonstrated reduced levels of RIG-I/DDX58, TBK-1, p-38, STAT1, pSTAT1, IRF-9 and OAS3. The biological significance of the vIRF-2 anti-IFN property was demonstrated by the rescue of encephalomyocarditis virus (EMCV) replication in vIRF-2 expressing cells treated with rIFN-α; EMCV was titred by plaque assay on L929 cells. These data confirm the role of KSHV vIRF-2 in negative regulation of the IFN-α/β innate immune response by a mechanism dependent on negative regulation of RIG-I/DDX58, STAT1, IRF-9 and OAS3.

Type of Work:Ph.D. thesis.
Supervisor(s):Blackbourn, David J.
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Cancer Sciences
Subjects:QR180 Immunology
QR355 Virology
Institution:University of Birmingham
ID Code:2843
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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