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