Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigation of the roles of MERS-CoV accessory proteins: ORF4a and ORF4b with the host(University of Liverpool, 2024-03-05) Almsaud, Mai; Hiscox, JulianCoronaviruses account for a high burden of respiratory disease in humans, ranging from common colds to severe acute respiratory syndrome. The emergence of severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome (MERS-CoV) since 2012 and, more recently, SARS-CoV-2 have attracted considerable attention worldwide. The persistence of sporadic MERS-CoV cases, along with the absence of vaccines and antivirals, and the strikingly high morbidity and mortality rate of 36%, underscore the ongoing risk posed by MERS-CoV to global health security. Nevertheless, gaining a comprehensive understanding of the pathogenic significance of MERS-CoV requires further research. As viral replication and infection rely on the host cell machinery for the viral life cycle, studying how cells respond to infection allows us to understand the mechanisms involved in either limiting virus infection or, when control fails, in pathogenesis. In contrast, viruses can encode specific proteins to counteract these host responses. MERS-CoV ORF4a and ORF4b are two such proteins and recognised for their roles in modulating the host immune system, thereby influencing viral pathogenicity. This study investigated the significant roles of MERS-CoV accessory proteins, ORF4a and ORF4b, using two distinct high-throughput methods: a label-free mass spectrometry proteomics approach and a transcriptomic approach based on long-read cDNA-PCR sequencing. The proteomic approach unveiled novel interactions between MERS-CoV ORF4a, ORF4b, and host factors. Complementing the LC-MS/MS analysis, long-read length Nanopore sequencing was employed to explore the transcriptomic landscape influenced by MERS-CoV ORF4b expression. This technique allowed for a comprehensive assessment of isoform usage and mRNA regulation, providing novel insights into the virus hijacking splicing machinery by ORF4b. The investigation of both viral proteins, ORF4a and ORF4b, was extended by comparing their relative strengths in subverting the innate IFN-β response with those of similar proteins encoded by other coronaviruses. This demonstrated the distinct potency of MERS-CoV ORF4a and ORF4b proteins in relation to the severity of MERS disease when compared to other coronaviruses. Collectively, this thesis contributes to the understanding of the interactions that MERS-CoV establishes with its host, highlighting the significance of accessory ORFs in pathogenesis, and emphasising their potential as subjects for ongoing monitoring.39 0