Functional analysis of the African swine fever virus MGF360 and MGF505 proteins, and its application to vaccine development

• Main Author: Connell, Samuel
• Format: Dissertation
• Language: English
• Published: University of Oxford 2021
• Subjects: African swine fever virus; Virology

African swine fever virus (ASFV) is a deadly haemorrhagic pathogen of swine. In recent years, ASFV has spread beyond Africa and continues to ravage European and Asian pig herds, posing a severe socio-economic threat. No vaccines are currently available leaving preventive and extermination measures as the only course of action. The virus' dsDNA genome is up to 190kb in length, and contains 5 clusters of paralogous genes known as Multigene family (MGF) genes close to both termini. Genes of MGF360 and MGF505 clusters have been implicated in the subversion of the innate immune system, and their absence is known to attenuate virulent isolates, however their specific mechanism of action is unknown. Here I have characterised the functions of MGF360-10L, 11L, 12L, 13L, 14L, MGF505-1R, 2R and 3R, by performing in silico and in vitro analysis. I revealed that these MGF genes inhibit the interferon signalling pathways at the level of the transcription factors or below. MGF360-12L and MGF505-1R were demonstrated to both contain a functional novel Cullin-RING-E3 ubiquitin ligase (CRL) recruitment motif, which was indispensable for the inhibitory action of MGF505- 1R on the interferon pathway. Moreover MGF505-1R reduced levels of endogenous p300, a cofactor essential in the transcription of type-I Interferon and other genes. Mutagenesis of the CRL motif restored p300 levels, indicating that MGF505-1R targets the p300 cofactor for CRL driven ubiquitination and subsequent proteasomal degradation. I applied the knowledge gained here to the rational development of vaccine candidates. Two profiles of MGF genes were deleted from the virulent Georgia07/1, and the impact on ex vivo ISG expression, and in vivo attenuation in pigs was assessed. This revealed the specific genes potentially key for attenuation. Overall, I have advanced knowledge on understanding the molecular mechanisms of the ASFV MGF360 and MGF505 proteins and identified two of those with critical roles during virus infection.