Abstract
Vaccinia virus (VACV) morphogenesis is controlled by a temporal cascade enacted by intermediate and late promoter sequences, in conjunction with virally encoded transcription factors. VACV morphogenesis culminates with the formation of two structurally and functionally distinct virion morphotypes from a single mature virus (MV) progenitor; the intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV are typified by their single membrane and the presence of the A26 protein, which is expressed at 10 hours post infection. EEVs are characterized by their double membrane and suite of virally encoded proteins, the most crucial of which is the F13 protein expressed at 4 hpi. Previous investigations have implicated the temporal expression of A26 protein as a regulatory switch, negatively regulating the formation of EEV during late times of infection. However, reverse genetic approaches have refuted this claim. Despite this, these studies do not consider the temporal organisation of key IMV and EEV proteins, in addition to their potential to regulate one another.The aim of this study was to investigate the concept of altering the temporal regulation of both A26 and F13 to generate novel phenotypes, exploring the mechanism underpinning IMV vs EEV balance. Recombinant viruses were generated with F13L and A26L alleles expressed under the control of either F13L or A26L promoter sequences. When A26L was introduced under the intermediate F13L promoter sequence a significant plaque size reduction was observed.In addition, a further deduction was observed when F13 expression was delayed under the late A26 promoter. Temporally advanced A26 expression significantly altered its rate of association with the key MV membrane protein A27, which is required for EEV formation. When A26 was expressed under the intermediate F13L promoter, incorporation of A26 on virions was significantly enhanced when compared to A26 expressed under its native promoter sequence.This was correlated with a small reduction in EEV formation. The data presented in this thesis reveals the A26 protein as a potential negative regulator of EEV formation.The temporal segregation of A26 from the EEV morphogenesis proteins; F13 and A27 underpins the transition from EEV to IMV production. In addition, this thesis introduces the concept of altering temporal regulation to explore the constraints of the poxviral genome and its ability to acquire variation. These findings will assist in the refinement of sequencing algorithms used to characterise novel pathogen populations.