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Title: Characterising retroviral restriction by TRIM proteins
Author: Fraser, S. J.
ISNI:       0000 0004 7429 0207
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Tripartite motif (TRIM) proteins are numerous in the human proteome, and a number of these molecules are known to restrict retroviral replication. TRIM5α (T5α) is one such factor. It targets the viral capsid and imposes a block to infection between entry and reverse transcription. Capsid recognition is mediated by the C-terminal B30.2 domain, which contains surface-exposed loops of high amino acid variability. Restriction is then effected via proteasome recruitment and the induction of innate immune cascades. Although T5α is well-characterised in this respect, other factors – such as the highly divergent TRIM1 (T1) – remain poorly understood. To further characterise the T1 restriction phenotype, chimeras of this protein and its non-restricting paralogue, T18, were generated by overlapping PCR. The restriction activities of the resulting molecules were then measured using an established flow cytometry assay. These experiments revealed that T1 also binds capsid via the B30.2 domain, although the majority of this region can be functionally replaced. Other aspects of T1 biology addressed in this work include the contribution of N-terminal components to restriction potency, and the relationship between protein expression level and restriction activity. Following a number of attempts to generate a functional chimera of T1 and 5α, the latter half of this thesis explores how the spacing between capsid-binding and effector domains can influence restriction activity. To this end, a panel of mutations were made in the linker 2 (L2) region of T5α, and their effects on restriction measured. These experiments revealed that even small changes in interdomain spacing can have profound phenotypic consequences. Collectively, this work reinforces the notion that TRIM family members share a common overall design, allowing individual components to be shuffled between them. At the same time, each molecule has been shaped by unique evolutionary pressures, which can render them sensitive even to relatively minor modifications.
Supervisor: Stoye, J. P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available