Restriction factors interfere with retroviral infection, blocking the viral life cycle after cell entry, but prior to integration into the chromosomal DNA. Fv1 restricts murine leukaemia virus (MLV), whereas TRIM5α (T5) restricts human immunodeficiency virus (HIV), and other retroviruses. The mechanism of restriction is still poorly understood, but a key factor is the interaction between restriction factors and the viral capsid (CA). Development of reliable and efficient binding assays would greatly enhance our understanding of this process. This thesis describes the development of a novel MLV binding assay. Such an assay has been described for HIV, using in vitro polymerised CA-NC and lysate from cells expressing T5 (Stremlau et al., 2006), however, initial attempts to use this method to explore binding of T5 found the assay to be somewhat unreliable. The interaction between T5 and HIV CA is thought to be one of low affinity, and so binding could be aided by using a higher concentration of T5. Following the purification of T21T5 fusion protein from baculovirus, it was possible to demonstrate an interaction with HIV CA-NC. In contrast to T5, binding of CypA, Fv1Cyp and T5Cyp to HIV CA-NC could be consistently demonstrated, regardless of whether cell lysate or purified protein was used. To generate an MLV binding assay, it was first necessary to develop a novel method for the in vitro polymerisation of CA. By incubating HIS-tagged CA protein with Ni-chelating lipid nanotubes it was possible generate CA-coated tubes. Analysis of these tubes by electron microscopy confirmed that the CA molecules were arranged in a regular lattice, and using these CA-coated tubes, it was possible to show an interaction with Fv1. Investigation of a number of Fv1 and CA variants revealed a good correlation between restriction and binding specificity. It was also possible to confirm that multimerisation of both Fv1 and CA were necessary requirements for a binding interaction to occur. With further development, it should be possible to use this assay to investigate the binding for other retroviral CAs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625391 |
| Date | January 2010 |
| Creators | Hilditch, L. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/765045/ |
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