The major structural protein of the retrovirus HIV-1 is called Gag and is expressed as a 55 kDa poly-protein with six contiguous domains. These are labelled from the N-terminus as MA, CA, SP1, NC, SP2 and P6. There are two distinct assembly steps in the lifecycle of HIV-1, termed immature and mature assembly, both of which are essential to the production of infectious viral progeny and are as such potential targets of therapeutic intervention. The immature assembly step involves self-association of, typically, 1000- 2500 copies of Gag in a nucleic acid-dependent manner, resulting in formation of a spherical capsid immediately below the host cell membrane. The resulting immature, noninfectious virions are released from the cell and the viral protease, PR, hydrolyses Gag into its component domains. MA remains at the membrane and NC remains in complex with the genome, whilst CA reassembles as a mature capsid with a conical shape and 5,7- Fullerene geometry. In the immature and mature state, CA forms a lattice in which N-CA is arranged as hexamers linked to one another by C-CA dimerisation, but the exact interfaces and CA conformations are different between the two states. In this thesis, experiments are described which seek to establish how a single protein, CA, can form two distinct lattices, and what the role of NC-nucleic acid interactions are in immature assembly. Several Gag mutants are studied using a combination of NMR spectroscopy, fluorescence spectroscopy, electron microscopy and in vitro virus capsid assembly assays. It is shown that the NC domain does not intrinsically effect any modulation of the C-CA domain at the level of the first intermediates in the assembly pathways, and that nucleic acid is required to link two Gag molecules together in order to promote immature assembly.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:509563 |
| Date | January 2010 |
| Creators | Knight, Michael John |
| Contributors | Werner, Jorn |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/150937/ |
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