Investigation into the mechanism of immature HIV-1 capsid assembly

The major structural protein of the retrovirus HIV-1 is called Gag and is expressed as a 55<br/>kDa poly-protein with six contiguous domains. These are labelled from the N-terminus as<br/>MA, CA, SP1, NC, SP2 and P6. There are two distinct assembly steps in the lifecycle of<br/>HIV-1, termed immature and mature assembly, both of which are essential to the<br/>production of infectious viral progeny and are as such potential targets of therapeutic<br/>intervention. The immature assembly step involves self-association of, typically, 1000-<br/>2500 copies of Gag in a nucleic acid-dependent manner, resulting in formation of a<br/>spherical capsid immediately below the host cell membrane. The resulting immature, noninfectious<br/>virions are released from the cell and the viral protease, PR, hydrolyses Gag into<br/>its component domains. MA remains at the membrane and NC remains in complex with<br/>the genome, whilst CA reassembles as a mature capsid with a conical shape and 5,7-<br/>Fullerene geometry. In the immature and mature state, CA forms a lattice in which N-CA<br/>is arranged as hexamers linked to one another by C-CA dimerisation, but the exact<br/>interfaces and CA conformations are different between the two states. In this thesis,<br/>experiments are described which seek to establish how a single protein, CA, can form two<br/>distinct lattices, and what the role of NC-nucleic acid interactions are in immature<br/>assembly. Several Gag mutants are studied using a combination of NMR spectroscopy,<br/>fluorescence spectroscopy, electron microscopy and in vitro virus capsid assembly assays.<br/>It is shown that the NC domain does not intrinsically effect any modulation of the C-CA<br/>domain at the level of the first intermediates in the assembly pathways, and that nucleic<br/>acid is required to link two Gag molecules together in order to promote immature<br/>assembly.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2010

Author dc:creator

• Knight, Michael John

Advisor dc:contributor.advisor

• Werner, Jorn M.