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Title: Structural analysis of retrovirus assembly and restriction factors by electron cryomicroscopy
Author: Acton, Oliver
ISNI:       0000 0004 8500 2418
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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In order to better understand and combat retroviral infection, molecular structures of complexes involved in the life cycle are required. Two important areas of retroviral biology for such studies involve retrovirus assembly and host cell restriction. During retroviral maturation, the gag polyprotein is processed by the retroviral protease into the membrane-associated matrix protein (MA), the genomeassociated nucleocapsid (NC) and the capsid protein (CA) that forms the shell surrounding the packaged viral genome. Whilst the structural organization of the HIV-1 capsid is the best studied, structures of capsids of other retroviral genera are important points of comparison. In addition, as a result past retroviral infections, the human genome contains endogenous retroviruses (ERVs), that although cannot transmit cell-to cell still assemble their CA into virus like particles. Here we present structural studies of CA derived from the endogenous retrovirus HERV-K (HML-2) that forms closed shell assemblies in vitro. SAMHD1 is a dNTP triphosphohydrolase that has been shown to restrict of HIV-1 infection in non-dividing myeloid-derived immune cells. SAMHD1 comprises an N-terminal SAM domain and central HD domain and in its inactive apo-state, exists in a monomer-dimer equilibrium. However, in the presence of GTP and substrate dNTPs, SAMHD1 forms a catalytically active tetramer that previous studies have shown can adopt multiple conformations. However, how these states relate to SAMHD1 conformation in the catalytic cycle of a live reaction remains unclear and furthermore, full-length structures of human SAMHD1 and role of the SAM domain in regulation and catalysis remain unknown. Recent advances in the field of electron cryo-microscopy now make it possible to address both of these topics. Using electron cryo-microscopy, highresolution fullerene-like assemblies of mature HML-2 capsid have been resolved. These maps reveal the basis for shell assembly and show how different conformations adopted by CA proteins at quasi-equivalent interhexamer and hexamer-pentamer interfaces generate the curved architecture of the shell. Cryomicroscopy studies of stabilised structures of the SAMHD1 probe the active tetramer at high resolution. The structures reveal the position of the SAM domain in the context of a catalytic tetramer and of bound substrates and allosteric activators, revealing quaternary states representative of physiological conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available