The human immunodeficiency virus type I (HIV-1) is a retrovirus that damages the human immune system, which is suppressed by a cellular factor APOBEC3G in non-permissive cells. The viral infectivity factor (Vif) can induce a poly-ubiquitination degradation of APOBEC3G to counteract the immune response by forming an E3 ubiquitin complex composed of cellular proteins Elongin B (EloB), Elongin C (EloC), Cullin 5 and Ring-Box protein. In this project, we solved the first structure for the Vif SOCS-box and EloBC complex in solution by Nuclear Magnetic Resonance, which shows that the proline-rich motif in the SOCS-box binds to the EloB carboxyl terminus by backbone interaction, based on weak van der Waals forces. Based on the results from cell assays it shows that the residues on the Vif proline-rich motif play an important role in viral infectivity; the proline-rich motif induced structure of the C-terminal tail has been demonstrated to be critical for EloB to perform further biological function, by a mechanism which allows HIV-1 to evade the immune response. In addition, expression and purification on full-length Vif in the presence of EloBC and core binding factor β (CBFβ) has been performed. These studies showed that soluble Vif in the tetramer is achieved when it is expressed at a low temperature with a low IPTG concentration. Solubility tests indicate that this complex must be kept in high salt concentration in order to prevent self-association. Comparison with previous studies on Vif expression and purification. This protocol enables one to obtain purified Vif from E. coli and opens the way to solve the structure by X-ray crystallography and extend our understanding on the Vif-induced APOBEC3G degradation mechanism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628425 |
| Date | January 2013 |
| Creators | Lu, Zhisheng |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/structural-studies-of-hiv1-vif-and-its-socsbox-domain(710df582-c838-4935-b6f6-c023195997c7).html |
Page generated in 0.002 seconds