Human Immunodeficiency virus type-l (HIV-l), has a number of regulatory genes in addition to the gag, pol, and env that are common to all replication competent retrovimses. It expresses six auxilialY proteins, tat, rev, Vi/, vpu, vpx and nef. Vif (Viral Infectivity Factor) is a 23 kDa basic protein of 192 aa. Vif has been shown to be essential for the modulation of virion infectivity in nom1al host cells and is believed to function by interacting with both viral and cellular proteins. More recent studies have focused on its involvement in controlling the encapsidation of cellular proteins ~f the APOBEC3 family. However earlier work of Vif suggested an involvement in viral morphogenesis and this was the main focus of the present study. Vifhas been shown to be associated with the viral nucleocapsid and to be specifically packed into HIV patiicles, either by interaction with viral RNA and/or Gag and GagPol precursors. This study had as its primary aim definition of the molecular interactions of Vif with the Gag precursor (Pr55GAG ), the viral Protease (RR), and the antiviral cellular proteins APOBEC3G/3F. An in vivo mammalian two-hybrid assay was used to study the interactions between Vif and both Pr55GAG and the viral PR. This found that Vif interacts with Pr55GAG • To begin mapping the positions of this interaction, a series of mutations were made in both proteins. Complimenting previous studies on Vif done at Warwick, amino acid 21 was found to be cmcial for the interaction between Vif and Pr55GAG in the mammalian two-hybrid assay. Interaction between Vif and Pr55GAG was fmiher confirmed using an independent in vitro GST pull-down assay. No interactions were found between Vif and PR and between PR and Pr55GAG • The second objective of this study was to analyse the molecular interactions of HIV-1 Vif, Pr55GAG , and PR with the APOBEC3 family of cellular proteins using both an in vivo mammalian two-hybrid assay and an in vitro GST pull-down assay. In the mammalian two-hybrid assay a direct interaction between Pr55GAG and both APOBEC3G and 3F was identified. These interactions were further confirmed using the GST pull-down assay. A direct interaction between Vif and the two APOBEC proteins APOBEC3G and 3F was also seen in vitro in the GST pull-down assay; however, these interactions could not be seen in vivo using the mammalian two hybrid assay. A third are~ of work was concemed with the high level expression of Vifin a bacterial expression system and purification of the protein for structural studies. This experimental work was complemented by computer based model building using a comparative modeling method. The aim of this work being to produce an atomic level resolution model for Vifwhich could be tested against the experimental results achieved in interaction site mapping studies. Building on earlier work done at Warwick a fourth area of this study involved in vivo experiments aimed at understanding the role of HIV-1 Vif iil resistance to protease inhibitors (Adekale et al., 2005). This involved establishing the molecular reagents to allow the generation of infectious molecular clones carrying various variants of Vifand the HIV-1 protease. Plasmid constructs were generated to allow the inse11ion of different variants of the PRgene into an infectious molecular clone building on the previously available strategy which allowed similar exchanges with the Vifgene.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:492525 |
| Date | January 2007 |
| Creators | Syed, Farhatullah |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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