An Investigation Into the Effect of Myristoylation on the Interactions Between HIV-1 Nef and Cellular Proteins

Boulton, Victoria J (1998) An Investigation Into the Effect of Myristoylation on the Interactions Between HIV-1 Nef and Cellular Proteins. PhD thesis, University of Glasgow.

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The nef gene is conserved across the primate lentiviruses. It is an auxiliary gene and encodes a myristoylated 27-34kDa protein expressed early in the viral replicative cycle. Data from in vivo macaque studies and analysis of long term non-progressor cohorts has implicated nef as a critical factor in the progression from latent HIV infection to full blown AIDS. However the exact mechanism of action of Nef remains controversial. These studies were undertaken to investigate Nef interactions with cellular proteins. The yeast-two-hybrid-system (YTHS) was modified to express Nef as an N-terminal fusion protein, allowing interactions to be evaluated for the authentically myristoylated Nef species. Screening of a Jurkat T-cell library identified a novel Nef interaction with an uncharacterised polypeptide (clone 39). Specificity screens confirmed the authenticity of the interaction and the polypeptide was further demonstrated to bind HIV-1 p55Gag. The cDNA sequence had no homologies to known genes but 99% homology to a human expression sequence tagged (EST) cDNA and 76% homology to a mouse EST-cDNA sequence, however no protein homologies were found to either of these sequences. A fragment of the clone 39 peptide sequence was found to have significant homology to cAMP phosphodiesterase, thus raising the interesting possibility that Nef may be capable of binding native phosphodiesterases causing the deregulation of cAMP levels typically associated with HIV infection. As cAMP is a key secondary messenger influencing a number of cellular signal transduction pathways a Nef interaction with phosphodiesterase might also explain the pleiotropic functions ascribed to Nef A highly conserved (PXX)3P proline motif in Nef had been previously been demonstrated to confer Nef binding to isolated Src homology 3 domains (SH3) of members of the Src tyrosine kinase family in vitro. The latter half of the studies described herein were therefore designed to determine whether Nef-SH3 complex formation was maintained in vivo in the context of the full length native proteins. Firstly Nef interactions with the T-lymphocyte specific kinase Lck were assessed. Nef was found to associate with Lck in a conformation-dependent manner. Nef binding was conferred by the open active conformation of Lck, indicative of the interaction occurring concomitantly with cell membrane receptor aggregation, possibly during viral entry. The Nef interaction with Lck was found to be mediated at least in part by the conserved (PXX)3P motif in Nef in conjunction with an N-terminal region in Nef Secondly, Nef interactions with primarily Hck but also Src and Fyn were investigated. For this purpose both the YTHS and baculovirus expression systems were utilised. Interactions with native Nef and the isolated Src homology domains SH3, SH2 and SH3-SH2 were evaluated in the YTHS, the resuhs of which demonstrated a conserved SH3 domain-mediated interaction with Nef for Hck and Src. No interaction was detected for Fyn. This result was corroborated by in vitro binding assays with the baculovirus expressed full length native proteins. The interactions between Nef and Hck was found to be conserved in vivo, however complex formation was independent of the Nef (PXX)3P motif Indeed deletion of a number of regions in Nef did not perturb the interaction, although unexpectedly the Bru laboratory isolate of Nef had severely impaired Hck binding ability. This suggested Hck binding was Nef isolate dependent and that subtle changes in amino acid sequence in targeted areas outside the (PXX)3P motif were sufficient to inhibit the association. Results from peptide substrate assays to assess the effect of Nef on Hck kinase activity revealed that the (PXX)3P motif was however critical for the Nef induced enhancement of Hck enzymatic activity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Mark Harris
Keywords: Genetics
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-75881
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:40
Last Modified: 19 Nov 2019 17:40

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