Mechanism and mediated mechanistic analysis of HIV Nef

Simmons, Alison (2004). Mechanism and mediated mechanistic analysis of HIV Nef. PhD thesis The Open University.



The nef gene of HIV is required for complete viral pathogenicity. As an accessory gene with no known paradigm its principle function has been enigmatic. Nef is capable of a variety of in vitro effects including CD4 downregulation, MHC I down regulation, alteration of T cell signalling activity and enhancement of viral infectivity. The effect of Nef on host CD4 T cell signalling paths is intriguing as it is the earliest expressed HIV gene and because HIV replicates to high levels in activated host cells. An effect of Nef on T cell activation state could dictate host T cell fate tipping the balance between productive viral infection and post-integration latency. Although Nef can form in vitro interactions with a variety of T cell signalling proteins in vivo evidence for signalling path activation has been inconclusive mainly due to the limitations of the experimental systems used to address the question.
An in vivo role for Nef in alteration of T cell activation state should lead to gene expression changes in the host cell. Large scale gene expression profiling was used to monitor T cell gene expression after controlled expression of Nef using the tetracycline inducible system. Nef induces a widespread transcriptional program in T cells. Comparison of this profile with reference T ceil activation states reveals a strong identity to the gene expression profile of Jurkat activated by anti-CD3 cross-linking. Gene expression profiling was used to explore the mechanism by which Nef mediates this effect. After cyclosporin treatment the profiie was attenuated. A similar effect was observed after Nef expression in Jurkat T cells genetically deficient in ZAP-70 and functionally deficient in TCRÇ. Expression of a mutant defective in a functional SH3 binding domain had a stronger effect on attenuation of gene induction. Thus gene expression profiling demonstrates definitively Nef is capable of activating T cells at the transcriptional level and that this effect is initiated high in the T cell signalling path.
Within the Nef expression profile differential expression of multiple genes with the ability to affect virai replication was noted. In a few instances it was possible to demonstrate the Nef mediated changes in mRNA levels were functionally significant. Proteomic analysis of Nef effect on T cells was initiated. 2D gel analysis of T cell lysates after Nef expression demonstrated a differential regulation of proteins in T cell lysates. The mechanism by which Nef initiates T cell signalling was explored using a similar approach. The proteins interacting with Nef in lipid rafts were identified after immunoprécipitation of HA tagged Nef from this subcellular compartment. 2D gel analysis reveals the presence of Nef changes the content of lipid rafts and that Nef co-immunoprecipitates with several proteins in the raft fraction. The identity of these interactors obtained by peptide microsequencing should give insight into the molecular mechanism by which Nef induces a CD4 T cell transcriptional programme.

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