Research objectives and content
A functional Nef gene is critical for the capacity of primate lentiviruses (HIV-1, HIV-2, and SlVs) to cause AIDS, but the relevant cellular functions and the molecular mechanisms of action of Nef are still unclear. Recent evidence, in particular the physical and functional SH3 domain-mediated binding of Nef to host cell protein tyrosine kinases, indicate that Nef functions by modulating cellular signal transduction pathways. The Src-family tyrosine kinases Hck and Lyn bind tightly to Nef, and in the case of Hck this interaction has been shown to result in induction of the catalytic activity of this tyrosine kinase leading to cellular activation and malignant transformation. Since Hck and Lyn are highly expressed in myeloid cells, these observations suggest that these proteins may be important mediators of the pathogenic function of Nef in HlV-infected monocyte/ macrophages in vivo. Functional studies on Nef-mutants which lack a functional SH3-binding site also suggest an important role for Nef/SH3-interactions in other cell types, including T cells, the principal target cells of HIV infection. However, despite a number of host cell proteins suggested to interact with Nef, a protein expressed in T-cells and showing Hck-like high-affinity SH3-mediated binding to Nef remains to be identified. Discovery of such a protein would be a major advance in understanding the biology of Nef and pathogenesis of HIV infection. Training content (objective, benefit and expected impact)
We have developed and successfully validated a system based on screening of gt11 lambda phage cDNA expression libraries with radiolabeled recombinant GST-Nef fusion protein. The aim of this postdoctoral project and the subject of this proposal is to utilize this system to identify and further characterize the relevant SH3-protein(s) associated with Nef in T-cells. Subsequently, novel molecular techniques will be applied for the study on protein-protein interactions between the HIV-1 encoded Nef and cellular partners, both in vitro and in vivo. Developing a detailed understanding of these issues could lead to new therapies for AIDS as well as novel aspects of cellular signal transduction mechanisms. This project will provide me (the applicant) with knowledge on new techniques and with experience on research in a highly competitive research field. The theoretical and practical skills mastered during this TMR period together with the experience acquired during my PhD will hopefully enable me to contribute to the ungoing research efforts to bring an end to the AIDS epidemic.
Links with industry / industrial relevance (22)
The understanding of the function of Nef at the molecular level may have significant relevance for the industry by enabling the development of novel anti-HlV therapies and vaccines. Together with industrial partners, Prof. Saksela is involved in the characterization of inhibitors of Nef function.