HIV-Nef enhances interleukin-2 production and phosphatidylinositol 3-kinase activity in a human T cell line

Objective: The Nei protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examin e the effects of Nef on T lymphocyte activation and associated signalling e vents. Design: A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, a nd either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates sign al transduction pathways and results in IL-2 production and IL-2 receptor a lpha-chain (CD25) expression. Cellular responses were examined in cells exp ressing either Nef or an irrelevant control protein. Methods: Activation of signalling was assessed by immunoblot analysis, or b y in-vitro phosphatidylinositol 3-kinase (P13K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expre ssion was examined using flow cytometry. Results: Infection of cells with recombinant vaccinia expressing HIV-nef re sulted in a marked increase in the production of IL-2 when cells were activ ated. The enhanced IL-2 response was accompanied by an increase in the leve l of P13K activity. IL-2 production remained sensitive to inhibition with t he P13K competitive inhibitor Ly294002, and to the fungal macrolide, rapamy cin. In contrast, CD25 expression was not affected, and there were no measu rable changes to nuclear factor kappa B (NF kappa B) activation pathways. Conclusion: Enhanced IL-2 production in stimulated T cells expressing HIV-N ef is associated with increased activation of PI3K-dependent signalling pat hways. The results support a model in which Nef affects HIV disease progres sion by distorting T cell responses. (C) 2000 Lippincott Williams & Wilkins .