HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF PROTEIN INHIBITS ACTIVATION PATHWAYS IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS AND T-CELL LINES

Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the los s of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from per ipheral blood mononuclear cells (PBMC) and CD4(+) T-cell lines. As bot h CD4 and the IL-2 receptor play crucial roles in antigen-driven helpe r T-cell signalling and T cell proliferation, respectively, the role o f Nef in the viral life cycle may be to perturb signalling pathways em anating from these receptors. However, the intracellular targets for N ef that result in receptor downregulation are unknown. Using a recombi nant glutathione S-transferase-full-length 27 kDa Nef (Nef 27) fusion protein, produced in Escherichia coli by translation from the first st art codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe c ytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction, vith at least seven host cell protein species ranging from 24 to 75 kD a. Immunoblotting identified four of these proteins as p56(lck), CD4, p53, and p44(mapk/erkl), all of which are intimately involved in intra cellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef 27 protein was introduced directly into PBMC by electroporation. Nef 27-treated PBMC showed reduced prolifera tive responsiveness to exogenous recombinant IL-2. Normally, stimulati on of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56(lck) activity and corresponding posttranslational modification of p56(lck). These changes were also inhibited by treatm ent of PBMC with Nef, suggesting that Nef interferes with activation o f p56(lck) and as a consequence of signalling via the IL-2 receptor. F urther evidence for Nef interfering with cell proliferation was the de creased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef 27, the alternat e 25-kDa isoform of Nef (Nef 25) produced by translation from the seco nd start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) w as shown to interact with only three cellular proteins of approximatel y 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identi fied as p56(lck). Also, proliferation and posttranslational modificati on of p56(lck) in response to IL-2 stimulation were not profoundly aff ected by treatment of PBMC with Nef 25 compared with Nef 27. However, Nef 25 did exhibit some activity since treatment of MT-2 cells with Ne f 25 resulted in decreased expression of c-myb. The effect of HIV-1 Ne f on cell activation and proliferation may partly be explained by its interaction with specific cellular proteins. Interaction of Nef with t hese proteins may modulate their activity such that the cellular respo nse to antigen or cytokines is severely altered resulting in the profo und immunodeficiency characteristic of HIV infection.