Section 9
Chapter 8,789

Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines

Greenway, A.; Azad, A.; McPhee, D.

Journal of Virology 69(3): 1842-1850


ISSN/ISBN: 0022-538X
PMID: 7853525
Accession: 008788509

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Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD-4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor downregulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef 27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3. as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56-lck, CD4, p53, and p44-mapk/erk1, all of which are intimately involved in intracellular 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 proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation 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 treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56-lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased 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 alternate 25-kDa isoform of Nef (Nef 25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56-lck. Also, proliferation and posttranslational modification of p56-lck in response to IL-2 stimulation were not profoundly affected 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 Nef 25 resulted in decreased expression of c-myb. The effect of HIV-1 Nef on cell activation and proliferation may partly be explained by its interaction with specific cellular proteins. Interaction of Nef with these proteins may modulate their activity such that the cellular response to antigen or cytokines is severely altered resulting in the profound immunodeficiency characteristic of HIV infection.

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