[Show abstract][Hide abstract] ABSTRACT: The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function
is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates
a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with
p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef
also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we
hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with
Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely
due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional
activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking
Journal of Virology 04/2002; 76(6):2692-702. DOI:10.1128/JVI.76.6.2692-2702.2002 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral protein R (Vpr), one of the accessory gene products encoded by the human immunodeficiency virus type 1 (HIV-1) genome, has a number of functions, including causing a growth arrest of HIV-1-infected cells and possibly the death of uninfected bystander cells. In microbial assay systems, the C-terminal portion of Vpr can cause cell death when added externally, and when expressed in yeast it causes growth arrest. In this study we have sought to obtain inhibitors of the Vpr functions that affect the microbial systems. Our first approach employed peptide display, which identified a number of sequences, including a heptapeptide sequence, GETRAPL, involved in binding to the C-terminus of Vpr. To determine whether GETRAPL could block the extracellular cytocidal activity of Vpr, the heptapeptide was synthesized and found to have some blocking activity in microbial assays. A second approach led to the finding that melittin inhibitors had activity against Vpr extracellular activities. In a third approach, compounds were tested against the Vpr-induced growth arrest. A number of compounds were found to abrogate the growth arrest, and some also inhibited Vpr's extracellular activity.
[Show abstract][Hide abstract] ABSTRACT: The Human Immunodeficiency Virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis. In order to determine more about the effects of Nef on basic cellular functions Nef was produced in yeast under a variety of conditions and in multiple cell types. Production of Nef caused cell death in acutely copper- or heat-stressed diploid cells. The N-terminal melittin-like region of Nef was involved in toxicity since a Trp5-->Ala change within Nef change caused increased toxicity. However, another determinant was also involved in toxicity since production of Nef20-206 was also still toxic. In each of these Nef-producing cells there was coincident membrane permeabilisation. These results suggest the possibility of a novel yeast bioassay for Nef inhibitors and that cells producing high levels of Nef may be selectively killed by stress.
Biochemistry and molecular biology international 10/1998; 46(2):277-86. DOI:10.1080/15216549800203792
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast. In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals, suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues during HIV infection.
[Show abstract][Hide abstract] ABSTRACT: It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.
Journal of Virology 11/1996; 70(10):6701-8. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 CD4+ 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 down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) 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 p56lck, CD4, p53, and p44mapk/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 Nef27 protein was introduced directly into PBMC by electroporation. Nef27-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 p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck 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 Nef27, the alternate 25-kDa isoform of Nef (Nef25) 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 p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)
Journal of Virology 04/1995; 69(3):1842-50. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Continuing controversy surrounds the cellular effects of the Nef protein of HIV-1, a nonstructural protein expressed by most isolates. Highly purified protein isoforms of MW 27 kDa (Nef 27) and 25 kDa (Nef 25), produced in Escherichia coli by translation from the first and second start codons of HIV-1 nef clone pNL4.3, respectively, were introduced into cells by a sophisticated electroporation technique which uses electric field rather than electric charge to transfer macromolecules across cell membranes. Electroporation of Nef 27 reduced the expression of cell surface CD4 by 30-50%, as measured by flow cytometry, on phytohemagglutinin (PHA)-activated PBMC as well as on a variety of CD4+ T-cell lines (MT-2, CEM, and Jurkat). Reduction in surface CD4 was observed in all cells of the CD4+ T-cell lines but only in the CD4+ cells of the mixed PBMC population. Electroporation of Nef 27 into MT-2 cells and PHA-activated PBMC also reduced the expression of IL-2R to background levels. Other cell surface antigens analyzed such as CD2, CD7, or transferrin receptor (TfR) were not affected by the introduction of HIV-1 Nef 27. In contrast to the effects of Nef 27, electroporation of Nef 25 into cells at equivalent concentrations did not affect the surface expression of CD4 and IL-2R. These data show that the HIV-1 clone pNL4.3 Nef 27 but not the Nef 25 isoform specifically decreases expression of two cell surface receptors important for antigen recognition of MHC class II antigens and for cell proliferation. Production of Nef 27 during HIV-1 infection of cells of the immune system may contribute to immunodeficiency even in the absence of direct viral cytopathic effects.