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Virus-specific CD8(+) T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag206-216 and Gag241-249 epitope-specific CD8(+) T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8(+) T-cell responses induced in all the 90-120-Ia(+) macaques on SIV replication remains unknown. Here, we identified three CD8(+) T-cell epitopes, Nef9-19, Nef89-97, and Nef193-203, associated with 90-120-Ia. Nef9-19 and Nef193-203 epitope-specific CD8(+) T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8(+) T cells, indicating that these CD8(+) T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Nef proteins of simian and human immunodeficiency viruses are known to directly bind and downregulate the CD4 receptor of infected cells. Recent results suggest that residues forming an alpha-helix N-cap in the CD4 cytoplasmic domain play a role in binding of CD4 to human immunodeficiency virus type 1 Nef protein. We determined the dissociation constants between Nef and several CD4 peptides that contain or do not contain the respective alpha-helix N-cap. Further, we compared helical secondary structure content of these CD4 peptide variants by circular dichroism spectroscopy. We conclude that presence of an alpha-helix in CD4 cytoplasmic domain increases CD4 affinity to Nef. In addition, the amino acid sequence of residues forming the helix N-cap influences CD4 affinity to Nef, too. Finally, the structural changes induced in Nef and CD4 upon binding to each other are investigated.

HIV-1 antigen Nef, Green Fluorescent Protein (GFP), and the fusion protein GFP-Nef ("Green Fluorescent Nef") were synthesized in bacterial cell-free system with continuous supply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane during incubation, the so-called continuous-exchange cell-free (CECF) system. The identity of synthesized proteins was confirmed by electrophoretic mobility, Western blotting, and GFP fluorescence. The system produced several nanomoles (hundreds of microg) of each protein per ml of the reaction mixture. Construction of GFP-fused proteins is considered as a general strategy for visualization and monitoring of cell-free production of the proteins that have no easily testable functions., (Copyright 2001 Academic Press.)

A growing body of literature suggests that the HIV accessory proteins Nef and Vpr could be involved in depletion of CD4(+) and non-CD4(+) cells and tissue atrophy, and in delaying the death of HIV-infected cells. Cell depletion is likely to be predominantly a bystander effect because the number of cells dying far outnumbers HIV-infected cells and is not confined to CD4(+) cells. The myristylated N-terminal region of Nef has severe membrane disordering properties, and when present in the extracellular medium causes rapid lysis in vitro of a wide range of CD4(+) and non-CD4(+) cells, suggesting a role for extracellular Nef in the depletion of bystander cells. A direct role for HIV-1 Nef in cytopathicity is supported by studies in HIV-infected Hu Liv/Thy SCID mice, in transgenic mice expressing nef gene alone, and in rhesus macaques infected with SIV/HIV chimeric virus containing HIV-1 nef. The N-terminal region of Nef has been directly implicated in development of simian AIDS. Extracellular Vpr and C-terminal fragments of Vpr cause membrane permeabilization and apoptosis of a wide range of CD4(+) and non-CD4(+) cells, and could also contribute to depletion of bystander cells. A direct in vivo role for Vpr in thymocyte depletion, thymic atrophy, and nephropathy is suggested in studies with vpr transgenic mice. Intracellular Nef and Vpr could help HIV-infected cells evade cell death by inhibiting apoptosis of infected cells and by avoiding virus-specific CTL response. Nef and Vpr are potential targets for therapeutic intervention and vaccine development, and strategies that prevent the death of bystander cells while promoting the early death of HIV-infected cells could arrest or retard progression to AIDS., (Copyright 2000 Academic Press.)

In addition to playing a crucial role in the pathogenesis of AIDS, HIV nef induces down-regulation of CD4 expression and TCR signaling and also regulates the sorting pathway in host T cells. To elucidate the Nef function in HIV progression, we searched for a cellular component which interacts with Nef. A human cDNA encoding a novel acyl-CoA thioesterase (hACTE-III) was isolated as an HIV nef-binding protein by yeast two-hybrid system. hACTE-III is homologous to E. coli thioesterase II but to none of the mammalian thioesterases and therefore belongs to a new type. hACTE-III exhibits enzymatic specificity for a broad range of fatty acyl-CoAs. The hACTE-III-binding region within Nef is localized in the central region (amino acids 109-152). hACTE-III greatly enhances its enzymatic activity upon direct binding to Nef. Considering that either Nef-overexpression or impaired fatty acid regulation induces alteration of subcellular morphology, the augmented hACTE-III function by Nef-binding might induce dysfunction of T cells.

The Nef protein of human immunodeficiency type 1 (HIV-1) has been implicated in diverse intracellular functions; however, extracellular functions have been less studied. Nef and the N-terminus of Nef possess membrane-perturbing and fusogenic activities in artificial membranes that also cause cytotoxicity to human cells, including lymphocytes. The present study investigates the toxicity of HIV-1 Nef peptides employing yeast and bacterial cells. The N-terminal portion of Nef was found to cause cell killing in Escherichia coli and in a variety of yeast cells. This activity was enhanced by myristylation of the Nef N-terminus, a modification that did not lead to toxicity in a control peptide. Cell death in yeast was due to permeabilization of the cell membrane as determined by the propidium iodide uptake of peptide-treated cells. Extracellular Nef, or its breakdown products, may have effects similar to the Nef peptides described here and could be responsible, at least in part, for the death of cells in lymphoid tissues during AIDS. Assays using yeast or bacteria are convenient, inexpensive, and robust and should be useful in further analysis and screening of inhibitors of this activity associated with HIV-1 Nef.

We prepared a series of nef mutant HIV-1 with a frameshift mutation at the Xho I site by up to 50 serial transfers into MT-4 cells. Here, we studied revertants. Immunofluorescence using an anti-Nef monoclonal antibody revealed that cells first became Nef antigen-positive at the 23rd passage. The percentage of Nef antigen-positive cells gradually increased and reached almost 100% by the 27th passage. The sequence of the provirus in the cells supported the generation of a revertant. This revertant mutated at the site immediately after the initially introduced frameshift mutation. This resulted in the substitution of only three amino acids and the insertion of two, which restored the proline-rich domain, a conserved region believed essential to viral replication, at the middle of Nef. Thus, the growth dominance of the revertant virus, compared with the original nef mutant, was directly demonstrated in vitro using serial passages consisting of mixed HIV-1 populations in a single cell.

Previously we reported that Nef protein from human immunodeficiency virus (HIV) induces proliferation of human peripheral blood mononuclear cells (PBMC). Herein, we show that HIV replicates in cells activated by Nef. PBMC proliferated in response to Nef and were capable of producing significant amounts of infectious virus upon infection. Polyclonal antibodies to Nef synthetic peptides blocked proliferation and neutralized infection. Characteristic of superantigens, T-cell proliferation in response to Nef required antigen-presenting cells and showed CD4 Vb preferences as previously shown. These findings suggest that Nef is a virally encoded T-cell superantigen and, as such, may be vital in the establishment of HIV infection in a new host and in subsequent disease pathogenesis.

Overlapping peptides corresponding to the entire Nef (HIVBRU) sequence were tested for their relative abilities to block binding of staphylococcal enterotoxins (SEs) to Raji cells. An internal sequence, Nef(123-160), blocked binding of two highly homologous SEs, SEA and SEE, while it was less effective against SEB and SEC1. Nef(123-160) bound directly class II DR antigens, as assessed by specific antibodies, and its binding was significantly inhibited by SEE, and also by SEA to a lesser degree. Purified Nef inhibited specific binding of SEA to Raji cells in a dose-dependent manner. Nef induced IL 2 production and proliferation by human mononuclear cells. Definitive expansion of specific V beta T cell populations was not observed, possibly due to lesser mitogenic activity of Nef relative to SEs. Thus, Nef may have mitogenic activity similar to that of superantigens.

A comparison of the nucleotide sequence of the human thyrotropin receptor (hTSH-R) with that of HIV-1 revealed 61% homology between a 161 base pair region encoding a unique portion of the hTSH-R and an immunogenic HIV-1 regulatory protein, nef. Amino acid analysis of this region shows 27% homology, including a segment in which 7/10 consecutive amino acids are identical. Sera from rabbits successfully immunized with a 16 amino acid portion of the hTSH-R (352-367, p1) was assessed for reactivity against a partially homologous nef peptide (nef-1) by ELISA, with a finding five-fold higher post-immunization values compared to pre-immune sera. The specificity of this response was verified with Western blot, using recombinant nef protein. An ELISA using nef-1 gave 64% higher values with sera from Graves' disease patients than with normal controls. This homology and immunologic cross-reactivity suggests an avenue through which a shared immune response against an HIV-1 related retrovirus could play a role in the pathogenesis of Graves' disease.