Apoptosis enhancement by the HIV-1 Nef protein

The HIV-1 nef gene, essential for AIDS pathogenesis, encodes a 27-kDa protein (Nef) whose biochemical and biological functions are unclear. It has been suggested that Nef expression contributes to the T cell depletion observed during the disease by promoting their apoptosis. We report that in CD4(+) human lymphoblastoid cell lines transfected with the nef cDNA obtained from three different HIV-1 strains, expression of the Nef protein enhances and accelerates the response to four unrelated apoptotic agents (staurosporine, anisomycin, camptothecin, and etoposide) but not to an anti-Fas agonist Ab. Nef reduces the expression of the anti-apoptotic proteins Bcl-2 and Bcl-X(L) and induces a striking enhancement of apoptotic hallmarks, including mitochondrial depolarization, exposure of phosphatidylserine on the cell surface, activation of caspase-3, and cleavage of the caspase target poly(ADP-ribose) polymerase. Interestingly, the peptide Z-Val-Ala-DL-Asp-fluoromethylketone (a broad-spectrum caspase inhibitor) reduces, but does not abolish, phosphatidylserine exposure, suggesting that Nef also activates a caspase-independent apoptotic pathway. Surprisingly, Nef expression increases DNA degradation but without causing oligonucleosomal fragmentation. An increased apoptotic response and down-modulation of Bcl-2/Bcl-X(L) following Nef expression are observed also in NIH-3T3 fibroblasts. These data show that Nef enhances programmed cell death in different cell types by affecting multiple critical components of the apoptotic machinery independently from the Fas pathway.     

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background

The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region.

Results

The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF??AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef??s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck.

Conclusion

Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.     

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.     

Remote protein homology detection using recurrence quantification analysis and amino acid physicochemical properties

Remote homology detection refers to the detection of structure homology in evolutionarily related proteins with low sequence similarity. Supervised learning algorithms such as support vector machine (SVM) are currently the most accurate methods. In most of these SVM-based methods, efforts have been dedicated to developing new kernels to better use the pairwise alignment scores or sequence profiles. Moreover, amino acids’ physicochemical properties are not generally used in the feature representation of protein sequences. In this article, we present a remote homology detection method that incorporates two novel features: (1) a protein's primary sequence is represented using amino acid's physicochemical properties and (2) the similarity between two proteins is measured using recurrence quantification analysis (RQA). An optimization scheme was developed to select different amino acid indices (up to 10 for a protein family) that are best to characterize the given protein family. The selected amino acid indices may enable us to draw better biological explanation of the protein family classification problem than using other alignment-based methods. An SVM-based classifier will then work on the space described by the RQA metrics. The classification scheme is named as SVM-RQA. Experiments at the superfamily level of the SCOP1.53 dataset show that, without using alignment or sequence profile information, the features generated from amino acid indices are able to produce results that are comparable to those obtained by the published state-of-the-art SVM kernels. In the future, better prediction accuracies can be expected by combining the alignment-based features with our amino acids property-based features. Supplementary information including the raw dataset, the best-performing amino acid indices for each protein family and the computed RQA metrics for all protein sequences can be downloaded from http://ym151113.ym.edu.tw/svm-rqa.     

Molecular design, functional characterization and structural basis of a protein inhibitor against the HIV-1 pathogenicity factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as "wrapping Nef", is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.     

A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.     

Thymosin alpha 1: amino acid homology with peptide T from the human immunodeficiency virus envelope

Thymosin alpha 1 has many effects on immune function and its absence in primary immunodeficiency states produce a clinical presentation similar to the one encountered in acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus (HIV), the etiologic agent of AIDS, binds to T4 helper/inducer lymphocytes through specific surface receptors which include the CD4 glycoprotein. Octapeptide T, a component of the HIV envelope, mediates the binding of HIV to its receptor. In this report, we draw attention to the similarity between the amino acid sequence of thymosin alpha 1 and peptide T and its analogues. This similarity can produce a cross-reactivity between thymosin alpha 1 and HIV and may be a factor in the pathophysiology of the acquired immuno-deficiency syndrome.     

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.     

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.     

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.     

Extracellular HIV-1 Nef protein modulates lytic activity and proliferation of human CD8+ T lymphocytes

The effect of extracellular HIV Nef (exNef) protein on the induction of lytic activity and proliferation of CD8+T lymphocytes from 18 donors was studied. At 10 ng/ml, exNef-induced a 2- to 8-fold enhancement of basal lytic activity in cells from all donors in an allogeneic induction assay, whereas it was ineffective at 100ng/ml. The extent of enhancement was inversely correlated with the basal level of lytic activity without exNef. Only in combination with PHA did both exNef concentrations stimulate proliferation, and in a manner inversely related to the effect of PHA alone. Thus, concentrations of exNef commonly found in sera of HIV-infected patients were found to modulate the induction of lytic activity and proliferation of CD8+ T lymphocytes in vitro, to an extent strongly dependent on the quite variable responsiveness of each donor. These findings point to Nef as a potential agent for modulating CD8+ T cell function in pathogenesis and therapy.     

Molecular evolution of protein: Internal homology in the amino acid sequence of calmodulin

Calmodulin is one of the calcium-binding proteins and is distributed widely in eukaryotes. The amino acid sequences were studied of calmodulin taken from bovine brain, scallop (Patinopecten), sea anemone (Metridium senile) and tetrahymena (Tetrahymena pyriformis). One notable feature of the primary structure of calmodulin is its internal homology. It can be subdivided into four domains with similar amino acid sequences. This homology implies that the primary structure of calmodulin has been elongated twice by intragenic duplication. Using this intragenic duplication model, the amino acid sequences of calmodulin from those four species were analyzed in detail. This kind of approach has proved very useful for investigation of the origin and evolution of this protein.     

HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution

The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV(mac239) Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.     

Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein

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 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

Dynamics of Physical Interaction between HIV-1 Nef and ASK1: Identifying the Interacting Motif(S)

FasL mediated preferential apoptosis of bystander CTLs while protection of infected CD4⁺T cells remains one of the hallmarks of immune evasion during HIV infection. The property of infected host cells to evade cell-autonomous apoptosis emanates from ability of HIV-1Nef -protein to physically interact with ASK-1 and thereby inhibit its enzymatic activity. The specific domains of HIV-1Nef through which it may interact with ASK1 and thereby impair the ASK1 activity remain unidentified so far and represent a major challenge towards developing clear understanding about the dynamics of this interaction. Using mammalian two hybrid screen in association with site directed mutagenesis and competitive inhibitor peptides, we identified constituent minimal essential domain (152 DEVGEANN 159) through which HIV-1Nef interacts with ASK1 and inhibits its function. Furthermore our study also unravels a novel alternate mechanism underlying HIV-1 Nef mediated ASK1 functional modulation, wherein by potentiating the inhibitory ser⁹⁶⁷ phosphorylation of ASK1, HIV-1Nef negatively modulated ASK1function.