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1.
Biochem Biophys Res Commun ; 625: 122-127, 2022 10 15.
Article in English | MEDLINE | ID: mdl-35961135

ABSTRACT

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.


Subject(s)
Gene Products, nef , HIV-1 , Death-Associated Protein Kinases , Gene Products, nef/physiology , HIV-1/genetics , Humans , Protein Kinases , Viral Proteins , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/genetics
2.
PLoS Pathog ; 14(8): e1007269, 2018 08.
Article in English | MEDLINE | ID: mdl-30125328

ABSTRACT

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.


Subject(s)
CD4-Positive T-Lymphocytes/virology , Gene Products, nef/physiology , HIV-1/physiology , Lymphoid Tissue/virology , Membrane Proteins/metabolism , Virus Replication/genetics , Animals , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Colobus/virology , HEK293 Cells , Humans , Jurkat Cells , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Simian Immunodeficiency Virus/genetics
4.
Curr HIV Res ; 9(7): 474-89, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22103831

ABSTRACT

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.


Subject(s)
Gene Products, nef/physiology , HIV/pathogenicity , Virion/pathogenicity , Virus Replication/physiology , Animals , Gene Products, nef/genetics , HIV/genetics , HIV-1 , Humans , Macaca mulatta , Mutation
5.
Curr HIV Res ; 9(7): 514-23, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22103835

ABSTRACT

The multifunctional Nef protein of primate lentiviruses is commonly considered an early viral factor that down-modulates various receptors from the cell surface and modulates several signaling pathways to facilitate viral immune evasion and to render the cell conducive for viral replication. However, Nef also acts during the late stages of infection, e.g. by increasing the infectivity of progeny virions. Just recently, it has become clear that many primate lentiviruses that have been detected in about 40 different monkey and ape species also use Nef to antagonize tetherin (BST2/CD317), a cellular factor that inhibits virus release by tethering nascent viral particles to the cell surface. Exceptions are some simian immunodeficiency viruses (SIVs) infecting Cercopithecus monkeys that employ their accessory Vpu protein to counteract the restriction by tetherin. Furthermore, pandemic HIV-1 group M strains switched from Nef to Vpu and HIV-2 group A isolates from Nef to Env after zoonotic transmission from chimpanzees and sooty mangabeys, respectively, to antagonize the tetherin restriction in humans. These evolutionary switches were most likely enforced by a deletion in the cytoplasmic domain of the human tetherin orthologue that confers resistance to Nef. Here, we summarize some of our current knowledge about Nef-mediated tetherin antagonism.


Subject(s)
Gene Products, nef/physiology , HIV/physiology , Lentivirus Infections/immunology , Lentiviruses, Primate/physiology , Primates/virology , Animals , Antigens, CD/physiology , Biological Evolution , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/physiology , HIV-1/physiology , HIV-2/physiology , Humans , Lentiviruses, Primate/pathogenicity , Species Specificity , Viral Regulatory and Accessory Proteins/physiology , Virus Release/physiology
6.
Curr HIV Res ; 9(7): 531-42, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22103837

ABSTRACT

A bewildering number of host cell proteins associated with Nef can be found in the literature and in the public protein interaction databases. However, only in a few of these cases, including binding of Nef to certain Src homology-3 (SH3) domain proteins, is the interaction understood in any molecular detail or even known to be direct. Indeed, SH3 binding capacity by Nef is required for many of the other protein interactions to take place, suggesting that a large proportion of the latter is indirectly coupled to Nef via an SH3 protein. Accordingly, the proline-rich SH3 binding site, the "PxxP motif", is one of the key functional determinants of Nef. It is highly conserved among the lentiviral Nef proteins, and mutations disrupting it abrogate the majority of the known effects of Nef on host cell physiology. This review summarizes the current understanding as well as the outstanding gaps in our knowledge regarding the relevant SH3 protein partners and SH3-dependent cellular functions of Nef. The roles of these interactions in the pathogenesis of AIDS and their potential as targets for antiviral drug development are also discussed.


Subject(s)
Gene Products, nef/physiology , HIV/physiology , Shc Signaling Adaptor Proteins/physiology , Simian Immunodeficiency Virus/physiology , src Homology Domains , Acquired Immunodeficiency Syndrome/immunology , Animals , Gene Products, nef/chemistry , HIV/pathogenicity , Humans , Macrophages/metabolism , Simian Immunodeficiency Virus/pathogenicity , T-Lymphocytes/metabolism
7.
Angiology ; 61(7): 669-78, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20566577

ABSTRACT

Human immunodeficiency virus (HIV)-infected patients have increased rates of atherosclerotic cardiovascular diseases because the highly active antiretroviral therapy (HAART) decreased the morbidity and mortality of the disease. Endothelial dysfunction is possibly the most plausible link between HIV infection and related expression of cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on the endothelial cells. HIV-1 accessory protein negative regulate factor (Nef) has been shown to be very important for high virus replication and disease progression. Nef could upregulate the expression of ICAM-1 in the pathogenesis of HIV infection. Here, we provide evidence that the HIV-1 Nef can transcriptionally induce the expression of ICAM-1 in stable expressed Nef vascular endothelial cells. Nef-induced ICAM-1 upregulation requires the activation of the downstream kinase extracellular signal-regulated kinase (ERK). Flow cytometry (FCM) results showed that the percentage of ICAM-1 positive cells in Nef-expressed cells and control cells was (35.3% +/- 2.2%) and (12.5% +/- 0.8%), respectively (P < .01). Furthermore, inhibition of Nef activity by ERK mitogen-activated protein kinase (MAPK) inhibitor effectively blocked ICAM-1 upregulation, suggesting that ERK MAPK activation is an important initiating event in Nef-mediated ICAM-1 expression in Nef-expressed cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.


Subject(s)
Endothelial Cells/metabolism , HIV Infections/genetics , Intercellular Adhesion Molecule-1/biosynthesis , nef Gene Products, Human Immunodeficiency Virus/physiology , Blotting, Western , Cells, Cultured , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Expression Regulation/physiology , Gene Products, nef/physiology , HIV Infections/metabolism , HIV-1 , Humans , Intercellular Adhesion Molecule-1/genetics , Jurkat Cells/physiology , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Signal Transduction , Transcription, Genetic , Transfection , Up-Regulation/physiology
8.
Trends Cell Biol ; 20(3): 160-9, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20071173

ABSTRACT

p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in antiviral immunity will be pivotal to evaluate thoroughly the potential of agents that inhibit Pak as a new class of anti-viral therapeutics.


Subject(s)
Virus Diseases/physiopathology , p21-Activated Kinases/physiology , Adenoviridae/physiology , Amino Acid Motifs , Amino Acid Sequence , Apoptosis/physiology , Enzyme Activation , Gene Products, nef/physiology , Hepadnaviridae/physiology , Herpesviridae/physiology , Humans , Molecular Sequence Data , Poxviridae/physiology , Sequence Alignment , Virus Internalization , Virus Replication/physiology
9.
J Virol ; 83(22): 11673-81, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19726508

ABSTRACT

The Vpu accessory gene that originated in the primate lentiviral lineage leading to human immunodeficiency virus type 1 is an antagonist of human tetherin/BST-2 restriction. Most other primate lentivirus lineages, including the lineage represented by simian immunodeficiency virus SIVagm from African green monkeys (AGMs), do not encode Vpu. While some primate lineages encode gene products other than Vpu that overcome tetherin/BST-2, we find that SIVagm does not antagonize physiologically relevant levels of AGM tetherin/BST-2. AGM tetherin/BST-2 can be induced by low levels of type I interferon and can potently restrict two independent strains of SIVagm. Although SIVagm Nef had an effect at low levels of AGM tetherin/BST-2, simian immunodeficiency virus SIVmus Vpu, from a virus that infects the related monkey Cercopithecus cephus, is able to antagonize even at high levels of AGM tetherin/BST-2 restriction. We propose that since the replication of SIVagm does not induce interferon production in vivo, tetherin/BST-2 is not induced, and therefore, SIVagm does not need Vpu. This suggests that primate lentiviruses evolve tetherin antagonists such as Vpu or Nef only if they encounter tetherin during the typical course of natural infection.


Subject(s)
Chlorocebus aethiops/virology , Simian Immunodeficiency Virus/physiology , Animals , Blotting, Western , Cell Line , Gene Products, nef/physiology , Interferon alpha-2 , Interferon beta-1b , Interferon-alpha/pharmacology , Interferon-beta/pharmacology , Molecular Sequence Data , Recombinant Proteins , Retroviridae Proteins/genetics , Retroviridae Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/drug effects , Viral Regulatory and Accessory Proteins/physiology
10.
Am J Physiol Lung Cell Mol Physiol ; 297(4): L729-37, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19648286

ABSTRACT

Golgi dysfunction has been previously investigated as a mechanism involved in monocrotaline-induced pulmonary hypertension (PAH). In the present study, we addressed whether Golgi dysfunction might occur in pulmonary vascular cells in idiopathic PAH (IPAH) and whether there might be a causal relationship between trafficking dysfunction and vasculopathies of PAH. Quantitative immunostaining for the Golgi tethers giantin and p115 on human lung tissue from patients with IPAH (n = 6) compared with controls demonstrated a marked cytoplasmic dispersal of giantin- and p115-bearing vesicular elements in vascular cells in the proliferative, obliterative, and plexiform lesions in IPAH and an increase in the amounts of these Golgi tethers/matrix proteins per cell. The causality question was approached by genetic means using human immunodeficiency virus (HIV)-Nef, a protein that disrupts endocytic and trans-Golgi trafficking. Macaques infected with a chimeric simian immunodeficiency virus (SIV) containing the HIV-nef gene (SHIV-nef), but not the nonchimeric SIV virus containing the endogenous SIV-nef gene, displayed pulmonary arterial vasculopathies similar to those in human IPAH. Giantin and p115 levels and their subcellular distribution in pulmonary vascular cells in lungs of SHIV-nef infected macaques (n = 4) were compared with SIV-infected (n = 3) and an uninfected macaque control. Only macaques infected with chimeric SHIV-nef showed pulmonary vascular lesions containing cells with dramatic cytoplasmic dispersal and an increase in giantin and p115. Specifically, the HIV-Nef-positive cells showed increased giantin, p115, and the activated transcription factor PY-STAT3. These data represent the first test of the Golgi dysfunction hypothesis in IPAH and place trafficking and Golgi disruption in the chain of causality of pulmonary vasculopathies in the macaque model.


Subject(s)
Endothelium, Vascular/pathology , Gene Products, nef/physiology , Golgi Apparatus/pathology , Hypertension, Pulmonary/pathology , Macaca/virology , Pulmonary Artery/pathology , Vascular Diseases/pathology , Animals , Case-Control Studies , Disease Models, Animal , Endothelium, Vascular/metabolism , Fluorescent Antibody Technique , Golgi Apparatus/metabolism , Golgi Matrix Proteins , HIV Infections/metabolism , HIV Infections/pathology , HIV Infections/virology , HIV-1/isolation & purification , Humans , Hypertension, Pulmonary/metabolism , Membrane Proteins/metabolism , Pulmonary Artery/metabolism , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/isolation & purification , Vascular Diseases/metabolism
11.
J Immunol ; 183(4): 2415-24, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19620308

ABSTRACT

HIV-1 Nef has been reported to disrupt MHC class II (MHCII)-mediated Ag presentation by a dual strategy that comprises a reduction in cell surface levels of peptide-loaded mature MHCII molecules and a up-regulation of immature MHCII molecules. We show that Nef achieves relocation of MHCII away from the cell surface in monocytic cells by both delaying its transport to the cell surface and by accelerating endocytic removal of cell surface MHCII to a lysosomal compartment. Nef-induced MHCII endocytosis is cholesterol-sensitive but clathrin- and dynamin-independent. Internalized MHCII molecules traverse the early endosomal system and colocalize with pinocytic cargo before reaching lysosomes. Nef-triggered MHCII endocytosis requires Rab5 activity and lyst function, whereas lysosomal trafficking of internalized MHCII molecules requires Rab7 activity. We further show that a similar pathway can remove peptide-MHCII complexes from the surface of monocytic cells not expressing Nef. Our data suggest that Nef uses mechanisms involved in normal MHCII recycling and turnover to mediate the delivery of cell surface MHCII to a lysosomal destination. Thus, Nef-mediated endocytosis of MHCII provides a novel perspective on the regulation of normal MHCII trafficking.


Subject(s)
Cell Membrane/metabolism , Endocytosis/immunology , Gene Products, nef/physiology , HIV-1/immunology , HLA-D Antigens/metabolism , Signal Transduction/immunology , Animals , Cell Line , Cell Membrane/immunology , Cell Membrane/virology , Cells, Cultured , Endocytosis/genetics , HIV-1/genetics , HLA-D Antigens/biosynthesis , HLA-D Antigens/physiology , Humans , Lysosomes/immunology , Lysosomes/metabolism , Lysosomes/virology , Mice , Mice, Inbred C57BL , Signal Transduction/genetics , U937 Cells
12.
Nat Rev Microbiol ; 7(6): 467-76, 2009 06.
Article in English | MEDLINE | ID: mdl-19305418

ABSTRACT

In the subset of primate lentiviruses that contain a vpu gene - HIV-1 and its simian precursors - the Nef protein has lost the ability to down-modulate CD3, block T cell activation and suppress programmed death. Vpu counteracts a host restriction factor induced by the inflammatory cytokine interferon-alpha. I propose that the acquisition of vpu may have allowed the viral lineage that gave rise to HIV-1 to evolve towards greater pathogenicity by removing the selective pressure for a protective Nef function that prevents damagingly high levels of immune activation.


Subject(s)
Evolution, Molecular , HIV-1/pathogenicity , Animals , Gene Products, nef/metabolism , Gene Products, nef/physiology , HIV-1/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Human Immunodeficiency Virus Proteins/physiology , Humans , Models, Biological , Simian Immunodeficiency Virus/metabolism , Simian Immunodeficiency Virus/pathogenicity , Viral Regulatory and Accessory Proteins/metabolism , Viral Regulatory and Accessory Proteins/physiology
13.
Traffic ; 9(11): 1925-35, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18764822

ABSTRACT

The Nef protein of HIV-1 removes the immune costimulatory proteins CD80 and CD86 from the cell surface by a unique clathrin- and dynamin-independent, actin-based endocytic pathway that deploys coupled activation of c-src and Rac. In this study, we show that, similar to major histocompatibility complex class I (MHCI), Nef subsequently reroutes CD80 and CD86 to the Golgi region. However, not only are CD80/CD86 internalized by a different mechanism from MHCI but also the vesicular pathway of Golgi delivery for CD80/CD86 is distinct from that employed for MHCI. While MHCI passes through early endosomal and sorting compartments marked by Rab5/early embryonic antigen 1 and ADP ribosylation factor 6, respectively, CD80 and CD86 enter endocytic vesicles that do not acquire conventional early endosomal markers but remain accessible to fluid probes. Rather than being delivered to preexisting Rab11-positive recycling compartments, these vesicles recruit Rab11 de novo. Rab11 activity is also necessary for the delivery of CD80/CD86 in these transitional vesicles to the Golgi region. These data reveal an unusual pathway of endocytic vesicular traffic to the Golgi and its recruitment in a viral immune evasion strategy.


Subject(s)
B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Endocytosis , Gene Products, nef/physiology , Golgi Apparatus/metabolism , HIV-1/physiology , rab GTP-Binding Proteins/physiology , ADP-Ribosylation Factor 6 , Humans , Phosphatidylinositol 3-Kinases/metabolism , U937 Cells
14.
J Virol ; 82(16): 7758-67, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18524831

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.


Subject(s)
Gene Expression Regulation, Viral , Gene Products, nef/biosynthesis , Gene Products, nef/physiology , Histocompatibility Antigens Class II/physiology , Adaptor Protein Complex 2/metabolism , Amino Acid Motifs , Antigen Presentation , Binding, Competitive , CD4-Positive T-Lymphocytes/metabolism , Cell Membrane/metabolism , Cytoplasm/metabolism , HeLa Cells , Histocompatibility Antigens Class II/chemistry , Humans , Leucine/chemistry , Leukocytes, Mononuclear/metabolism , Models, Biological
16.
J Virol ; 82(6): 2918-29, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18094167

ABSTRACT

Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and betaPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or betaPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a betaPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or betaPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex.


Subject(s)
Gene Products, nef/physiology , HIV-1/physiology , Proto-Oncogene Proteins c-vav/metabolism , p21-Activated Kinases/metabolism , Base Sequence , Cell Membrane/metabolism , Protein Binding , RNA Interference
18.
J Virol ; 81(20): 11426-40, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17670831

ABSTRACT

Primary CD4(+) T lymphocytes, supporting in vitro human immunodeficiency virus type 1 (HIV-1) replication, are destined to die by apoptosis. We explored the initial molecular events that act upstream from mitochondrial dysfunction in CD4(+) T lymphocytes exposed to the HIV-1(LAI) strain. We tracked by immunofluorescence the cells expressing the p24 viral antigen and used Percoll density gradients to isolate a nonapoptotic CD4(+) T-cell subset with a high inner mitochondrial transmembrane potential (DeltaPsim) but no outer mitochondrial membrane (OMM) rupture. In most p24(+) (but not bystander p24(-)) cells of this subset, the lysosomes were undergoing limited membrane permeabilization, allowing the lysosomal efflux of cathepsins (Cat) to the cytosol. This was also induced by HIV-1 isolates from infected patients. Using pepstatin A to inhibit Cat-D enzymatic activity and Cat-D small interfering RNA to silence the Cat-D gene, we demonstrate that once released into the cytosol, Cat-D induces the conformational change of Bax and its insertion into the OMM. Inhibition of Cat-D activity/expression also conferred a transient survival advantage upon productively HIV-1-infected cells, indicating that Cat-D is an early death factor. The transfection of activated CD4(+) T lymphocytes with a Nef expression vector rapidly induced the permeabilization of lysosomes and the release of Cat-D, with these two events preceding OMM rupture. These results reveal a previously undocumented mechanism in which Nef acts as an internal cytopathic factor and strongly suggest that this viral protein may behave similarly in the context of productive HIV-1 infection in CD4(+) T lymphocytes.


Subject(s)
Apoptosis , CD4-Positive T-Lymphocytes/virology , Gene Products, nef/physiology , HIV-1/pathogenicity , Lysosomes/virology , CD4-Positive T-Lymphocytes/pathology , Cells, Cultured , HIV Infections/pathology , Humans , Intracellular Membranes/virology , Lysosomes/ultrastructure , Permeability
19.
Curr Drug Discov Technol ; 4(1): 12-23, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17630924

ABSTRACT

Human immunodeficiency virus (HIV)-1 Nef is a regulatory protein critically involved in AIDS pathogenesis. We previously demonstrated that extracellular Nef is efficiently internalized by human primary monocyte-derived macrophages (MDMs), thereby activating a number of transcription factors including STATs, MAPKs, IRF-3, and NF-kappaB. Such an activation state leads to the release of inflammatory factors whose paracrine effects deserve deep consideration. Here, we demonstrate that quiescent CD4 lymphocytes undergo cell activation when cultivated in supernatants from autologous MDMs treated with extracellular wt Nef but not with its counterpart mutated in the (72)PxxP(75) polyproline domain. Of a pathogenetic relevance, this effect coupled with the sensitization of quiescent CD4 lymphocytes to HIV-1 infection. By microarray assay, we found that the CCL24/eotaxin-2 gene was up-regulated in MDMs treated with wt Nef but not with the (72)AxxA(75) mutant. In addition, the higher transcription activity correlated with a significant increase of the CCL24/Eotaxin-2 release. Finally, we observed that anti-CCL24/eotaxin-2 antibodies efficiently neutralized the stimulatory effect on CD4 lymphocytes of supernatants from MDMs treated with extracellular Nef. Overall, these data support the idea that CCL24/eotaxin-2 is part of the mechanism of CD4 lymphocyte activation paracrinally induced by Nef.


Subject(s)
Chemokines, CC/physiology , Gene Products, nef/physiology , HIV-1/pathogenicity , Adult , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Chemokine CCL24 , Chemokines, CC/biosynthesis , Gene Products, nef/genetics , Gene Products, nef/metabolism , HIV-1/physiology , Humans , Leukocytes, Mononuclear , Lymphocyte Activation , Macrophages/metabolism , Macrophages/virology , Male , Mutation , Oligonucleotide Array Sequence Analysis , Paracrine Communication , Peptides/genetics , Transcription, Genetic , Virus Replication , nef Gene Products, Human Immunodeficiency Virus
20.
Proc Natl Acad Sci U S A ; 104(16): 6812-7, 2007 Apr 17.
Article in English | MEDLINE | ID: mdl-17412836

ABSTRACT

Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We now show that dynamin 2 (Dyn2), a key regulator of vesicular trafficking, is a binding partner of Nef that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, because it was diminished in clathrin-depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation but not clathrin-independent endocytosis. Together, these findings imply that the infectivity enhancement activity of Nef depends on Dyn2- and clathrin-mediated membrane invagination events.


Subject(s)
Dynamin II/physiology , Gene Products, nef/physiology , HIV-1/pathogenicity , Animals , COS Cells , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Gene Products, nef/deficiency , Gene Products, nef/genetics , HIV Infections/metabolism , HIV-1/physiology , Humans , Jurkat Cells , Protein Binding/physiology , Virion/pathogenicity , Virion/physiology , nef Gene Products, Human Immunodeficiency Virus
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