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Didier Trono - One of the best experts on this subject based on the ideXlab platform.

  • the hiv 1 Nef Protein and phagocyte nadph oxidase activation
    Journal of Biological Chemistry, 2002
    Co-Authors: Frederik Vilhardt, Didier Trono, Olivier Plastre, Makoto Sawada, Kazuo Suzuki, Maciej Wiznerowicz, Etsuko Kiyokawa, Karlheinz Krause
    Abstract:

    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.

  • HIV-1 Nef Protein binds to the cellular Protein PACS-1 to downregulate class I major histocompatibility complexes
    Nature Cell Biology, 2000
    Co-Authors: Vincent Piguet, Aram Mangasarian, Christelle Borel, Nicolas Demaurex, Gary Thomas, Didier Trono
    Abstract:

    Major-histocompatibility-complex (MHC) Proteins are used to display, on the surface of a cell, peptides derived from foreign material - such as a virus - that is infecting that cell. Cytotoxic T lymphocytes then recognize and kill the infected cell. The HIV-1 Nef Protein downregulates the cell-surface expression of class I MHC Proteins, and probably thereby promotes immune evasion by HIV-1. In the presence of Nef, class I MHC molecules are relocalized from the cell surface to the trans-Golgi network (TGN) through as-yet-unknown mechanisms. Here we show that Nef-induced downregulation of MHC-I expression and MHC-I targeting to the TGN require the binding of Nef to PACS-1, a molecule that controls the TGN localization of the cellular Protein furin. This interaction is dependent on Nef's cluster of acidic amino acids. A chimaeric integral membrane Protein containing Nef as its cytoplasmic domain localizes to the TGN after internalization, in an acidic-cluster- and PACS-1-dependent manner. These results support a model in which Nef relocalizes MHC-I by acting as a connector between MHC-I's cytoplasmic tail and the PACS-1-dependent Protein-sorting pathway.

  • The Nef Protein of HIV-1 associates with rafts and primes T cells for activation
    Proceedings of the National Academy of Sciences of the United States of America, 2000
    Co-Authors: Jen-kuei Wang, Etsuko Kiyokawa, Eric Verdin, Didier Trono
    Abstract:

    The Nef Protein is an important virulence factor of primate lentiviruses, yet the mechanisms by which it exerts this influence are imperfectly understood. Here, using an inducible system, we demonstrate that Nef increases IL-2 secretion from T cells stimulated via CD3 or CD28. This effect requires the conservation of the Nef myristoylation signal and SH3-binding proline-based motif. Together with several Proteins involved in the initiation and propagation of T cell signaling, Nef associates with membrane microdomains known as rafts. The Nef-mediated superinduction of IL-2 reflects the activation of both NFAT and NFkappaB. Accordingly, Nef also enhances HIV-1 transcription in response to CD3 or CD28 stimulation. Nef-induced IL-2 hyperresponsiveness is also observed in primary CD4 lymphocytes. Overall, these data suggest that Nef acts at the level of rafts to prime T cells for activation. Likely consequences of this effect are the promotion of HIV-1 replication and the facilitation of virus spread.

  • The Nef Protein of primate lentiviruses.
    Reviews in medical virology, 1999
    Co-Authors: Vincent Piguet, Didier Trono
    Abstract:

    The Nef Protein of primate lentiviruses acts as an important virulence factor in vivo both in monkeys and in humans. Among a human cohort of long-term non-progressors, several Nef defective HIV1 viruses have been isolated, indicating that Nef may accelerate HIV progression and disease in humans. Additionally, a Nef-deleted SIV virus has low titres in rhesus monkeys and the animals develop AIDS at a much slower rate. In vitro, Nef can exert at least three kinds of effects: it downregulates CD4 and MHC class I, it stimulates virion infectivity and it alters signal transduction pathways. To accomplish this, Nef interacts with a series of cellular partners including CD4, components of the adaptor complexes AP-1 and AP-2, and several Protein kinases, Nef often functioning as a connector between targets and effectors. The high degree of understanding of at least some aspects of Nef action, as well as the importance of this viral gene product for disease induction, identify Nef as a valuable target for the development of novel antiviral therapies. Moreover, the possibility of developing vaccines using attenuated viruses with deletions in Nef and other crucial genes raises the possibility that the AIDS epidemic might one day be restrained.

  • the Nef Protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix
    Journal of Virology, 1997
    Co-Authors: Simon Swingler, Philippe Gallay, Diana Camaur, Jinping Song, Arie Abo, Didier Trono
    Abstract:

    The human immunodeficiency virus type 1 matrix (MA) Protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a Protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.

Margherita Doria - One of the best experts on this subject based on the ideXlab platform.

  • the human immunodeficiency virus type 1 vpr Protein upregulates pvr via activation of the atr mediated dna damage response pathway
    Journal of General Virology, 2013
    Co-Authors: Lia Vassena, Erica Giuliani, Giulia Matusali, Eric A Cohen, Margherita Doria
    Abstract:

    Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr Protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same Protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the Protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4+ T lymphocytes and inhibits the PVR downregulating activity of the viral Nef Protein.

  • the hiv 1 Nef Protein has a dual role in t cell receptor signaling in infected cd4 t lymphocytes
    Virology, 2011
    Co-Authors: Francesca Neri, Marina Potestà, Giorgia Giolo, Stefania Petrini, Margherita Doria
    Abstract:

    Abstract The phenotypic changes that are induced by immune activation in CD4 + T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef Protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4 + T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4 + T lymphocytes.

  • human immunodeficiency virus 1 Nef Protein downmodulates the ligands of the activating receptor nkg2d and inhibits natural killer cell mediated cytotoxicity
    Journal of General Virology, 2007
    Co-Authors: Cristina Cerboni, Francesca Neri, Nicoletta Casartelli, Alessandra Zingoni, David Cosman, Paolo Rossi, Angela Santoni, Margherita Doria
    Abstract:

    Natural killer (NK) cells are a major component of the host innate immune defence against various pathogens. Several viruses, including Human immunodeficiency virus 1 (HIV-1), have developed strategies to evade the NK-cell response. This study was designed to evaluate whether HIV-1 could interfere with the expression of NK cell-activating ligands, specifically the human leukocyte antigen (HLA)-I-like MICA and ULBP molecules that bind NKG2D, an activating receptor expressed by all NK cells. Results show that the HIV-1 Nef Protein downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, with a stronger effect on the latter molecule. The activity on MICA and ULBP2 is well conserved in Nef Protein variants derived from HIV-1-infected patients. In HIV-1-infected cells, cell-surface expression of NKG2D ligands increased to a higher extent with a Nef-deficient virus compared with wild-type virus. Mutational analysis of Nef showed that NKG2D ligand downmodulation has structural requirements that differ from those of other reported Nef activities, including HLA-I downmodulation. Finally, data demonstrate that Nef expression has functional consequences on NK-cell recognition, causing a decreased susceptibility to NK cell-mediated lysis. These findings provide a novel insight into the mechanisms evolved by HIV-1 to escape from the NK-cell response.

  • CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression
    Journal of virology, 2003
    Co-Authors: Nicoletta Casartelli, Paolo Rossi, Gigliola Di Matteo, Marina Potestà, Margherita Doria
    Abstract:

    The human immunodeficiency virus type 1 (HIV-1) Nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef Protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of Nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived Nef alleles were analyzed for their steady-state expression of a Nef Protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived Nef alleles, compared to Nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived Nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary Nef alleles not correlated with clinical progression. Defective Nef Proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.

Yves Collette - One of the best experts on this subject based on the ideXlab platform.

  • Protein Protein interaction inhibition 2p2i combining high throughput and virtual screening application to the hiv 1 Nef Protein
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: S Betzi, Stefan Arold, Audrey Restouin, Sandrine Opi, Isabelle Parrot, Francoise Guerlesquin, Xavier Morelli, Yves Collette
    Abstract:

    Protein–Protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, Protein–Protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of “hot spots” and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef Protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef–SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules.

  • The primate lentivirus-encoded Nef Protein can regulate several steps of the viral replication cycle.
    Virology, 1999
    Co-Authors: Yves Collette, Daniel Olive
    Abstract:

    The primate lentiviruses encode a Protein, Nef, which is required for efficient viral replication in their host. Several biological activities of Nef identified in vitro may contribute to this requirement in vivo, including receptor modulation and interference with cellular signaling pathways. We show that HIV- and SIV-encoded Nef can enhance virus production within a single viral replication cycle, not only by increasing viral infectivity, as previously reported, but also by acting through the efficiency of viral transcription and of viral release.

  • human immunodeficiency virus type 1 Nef Protein sensitizes cd4 t lymphoid cells to apoptosis via functional upregulation of the cd95 cd95 ligand pathway
    Blood, 1999
    Co-Authors: Giorgio Zauli, Daniel Olive, Davide Gibellini, Paola Secchiero, Helene Dutartre, Silvano Capitani, Yves Collette
    Abstract:

    Many viruses have evolved genes encoding Proteins that regulate cell death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef Protein alters T-cell development and signaling and is required for optimal viral replication and pathogenicity in vivo. To analyze the interference of Nef with cell survival, we used both regulated and constitutively expressed Nef alleles in stably transfected T-cell lines. Nef-expressing cells were sensitized to cell death by apoptosis, which was specifically exacerbated by an anti-CD95 IgM monoclonal antibody (MoAb). Flow cytometric analysis showed that the surface expression of both CD95 and CD95 ligand (CD95L) was upregulated by endogenous Nef expression. Nef-mediated apoptosis was almost completely suppressed by the addition in culture of an anti-CD95 Fab′ IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly, mutation of a proline motif in the core region of the Nef gene, which disrupts its ability to interact with cellular kinases and reduces HIV-1 replication in vitro, completely abrogated the Nef-mediated induction of apoptosis as well as its ability to upregulate surface CD95 and CD95L. These findings may provide molecular insight into the role of endogenous Nef in the T-cell depletion observed in vivo, particularly HIV-specific cytotoxic CD8+ T cells.

  • The Human Immunodeficiency Virus Type 1 Nef Protein Binds the Src-Related Tyrosine Kinase Lck SH2 Domain Through a Novel Phosphotyrosine Independent Mechanism
    Virology, 1998
    Co-Authors: Helene Dutartre, Mark Harris, Daniel Olive, Yves Collette
    Abstract:

    Primate lentiviruses encode for an unique Nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular Proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the Nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion Proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef Protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral Protein.

Mark Marsh - One of the best experts on this subject based on the ideXlab platform.

  • The Nef Protein of the macrophage tropic HIV-1 strain AD8 counteracts human BST-2/tetherin
    Viruses, 2020
    Co-Authors: Sebastian Giese, Scott P. Lawrence, Michela Mazzon, Bernadien M. Nijmeijer, Mark Marsh
    Abstract:

    Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu Proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef Protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef Proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory Protein other than Vpu to antagonise human tetherin.

  • The Nef Protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin
    2020
    Co-Authors: Sebastian Giese, Scott P. Lawrence, Michela Mazzon, Bernadien M. Nijmeijer, Mark Marsh
    Abstract:

    Abstract Bst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu Proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef Protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef Proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory Protein other than Vpu to antagonise human tetherin.

Dieter Willbold - One of the best experts on this subject based on the ideXlab platform.

  • brain transcriptome wide screen for hiv 1 Nef Protein interaction partners reveals various membrane associated Proteins
    PLOS ONE, 2012
    Co-Authors: Ellen C Kammula, Dieter Willbold, Jessica Motter, Alexandra Gorgels, Esther Jonas, Silke Hoffmann
    Abstract:

    HIV-1 Nef Protein contributes essentially to the pathology of AIDS by a variety of Protein-Protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane Proteins, membrane-associated Proteins or other Proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane Proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and Protein trafficking or quality control. For a subfraction of the hereby identified Proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  • hiv 1 Nef mimics an integrin receptor signal that recruits the polycomb group Protein eed to the plasma membrane
    Molecular Cell, 2004
    Co-Authors: Vanessa Witte, Bernd Laffert, Olaf Rosorius, Peter Lischka, Katja Blume, Gunther Galler, Andrea Stilper, Dieter Willbold, Paola Daloja, Michael Sixt
    Abstract:

    The Nef Protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) Protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

  • the n terminus of Nef from hiv 1 siv associates with a Protein complex containing lck and a serine kinase
    Immunity, 1997
    Co-Authors: Andreas Baur, Bernd Laffert, Dieter Willbold, Gabriele Sass, Cecilia Chengmayer, Matija B Peterlin
    Abstract:

    The Nef Protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiProtein complex at the far N-terminus of the viral Protein. One of the kinases was identified as Lck, whereas the second Protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef Proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef Proteins, inhibited association of the NAKC and significantly reduced virion infectivity.