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(1/788) HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines.

Vaccine strategies designed to elicit strong cell-mediated immune responses to HIV Ags are likely to lead to protective immunity against HIV infection. Dendritic cells (DC) are the most potent APCs capable of priming both MHC class I- and II-restricted, Ag-specific T cell responses. Utilizing a system in which cultured DC from HIV-seronegative donors were used as APC to present HIV-1 Ags to autologous T cells in vitro, the strength and specificity of primary HIV-specific CTL responses generated to exogenous HIV-1 Nef protein as well as intracellularly expressed nef transgene product were investigated. DC expressing the nef gene were able to stimulate Nef-specific CTL, with T cells from several donors recognizing more than one epitope restricted by a single HLA molecule. Primary Nef-specific CTL responses were also generated in vitro using DC pulsed with Nef protein. T cells primed with Nef-expressing DC (via protein or transgene) were able to lyse MHC class I-matched target cells pulsed with defined Nef epitope peptides as well as newly identified peptide epitopes. The addition of Th1-biasing cytokines IL-12 or IFN-alpha, during priming with Nef-expressing DC, enhanced the Nef-specific CTL responses generated using either Ag-loading approach. These results suggest that this in vitro vaccine model may be useful in identifying immunogenic epitopes as vaccine targets and in evaluating the effects of cytokines and other adjuvants on Ag-specific T cell induction. Successful approaches may provide information important to the development of prophylactic HIV vaccines and are envisioned to be readily translated into clinical DC-based therapeutic vaccines for HIV-1.  (+info)

(2/788) Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo.

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.  (+info)

(3/788) Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes.

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.  (+info)

(4/788) The acidic region and conserved putative protein kinase C phosphorylation site in Nef are important for SIV replication in rhesus macaques.

Variants of the pathogenic SIVmac239 clone with changes in Nef were analyzed to assess the functional relevance of two highly conserved regions in Nef in vitro and in vivo. Changes in a region with an acidic charge (Aci-Nef), or a potential protein kinase C phosphorylation site (PKC-Nef), impaired the ability of Nef to down-regulate CD4 and MHC class I surface expression and to alter CD3-initiated signal transduction in Jurkat T cells. The Aci-Nef, but not the PKC-Nef, associated with the previously described p65 phosphoprotein. SIV containing Aci-Nef, but not SIV containing PKC-Nef, showed reduced infectivity and replication in cell culture systems. One of two rhesus macaques infected with the PKC-Nef mutant virus showed rapid reversion and progressed to disease. In the second animal no reversions and nonprogressive infection was observed. In one of two macaques infected with the Aci-Nef variant, the mutations were stable during the first 40 weeks after infection. Thereafter, variants evolved in which up to six of the eight mutated positions in Nef were reverted and functional activity in vitro was partially restored. These changes occurred concomitantly with increasing viral load and disease progression. The second animal infected with the Aci-Nef variant showed no reversions and remained asymptomatic. Our study suggests that the acidic region and conserved PKC phosphorylation site in Nef are important for SIV replication in rhesus macaques and for several in vitro Nef functions. An almost wild-type activity in in vitro infectivity and replication assays seems insufficient to confer a full nef-positive phenotype in vivo.  (+info)

(5/788) HIV-1 Nef plays an essential role in two independent processes in CD4 down-regulation: dissociation of the CD4-p56(lck) complex and targeting of CD4 to lysosomes.

Human immunodeficiency virus type 1 (HIV-1) Nef down-regulates CD4 by triggering rapid endocytosis of cell surface CD4. To better understand how Nef induces CD4 down-regulation, we generated a series of Nef mutants with small in-frame deletions in the coding region. Three classes of mutants were obtained. The first class produces neither CD4 down-regulation nor dissociation of the CD4-p56(lck) complex. The second class induces CD4 down-regulation in cells lacking p56(lck) expression, but not in cells with p56(lck);these mutants fail to dissociate CD4 from p56lck. These results show that Nef-mediated CD4 dissociation from p56(lck) is important for CD4 down-regulation. The third class of mutants is able to dissociate the CD4-p56(lck) complex but fails to down-regulate surface CD4; internalized CD4 molecules are recycled back to the cell surface. This result suggests that Nef diverts the CD4 recycling pathway to a degradative pathway. We also demonstrate that Nef associates with phosphatidylinositol-3-kinase (PI3K) activity, which is known to be involved in several aspects of membrane trafficking. However, Nef mutants that cause internalized CD4 to be recycled do not associate with PI3K activity; thus Nef-associated PI3K activity might be involved in the latter process of targeting CD4 to a degradative pathway. We conclude that HIV-1 Nef plays a critical role in multiple processes in CD4 down-regulation: (i) disrupting the CD4-p56(lck) complex on the cell surface to allow CD4 internalization and (ii) diverting the internalized CD4 to a lysosomal pathway for its degradation, likely through a PI3K activity.  (+info)

(6/788) Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.  (+info)

(7/788) HIV-1 nef expression inhibits the activity of a Ca2+-dependent K+ channel involved in the control of the resting potential in CEM lymphocytes.

The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.  (+info)

(8/788) The cellular kinase binding motifs (PxxP and RR) in human immunodeficiency virus type 1 Nef protein are dispensable for producer-cell-dependent enhancement of viral entry.

We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) Nef is required for enhancing viral infectivity by increasing the efficiency of viral entry in a producer-cell-dependent manner, suggesting the possible involvement of a cellular factor(s) in the enhancement of viral entry. Moreover, it has been reported that a proline-rich (PxxP) motif and an Arg-Arg (RR) motif in HIV-1 Nef bind to the SH3 domain of the Src-family tyrosine kinase Hck and to a serine/threonine kinase, respectively. To address whether these cellular kinase binding motifs, PxxP and RR, could be involved in virus producer-cell-dependent enhancement of viral entry, we constructed two nef mutant proviral clones in which these motifs were mutated. The results show that the HIV-1 Nef PxxP motif, which significantly influenced viral infectivity, and the RR motif, which modestly affected viral infectivity, were both dispensable for enhanced viral entry, thus suggesting that another interaction of Nef with a cellular factor(s) is involved in the efficiency of viral entry.  (+info)