RESUMEN
Resting human CD4 T cells are highly resistant to transfection or infection with lentiviral vectors derived from the human immunodeficiency virus. We now describe a flexible and efficient approach involving virus-like particles containing simian immunodeficiency virus lentiviral gene product protein X and pseudotyping with CXCR4-tropic HIV Env. This method permits effective genetic manipulation of these cells while preserving their naturally quiescent state. This technology can also be extended to primary lymphoid cultures where authentic cellular composition and functional relationships are preserved.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética , Proteínas Virales/genética , Animales , Linfocitos T CD4-Positivos/patología , Vectores Genéticos , VIH-1/genética , Humanos , Lentivirus/genética , Receptores CXCR4/genética , Receptores CXCR4/uso terapéutico , Virus de la Inmunodeficiencia de los Simios , Proteínas Virales/administración & dosificación , Proteínas Virales/uso terapéutico , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/uso terapéuticoRESUMEN
Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.
Asunto(s)
Núcleo Celular/metabolismo , Fase G2 , Productos del Gen vpr/fisiología , VIH-1/fisiología , Lamina Tipo B , Membrana Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/virología , Ciclina B/metabolismo , Ciclina B1 , Citoplasma/metabolismo , Productos del Gen vpr/genética , Células HeLa , Humanos , Laminas , Macrófagos/virología , Microscopía Fluorescente , Microscopía por Video , Mitosis , Mutación , Membrana Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Integración Viral , Fosfatasas cdc25/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Productos del Gen vpr/fisiología , VIH-1/fisiología , Macrófagos/virología , Carga Viral , Ciclo Celular , Humanos , Tejido Linfoide/virología , Receptores CCR5/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The nuclear expression and action of the nuclear factor kappa B (NF-kappaB) transcription factor requires signal-coupled phosphorylation and degradation of the IkappaB inhibitors, which normally bind and sequester this pleiotropically active factor in the cytoplasm. The subsequent molecular events that regulate the termination of nuclear NF-kappaB action remain poorly defined, although the activation of de novo IkappaBalpha gene expression by NF-kappaB likely plays a key role. Our studies now demonstrate that the RelA subunit of NF-kappaB is subject to inducible acetylation and that acetylated forms of RelA interact weakly, if at all, with IkappaBalpha. Acetylated RelA is subsequently deacetylated through a specific interaction with histone deacetylase 3 (HDAC3). This deacetylation reaction promotes effective binding to IkappaBalpha and leads in turn to IkappaBalpha-dependent nuclear export of the complex through a chromosomal region maintenance-1 (CRM-1)-dependent pathway. Deacetylation of RelA by HDAC3 thus acts as an intranuclear molecular switch that both controls the duration of the NF-kappaB transcriptional response and contributes to the replenishment of the depleted cytoplasmic pool of latent NF-kappaB-IkappaBalpha complexes.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas I-kappa B , FN-kappa B/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células COS , Proteína de Unión a CREB , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Citoplasma/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histona Acetiltransferasas , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Ratones , Inhibidor NF-kappaB alfa , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Factor de Transcripción ReIA , Factores de Transcripción , Factor de Necrosis Tumoral alfa/farmacología , Factores de Transcripción p300-CBPRESUMEN
In vivo infection of lymphatic tissues by the human immunodeficiency virus type 1 (HIV-1) leads to enhanced apoptosis, which prominently involves uninfected bystander cells. Increased killing of such bystander cells is mediated in part through Nef induction of Fas ligand (FasL) expression on the surface of the virally infected T cells. The subsequent interaction of FasL with Fas (CD95) displayed on neighbouring cells, including HIV-1-specific cytotoxic T lymphocytes, may lead to bystander cell killing and thus forms an important mechanism of immune evasion. As HIV-1 also enhances Fas expression on virally infected cells, it is unclear how these hosts avoid rapid cell-autonomous apoptosis mediated through cis ligation of Fas by FasL. Here we show that HIV-1 Nef associates with and inhibits apoptosis signal-regulating kinase 1 (ASK1), a serine/threonine kinase that forms a common and key signalling intermediate in the Fas and tumour-necrosis factor-alpha (TNFalpha) death-signalling pathways. The interaction of Nef with ASK1 inhibits both Fas- and TNFalpha-mediated apoptosis, as well as the activation of the downstream c-Jun amino-terminal kinase. Our findings reveal a strategy by which HIV-1 Nef promotes the killing of bystander cells through the induction of FasL, while simultaneously protecting the HIV-1-infected host cell from these same pro-apoptotic signals through its interference with ASK1 function.
Asunto(s)
Productos del Gen nef/fisiología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Apoptosis , Catálisis , Línea Celular , Inhibidores Enzimáticos , Proteína Ligando Fas , Humanos , Células Jurkat , MAP Quinasa Quinasa Quinasa 5 , Glicoproteínas de Membrana/metabolismo , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The Nef protein of the type 1 human immunodeficiency virus (HIV-1) plays a key although poorly understood role in accelerating the progression of clinical disease in vivo. Nef exerts several biological effects in vitro, including enhancement of virion infectivity, downregulation of CD4 and major histocompatibility complex class I receptor expression, and modulation of various intracellular signaling pathways. The positive effect of Nef on virion infectivity requires its expression in the producer cell, although its effect is manifested in the subsequent target cell of infection. Prior studies suggest that Nef does not alter viral entry into target cells; nevertheless, it enhances proviral DNA synthesis, arguing for an action of Nef at the level of viral uncoating or reverse transcription. However, these early studies discounting an effect of Nef on virion entry may be confounded by the recent finding that HIV enters cells by both fusion and endocytosis. Using epifluorescence microscopy to monitor green fluorescent protein-Vpr-labeled HIV virion entry into HeLa cells, we find that endocytosis forms a very active pathway for virus uptake. Virions entering via the endocytic pathway do not support productive infection of the host cell, presumably reflecting their inability to escape from the endosomes. Conversely, our studies now demonstrate that HIV Nef significantly enhances CD4- and chemokine receptor-dependent entry of HIV virions into the cytoplasmic compartment of target cells. Mutations in Nef either impairing its ability to downregulate CD4 or disrupting its polyproline helix compromise virion entry into the cytoplasm. We conclude that Nef acts at least in part as a regulator of cytosolic viral entry and that this action contributes to its positive effects on viral infectivity.
Asunto(s)
Citoplasma/virología , Productos del Gen nef/metabolismo , VIH-1/fisiología , Virión/fisiología , Antígenos CD4/metabolismo , Productos del Gen vpr/genética , Productos del Gen vpr/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Productos del Gen vpr/fisiología , VIH-1/fisiología , Carioferinas , Receptores Citoplasmáticos y Nucleares , Secuencia de Aminoácidos , Proteínas Portadoras/fisiología , Ácidos Grasos Insaturados/farmacología , Productos del Gen vpr/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana , Proteína Exportina 1RESUMEN
Nerve growth factor binds to the TrkA and p75(NTR) (p75) and generates signals leading to neuronal cell survival, differentiation, and programmed cell death. Here we describe a series of experiments involving selective activation of either TrkA or p75 in which distinct cell-signaling intermediates promote different cellular consequences. We analyzed pheochromocytoma 12 (PC12) cells stably expressing chimeras consisting of the extracellular domain of PDGF receptor (PDGFR) fused to the transmembrane and cytoplasmic segments of p75 or TrkA. Because PC12 cells lack endogenous PDGFR, addition of PDGF to these cell lines permits selective activation of the p75 or TrkA responses without stimulating endogenous receptors. Although both p75 and TrkA activated nuclear factor-kappaB (NF-kappaB), we show that distinct proximal-signaling intermediates are used by each receptor. A dominant-negative mutant of TRAF6 blocked p75- but not TrkA-mediated induction of NF-kappaB. Conversely a dominant-negative mutant of Shc inhibited TrkA but not p75 activation of NF-kappaB. Both of these distinct signaling pathways subsequently converge, leading to activation of the IkappaB kinase complex. Moreover, the activation of NF-kappaB by these distinct pathways after stimulation of either TrkA or p75 leads to different physiological consequences. Blocking p75-mediated activation of NF-kappaB by ecdysone-inducible expression of a nondegradable mutant of IkappaBalpha significantly enhanced apoptosis. In contrast, blocking NF-kappaB induction via TrkA significantly inhibited neurite process formation in PC12 cells. Together these findings indicate that, although both of these receptors lead to the activation of NF-kappaB, they proceed via distinct proximal-signaling intermediates and contribute to different cellular outcomes.
Asunto(s)
Proteínas I-kappa B , FN-kappa B/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuritas/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Genes Dominantes , Inhibidor NF-kappaB alfa , FN-kappa B/farmacología , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Generation of the NF-kappaB p50 transcription factor is mediated by the proteasome. We found previously that p50 is generated during translation of the NFKB1 gene and that this cotranslational processing allows the production of both p50 and p105 from a single mRNA. We now demonstrate that the Rel homology domain in p50 undergoes cotranslational dimerization and that this interaction is required for efficient production of p50. We further show that this coupling of dimerization and proteasome processing during translation uniquely generates p50-p105 heterodimers. Accordingly, after the primary cotranslational event, additional posttranslational steps regulate p50 homodimer formation and the intracellular ratio of p50 and p105. This cellular strategy places p50 under the control of the p105 inhibitor early in its biogenesis, thereby regulating the pool of p50 homodimers within the cell.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Dimerización , Endopeptidasa K/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , Factor de Transcripción ReIB , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
NF-kappa B has been implicated in the survival and differentiation of PC12 cells. In this study, we examined the effect of the NF-kappa B-inducing kinase (NIK) on these processes. When inducibly expressed in PC12 cells, a kinase-proficient but not -deficient form of NIK promoted neurite process formation and mediated anti-apoptotic signaling. As expected, NIK expression led to I kappa B kinase activation and induced nuclear translocation of NF-kappa B. However, NIK-induced neurite outgrowth was only partially blocked by concomitant expression of a nondegradable form of I kappa B alpha that completely blocks NF-kappa B induction. In search of additional signaling pathways activated by NIK, we now demonstrate that NIK activates MEK1 phosphorylation and induces the Erk1/Erk2 MAPK pathway. Treatment of PC12 cells with PD98059, a MEK1 inhibitor, potently blocked neurite process formation; however, a dominantly interfering mutant of the upstream Shc adapter failed to alter this response. These findings reveal a new function for NIK as a MEK1-dependent activator of the MAPK pathway and implicate both the I kappa B kinase and MAPK signaling cascades in NIK-induced differentiation of PC12 cells.
Asunto(s)
Apoptosis/fisiología , Diferenciación Celular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Secuencia de Bases , Oligonucleótidos , Células PC12 , Fosforilación , Ratas , Transducción de Señal , Quinasa de Factor Nuclear kappa BRESUMEN
Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G(2) cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-kappaB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G(2) cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.
Asunto(s)
Productos del Gen vpr/metabolismo , VIH-1/fisiología , Receptores de Glucocorticoides/metabolismo , Secuencia de Aminoácidos , Línea Celular , Fase G2 , Productos del Gen vpr/química , VIH-1/metabolismo , Humanos , Leucina Zippers , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Coactivador 2 del Receptor Nuclear , Unión Proteica , Estructura Secundaria de Proteína , Factores de Transcripción/metabolismo , Activación Transcripcional , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.
Asunto(s)
Núcleo Celular/metabolismo , Fase G2 , Productos del Gen vpr/química , Productos del Gen vpr/metabolismo , VIH-1/química , Secuencia de Aminoácidos , Western Blotting , Núcleo Celular/virología , Dicroismo Circular , Dimerización , Productos del Gen vpr/síntesis química , Productos del Gen vpr/aislamiento & purificación , VIH-1/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Macrófagos/citología , Macrófagos/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Transporte de Proteínas , Dispersión de Radiación , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trifluoroetanol/farmacología , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The NF-kappaB/Rel family of eukaryotic transcription factors plays an essential role in the regulation of inflammatory, antiapoptotic, and immune responses. NF-kappaB is activated by many stimuli including costimulation of T cells with ligands specific for the T-cell receptor (TCR)-CD3 complex and CD28 receptors. However, the signaling intermediates that transduce these costimulatory signals from the TCR-CD3 and CD28 surface receptors leading to nuclear NF-kappaB expression are not well defined. We now show that protein kinase C-theta (PKC-theta), a novel PKC isoform, plays a central role in a signaling pathway induced by CD3-CD28 costimulation leading to activation of NF-kappaB in Jurkat T cells. We find that expression of a constitutively active mutant of PKC-theta potently induces NF-kappaB activation and stimulates the RE/AP composite enhancer from the interleukin-2 gene. Conversely, expression of a kinase-deficient mutant or antisense PKC-theta selectively inhibits CD3-CD28 costimulation, but not tumor necrosis factor alpha-induced activation of NF-kappaB in Jurkat T cells. The induction of NF-kappaB by PKC-theta is mediated through the activation of IkappaB kinase beta (IKKbeta) in the absence of detectable IKKalpha stimulation. PKC-theta acts directly or indirectly to stimulate phosphorylation of IKKbeta, leading to activation of this enzyme. Together, these results implicate PKC-theta in one pathway of CD3-CD28 costimulation leading to NF-kappaB activation that is apparently distinct from that involving Cot and NF-kappaB-inducing kinase (NIK). PKC-theta activation of NF-kappaB is mediated through the selective induction of IKKbeta, while the Cot- and NIK-dependent pathway involves induction of both IKKalpha and IKKbeta.
Asunto(s)
Antígenos CD28/metabolismo , Complejo CD3/metabolismo , Isoenzimas/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Quinasa I-kappa B , Isoenzimas/genética , Células Jurkat , Mutación , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Transducción de SeñalRESUMEN
Signal-induced nuclear expression of the eukaryotic NF-kappaB transcription factor involves the stimulatory action of select mitogen-activated protein kinase kinase kinases on the IkappaB kinases (IKKalpha and IKKbeta) which reside in a macromolecular signaling complex termed the signalsome. While genetic studies indicate that IKKbeta is the principal kinase involved in proinflammatory cytokine-induced IkappaB phosphorylation, the function of the equivalently expressed IKKalpha is less clear. Here we demonstrate that assembly of IKKalpha with IKKbeta in the heterodimeric signalsome serves two important functions: (i) in unstimulated cells, IKKalpha inhibits the constitutive IkappaB kinase activity of IKKbeta; (ii) in activated cells, IKKalpha kinase activity is required for the induction of IKKbeta. The introduction of kinase-inactive IKKalpha, activation loop mutants of IKKalpha, or IKKalpha antisense RNA into 293 or HeLa cells blocks NIK (NF-kappaB-inducing kinase)-induced phosphorylation of the IKKbeta activation loop occurring in functional signalsomes. In contrast, catalytically inactive mutants of IKKbeta do not block NIK-mediated phosphorylation of IKKalpha in these macromolecular signaling complexes. This requirement for kinase-proficient IKKalpha to activate IKKbeta in heterodimeric IKK signalsomes is also observed with other NF-kappaB inducers, including tumor necrosis factor alpha, human T-cell leukemia virus type 1 Tax, Cot, and MEKK1. Conversely, the theta isoform of protein kinase C, which also induces NF-kappaB/Rel, directly targets IKKbeta for phosphorylation and activation, possibly acting through homodimeric IKKbeta complexes. Together, our findings indicate that activation of the heterodimeric IKK complex by a variety of different inducers proceeds in a directional manner and is dependent on the kinase activity of IKKalpha to activate IKKbeta.
Asunto(s)
Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Dimerización , Activación Enzimática , Productos del Gen tax/farmacología , Células HeLa , Humanos , Quinasa I-kappa B , Quinasas Quinasa Quinasa PAM/metabolismo , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Cuaternaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
nfkb2 encodes two members of the NF-kappa B/Rel family of proteins: p52 and p100. The p100 polypeptide has been proposed to serve as a precursor of p52, which corresponds to the N-terminal half of p100. While p52 functions as a Rel transcription factor, the larger p100 protein acts as a cytoplasmic inhibitor of select NF-kappa B/Rel transcription factor complexes. Because of their distinct functions, we have studied the biochemical basis for the production of these two nfkb2-derived gene products. Like the p50 product of the nfkb1 gene, p52 is principally generated in a cotranslational manner involving proteolytic processing by the proteasome. The generation of p52 is dependent on a glycine-rich region (GRR) located upstream of the p52 C-terminus, and repositioning of this GRR alters the location of proteasome processing. In most cells, small amounts of p52 are produced relative to the levels of p100, unlike the usually balanced production of nfkb1-derived p50 and p105. Using p100/p105 chimeras containing different segments of the nfkb1 and nfkb2 genes, we have found that diminished p52 processing is a property conferred by peptide sequences located downstream of the GRR, flanking the site of p52 processing.
Asunto(s)
FN-kappa B/biosíntesis , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Subunidad p52 de NF-kappa B , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , TransfecciónRESUMEN
The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.
Asunto(s)
Antígenos CD4/biosíntesis , Regulación hacia Abajo/inmunología , Productos del Gen nef/inmunología , Leucina/metabolismo , Virus de la Inmunodeficiencia de los Simios/inmunología , Tirosina/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Antígenos CD8/genética , Línea Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Productos del Gen nef/genética , VIH-1/genética , VIH-1/inmunología , Humanos , Leucina/genética , Macaca , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo , Tirosina/genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The proto-oncogene Cot/Tpl-2 encodes a MAP3K-related serine-threonine kinase. Expression of wild type Cot activates the IkappaB kinases (IKK) leading to induction of NF-kappaB. Conversely, expression of kinase-deficient Cot inhibits CD3/CD28 but not TNF alpha induction of NF-kappaB. These findings suggest the selective involvement of Cot/Tpl-2 or a closely related kinase in the CD3/CD28 costimulatory pathway leading to induced nuclear expression of NF-kappaB. In contrast, a kinase-deficient mutant of the NF-kappaB-inducing kinase (NIK) inhibits both CD3/CD28 and TNF alpha signaling, indicating that these pathways converge at or prior to the action of NIK. Consistent with such a sequential function of these two kinases, Cot physically assembles with and phosphorylates NIK in vivo.
Asunto(s)
Antígenos CD28/metabolismo , Complejo CD3/metabolismo , Quinasas Quinasa Quinasa PAM , FN-kappa B/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Inducción Enzimática , Humanos , Quinasa I-kappa B , Células Jurkat , Proto-Oncogenes Mas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Quinasa de Factor Nuclear kappa BRESUMEN
Human immunodeficiency virus 1 (HIV-1) Nef downregulates surface expression of CD4, an integral component of the functional HIV receptor complex, through accelerated endocytosis of surface receptors and diminished transport of CD4 from the Golgi network to the plasma membrane. HIV-1 Nef also diminishes surface expression of major histocompatibility complex (MHC) class I antigens. In the case of HIV-2 and simian immunodeficiency virus 1 (SIV-1) Nef, aminoterminal tyrosine-based motifs mediate the binding of Nef to the AP-1 and AP-2 adaptors and this interaction appears to be required for CD4 downregulation. As these tyrosine motifs are not present in the HIV-1 Nef protein, the molecular basis for the presumed interaction of Nef with components of the endocytic machinery is unknown. Here, we identify a highly conserved dileucine motif in HIV-1 Nef that is required for downregulation of CD4. This motif acts as an internalization signal in the context of a CD8-Nef chimera or in a fusion of the interleukin-2 receptor alpha with an 11-amino-acid region from Nef containing the dileucine motif. Finally, HIV-1 Nef binds to the AP-1 adaptor, both in vitro and in vivo, in a dileucine-dependent manner. We conclude that this conserved dileucine motif in HIV-1 Nef serves as a key interface for interaction with components of the host protein trafficking machinery. Our findings also reveal an evolutionary difference between HIV-1 and HIV-2/SIV in which the Nef proteins utilize structurally distinct motifs for binding cellular adaptors.
Asunto(s)
Antígenos CD4/metabolismo , Clatrina/metabolismo , Regulación hacia Abajo , Productos del Gen nef/metabolismo , VIH-1/metabolismo , Leucina/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Sitios de Unión , Productos del Gen nef/genética , Células HeLa , Humanos , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.