RESUMO
HIV-1 envelope spike (Env) is a heavily glycosylated, type I membrane protein that mediates fusion of viral and cell membranes to initiate infection. It is also a primary target of neutralizing antibodies and thus an important candidate for vaccine development. We have recently reported a nuclear magnetic resonance structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in a membrane-like environment. Taking HIV-1 as an example, we discuss here how a TM domain can anchor, stabilize, and modulate a viral envelope spike and how its high-resolution structure can contribute to understanding viral membrane fusion and to immunogen design.
Assuntos
Proteína gp160 do Envelope de HIV/química , HIV-1/química , Sequência de Aminoácidos , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Fusão de Membrana/fisiologia , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de Aminoácidos , Vacinas Sintéticas/químicaRESUMO
BACKGROUND: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env. RESULTS: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps. CONCLUSIONS: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions.
Assuntos
Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Vírion/isolamento & purificação , Vírion/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/imunologia , Humanos , Imunoensaio , Ligação Proteica , Vírion/imunologiaRESUMO
BACKGROUND: Mutant HIV (HIV-Env-Tr712) lacking the cytoplasmic tail of the viral glycoprotein (Env-CT) exhibits a cell-type specific replication phenotype such that replicative spread occurs in some T-cell lines (referred to as permissive cells) but fails to do so in most T-cell lines or in PBMCs (referred to as non-permissive cells). We aim to gain insight on the underlying requirement for the Env-CT for viral spread in non-permissive cells. RESULTS: We established that in comparison to HIV-Wt, both cell-free and cell-to-cell transmission of mutant HIV-Env-Tr712 from non-permissive cells were severely impaired under naturally low infection conditions. This requirement for Env-CT could be largely overcome by using saturating amounts of virus for infection. We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype. Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells. CONCLUSIONS: During natural infection conditions, the HIV-Env-CT is critically required for viral transmission in cultures of non-permissive cells by both cell-free and cell-to-cell routes and is instrumental for Gag accumulation to the VS. The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.
Assuntos
Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/patogenicidade , Linfócitos T/virologia , Fatores de Virulência/fisiologia , Linhagem Celular , Proteína gp160 do Envelope de HIV/genética , HIV-1/crescimento & desenvolvimento , Humanos , Fatores de Virulência/genética , Replicação ViralRESUMO
Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/química , HIV-1/fisiologia , Humanos , Camundongos , Microscopia Imunoeletrônica , Testes de Neutralização , Vírion/imunologiaRESUMO
In this study, the relationship between biological phenotypes, coreceptor usages, and sequence patterns of V1V2 or V3 regions on HIV-1 envelope gp 120 was carefully analyzed based on the existing isolates in the Los Alamos National Laboratory sequence database. Obviously, SI/NSI phenotypes were closely linked to the capability of HIV-1 to use coreceptor CXCR4, but not CCR5. Moreover, compared to NSI or R5 isolates, SI or X4 HIV-1 isolates significantly prefer higher net charges and the loss of the N-linked glycosylation site in the V3 loop, but they show no preference in either net charge or N-linked glycosylation of the V1V2 region. In addition, no significant relationship between V1V2 length and virus tropism or phenotypes was observed.
Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , Glicosilação , Proteína gp160 do Envelope de HIV/fisiologia , Humanos , Fenótipo , Receptores CCR5/fisiologia , TropismoRESUMO
The human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) has evolved to limit its overall immunogenicity by extensive glycosylation. Only a few studies dealing with glycosylation sites have taken into account available 3D data in a global approach. We compared primary env sequences from patients with acute HIV-1 infection. Conserved N-glycosylation sites were placed on the gp120-3D model. Based on vicinity, we defined glycosylation clusters. According to these clusters, we engineered plasmids encoding deglycosylated gp160 mutants. We also constructed mutants corresponding to nonclustered glycans or to the full deglycosylation of the V1 or V2 loop. After in vitro expression, mutants were tested for functionality. We also compared the inhibition of pseudotyped particles infection by human-neutralizing sera. Generally, clustered and nonclustered mutants were affected similarly. Silencing of more than one glycan had deleterious effects, independently of the type of sugar removed. However, some mutants were moderately affected by glycans removal suggesting a distinct role for these N-glycans. Additionally, compared to the wild-type pseudovirus, two of these mutants were neutralized at higher sera dilutions strengthening the importance of the location of specific N-glycans in limiting the neutralizing response. These results could guide the selection of env mutants with the fewest antigenic and functional alterations but with enhanced neutralization sensitivity.
Assuntos
Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Sequência de Aminoácidos , Antígenos CD4/metabolismo , Glicosilação , Proteína gp120 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/química , HIV-1/imunologia , Humanos , Fusão de Membrana , Modelos Biológicos , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
Human immunodeficiency virus has evolved several redundant mechanisms to remove its receptor, the CD4 molecule, from the cell surface. Indeed, HIV-1 encodes three proteins, Nef, Vpu and Env, that have a profound effect on CD4 trafficking and catabolism. Given this functional convergence, it is believed that cell surface CD4 regulation constitutes an important determinant of viral replication and pathogenesis in vivo. This review highlights recent progress made in our understanding of the molecular mechanisms underlying the down-regulation of the CD4 receptor by HIV-1 and describes our current comprehension of the role of CD4 down-regulation during HIV-1 infection.
Assuntos
Antígenos CD4/metabolismo , Regulação para Baixo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Produtos do Gene nef/fisiologia , Genes Virais , Genes nef , Genes vpu , Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Proteínas Virais/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
To better understand CXCR4 function on macrophages and the relationship between coreceptor use and macrophage tropism among diverse HIV-1 isolates, we analyzed macrophage pathways involved in Env-mediated fusion, productive HIV-1 infection, and chemokine-elicited signaling. We found that both CXCR4 and CCR5 transduced intracellular signals in monocyte-derived macrophages, activating K+ and Cl- ion channels and elevating intracellular calcium in response to their chemokine ligands stromal cell-derived factor-1alpha and macrophage inflammatory protein-1beta, respectively. The prototype T-tropic X4 strain IIIB infected macrophages poorly, and this was associated with failure of the IIIB Env to fuse efficiently with target macrophages despite functional CXCR4. In contrast, several primary X4 isolates mediated efficient CXCR4-dependent fusion and productive macrophage infection. Several R5X4 strains could fuse with and infect macrophages through both CCR5 and CXCR4. Thus, macrophages express functional CXCR4 and CCR5 but primary and prototype X4 isolates differ in their ability to utilize macrophage CXCR4. Isolates classified as X4 based on coreceptor use may be phenotypically either T-tropic or dual-tropic and, conversely, phenotypically dual-tropic isolates may be either R5X4 or X4 based on coreceptor use.
Assuntos
Quimiocinas/fisiologia , Infecções por HIV/virologia , HIV-1/fisiologia , Macrófagos/fisiologia , Receptores CXCR4/fisiologia , Transdução de Sinais/fisiologia , Fusão Celular/fisiologia , Canais de Cloreto/fisiologia , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/fisiologia , Infecções por HIV/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Canais de Potássio/fisiologia , Receptores CCR5/biossíntese , Receptores CCR5/fisiologia , Receptores CXCR4/biossíntese , TransfecçãoRESUMO
Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide chain, during its transport, into the lumen of the endoplasmic reticulum (ER). We have analyzed the kinetics of the cleavage of the HIV-1 Env protein signal sequence from gp160 and gp120 in HeLa, BHK, and Jurkat cells. Furthermore, we have determined the effects of this cleavage on the association of the gp160 and gp120 glycoproteins with the ER protein calnexin and the effects of the signal sequence cleavage on protein folding. The cleavage of the HIV-1 Env protein signal sequence on both gp160 and gp120 occurred very slowly in all three cell lines with a t(1/2) of 45-60 min. The core glycosylated and signal-sequence-retained forms of gp160 and gp120 associated with calnexin while the signal-sequence-cleaved forms of gp160 and gp120 had disassociated from calnexin and correctly folded as determined by their ability to associate with the CD4 cellular receptor. Further analysis of the folding state of gp160 and gp120 in nonreducing SDS-PAGE revealed that the signal-sequence-retained and calnexin-associated forms of gp160 and gp120 migrated as broad, diffuse bands, whereas the signal-sequence-cleaved or CD4-associated forms of gp160 and gp120 migrated as single sharper bands. The cause of this retardation in the rate of folding and intracellular transport of HIV-1 glycoproteins was localized to their signal sequences by fusing the vesicular stomatitis virus G protein with the HIV-1 Env protein signal sequence and expressing this chimeric protein in mammalian cells. The HIV-1 Env protein signal sequence on the VSV-G protein also confers a reduced rate of cleavage and slow intracellular transport and folding of the chimeric G protein. These results provide direct evidence that in vivo the HIV-1 glycoprotein signal sequence inhibits the folding of HIV-1 Env protein. Our data also suggest a direct correlation between the rate of the signal sequence cleavage and protein folding.
Assuntos
Regulação para Baixo/fisiologia , Glicoproteínas/metabolismo , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Calnexina , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/fisiologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Células HeLa , Humanos , Hidrólise , Células Jurkat , Proteínas de Membrana/fisiologia , Chaperonas Moleculares/fisiologia , Fosfoproteínas/fisiologia , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismoRESUMO
The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.
Assuntos
Antígenos CD4/fisiologia , Fusão Celular , Genes Reporter , Proteína gp160 do Envelope de HIV/fisiologia , Luciferases/genética , Proteínas Luminescentes/genética , Macrófagos/virologia , Receptores CCR5/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Fusão Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Humanos , Luciferases/biossíntese , Proteínas Luminescentes/biossíntese , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sensibilidade e Especificidade , TransfecçãoRESUMO
HIV-1 gp120/gp160 is known to disturb the activity of p56lck, protein kinase C (PKC) and Ca2+ homeostasis in T lymphocytes. We found that gp160 decreases the Kv1.3 current of Jurkat E6.1 cells probably by increasing the PKC-dependent phosphorylation of Kv channel protein after 5 days. This decrease is dose-dependent. In contrast, gp160 did not decrease the Kv1.3 current of the JCaM1.6 cell line, a p56(lck)-defective Jurkat cell line. This shows that p56lck was at the beginning of the events which induced the Kv1.3 current decrease. As a consequence of this decrease, Jurkat E6.1 cells were depolarized and exhibited a volume increase.
Assuntos
Proteína gp160 do Envelope de HIV/fisiologia , Ativação do Canal Iônico , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio , Proteína gp160 do Envelope de HIV/metabolismo , Humanos , Células Jurkat , Canal de Potássio Kv1.3 , FosforilaçãoRESUMO
Recent results show that the HIV-1 protein gp120 can enhance N-methyl-D-aspartate receptor-mediated release of noradrenaline from CNS nerve endings. We now investigate the mechanism of this action, including the structural determinants of the gp120 effect and the nature of its binding sites. The N-methyl-D-aspartate-evoked release of [3H]noradrenaline from rat hippocampal synaptosomes was potentiated similarly by gp120 and gp160; gp41 was ineffective. The regions of gp120 involved appear to be outside the CD4-binding domain of the protein, because gp120 retained its activity after pretreatment with N-carbomethoxycarbonyl-D-prolyl-D-phenylalanine, a compound known to inhibit binding of gp120 to CD4 receptors. Moreover, sequences of gp120 critical for binding to CD4 did not mimic the effect of gp120. Preincubation of synaptosomes with anti-galactocerebroside antibodies did not affect gp120 activity. The protein effect was retained by peptides mimicking its V3 sequence, including the cyclic V3 "universal peptide" and the linear V3 sequence BRU-C-34-A, but not RP-135 (a central portion of BRU-C-34-A). The block of the N-methyl-D-aspartate-induced [3H]noradrenaline release by 7-chlorokynurenate, an antagonist at the N-methyl-D-aspartate receptor glycine site, was competitively reversed by glycine, by V3 and by BRU-C-34-A. When added with N-methyl-D-aspartate, V3 was three to four orders of magnitude more potent than glycine (EC50 values: about 20 pM and 150 nM, respectively) in enhancing [3H]noradrenaline release. Gp120 did not release glycine or serine from synaptosomes, thus excluding indirect actions through these agents. To conclude, gp120 may act following recognition by its V3 sequence of a high-affinity site possibly coincident with the glycine site of N-methyl-D-aspartate receptors present on hippocampal terminals of noradrenergic neurons. Considering the importance of N-methyl-D-aspartate receptor activation and of noradrenaline in cognitive processes, the effects of gp120 and V3 described here may be relevant to the pathology of AIDS dementia.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1 , Hipocampo/metabolismo , Norepinefrina/metabolismo , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD4/metabolismo , Galactosilceramidas/metabolismo , Proteína gp160 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/fisiologia , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
The mechanism by which anti-fusion regulatory protein-1 (FRP-1) monoclonal antibody (mAb) induced cell fusion was investigated using U2ME-7 cells that are CD4+U937 cells transfected with the HIV gp160 gene. Protein kinase inhibitors (H-7, H-89, herbimycin A and genistein) suppressed cell fusion of Cd+U2ME-7 cells induced by anti-FRP-1 mAb. H-7 and H-89 also inhibited the cell aggregation, but herbimycin A and genistein did not. Intriguingly, only when herbimycin A was added either before or simultaneously with addition of anti-FRP-1 mAb, was cell fusion suppressed, suggesting that tyrosine kinase is related with the initial step of polykaryocyte formation. Anti-FRP-1 mAb induced the rapid tyrosine phosphorylation of multiple cellular proteins. These effects occurred within 1 min and returned to near baseline by 60 min. The rapid tyrosine phosphorylation was suppressed by herbimycin A and genistein. Although it remains to be determined which protein tyrosine kinase(s) is involved in this response, pp130 tyrosine phosphorylation appears to be a specific and early signal transmitted after the interaction of FRP-1 with a specific antibody. pp130 was present in the cytosol fraction and was distinct from pp125FAK, p130CAS, vinculin, and beta 1-integrin. Thus, our study may present evidence for a novel pathway of protein tyrosine kinases that phosphorylate specific, still unknown protein substrates during polykaryocyte formation.
Assuntos
Antígenos CD/fisiologia , Proteínas de Transporte/fisiologia , Proteína gp160 do Envelope de HIV/fisiologia , Proteínas Tirosina Quinases/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Benzoquinonas , Proteínas de Transporte/imunologia , Fusão Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Proteína-1 Reguladora de Fusão , Genes Virais , Genisteína/farmacologia , Proteína gp160 do Envelope de HIV/genética , Humanos , Lactamas Macrocíclicas , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , Rifabutina/análogos & derivados , Transdução de Sinais , Transfecção , Tirosina/metabolismoRESUMO
The human immunodeficiency virus type 1 (HIV-1) employs a number of complex strategies to interfere with the synthesis, stability, and subcellular localization of its specific cellular receptor CD4. To define better the mechanisms of inhibition of CD4 expression, we used a rabbit reticulocyte lysate in vitro system, in which cDNAs derived from HIV-1-infected cells were used to generate mRNA for the Tat, Vpu, and gp160 envelope proteins that were translated together with CD4-encoding mRNA. In the presence of microsomal membranes, we observed that cotranslation of Env mRNA resulted in a dose-dependent inhibition of CD4 translation. This effect was enhanced further when an mRNA-encoding Vpu in addition to Env mRNA was utilized. However, the activity of Vpu was mostly post-translational, since translation of Vpu alone, but not Env, was able to destabilize CD4 molecules presynthesized into microsomes. The Env-mediated inhibitory effect was specifically targeted at CD4 and did not affect the synthesis or stability of the CD8 molecule. Interestingly, mutated CD4 species, with a 20-fold lower affinity for HIV-1 Env than wild-type, were less sensitive to cotranslational inhibition. Our report identifies the envelope as the HIV-1 protein responsible for down-regulation of CD4 translation. We further propose a mechanism whereby direct interactions between gp160 and nascent CD4 molecules can cause interference with and premature termination of CD4 protein elongation.
Assuntos
Antígenos CD4/genética , Proteína gp160 do Envelope de HIV/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Animais , Sítios de Ligação , Sistema Livre de Células , Genes Virais , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Mutação , RNA Mensageiro/genética , Coelhos , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Human immunodeficiency virus binds to CD4+ T lymphocyte by the interaction, in part, between its gp120 envelope glycoprotein and the CD4 molecule. We and others have reported that the lipid kinase phosphatidylinositol-3-kinase (PI3-kinase) is associated with the CD4-p56lck complex and can be activated by various CD4 ligands. In a previous report we showed that the gp160 envelope down-regulates lymphocyte function-associated antigen-1 (LFA-1)-dependent adhesion between CD4+ T cells and B cells. This down-regulation was shown to be p56lck-dependent. Here we investigate the role of PI3-kinase in the inhibition of adhesion induced by gp160 binding to CD4. We found that gp160 activates the PI3-kinase of HUT78 CD4+ T cell lines in a way dependent on CD4-p56lck association, since no activation was detected when the interaction between CD4 and p56lck was disrupted. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002 and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by gp160. These results strongly suggest that PI3-kinase activation induced by gp160 leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. Inhibition by gp160 of cytoskeleton rearrangement-dependent, anti-CD3-mediated T cell adhesion to B cells was blocked by neutralization of PI3-kinase activity, while inhibition of cytoskeleton rearrangement-independent, Mg(2+)-induced T cell adhesion was not. These results emphasize the role of PI3-kinase in the regulation of cytoskeleton structure. It is proposed that gp160 activates both p56lck and PI3-kinase which lead to a cytoskeleton organization unfavorable for LFA-1 function.
Assuntos
Linfócitos T CD4-Positivos/citologia , Adesão Celular , Proteína gp160 do Envelope de HIV/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Antígenos CD4/fisiologia , Linfócitos T CD4-Positivos/enzimologia , Cromonas/farmacologia , Citoesqueleto/fisiologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Morfolinas/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais , WortmaninaAssuntos
Apoptose/fisiologia , Proteína gp160 do Envelope de HIV , Infecções por HIV , Animais , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos , Cálcio/metabolismo , Calmodulina/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp160 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/fisiologia , HumanosRESUMO
Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.
Assuntos
Alanina , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Epitopos Imunodominantes/química , Zíper de Leucina , Sequência de Aminoácidos , Proteína gp160 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese , Relação Estrutura-Atividade , Montagem de VírusRESUMO
We have isolated new MAbs directed against the human fusion regulatory protein 1 (FRP-1; CD98) molecule using human FRP-1-expressing L929 cells as antigens. The biological activities, and in particular the human immunodeficiency virus (HIV)-mediated fusion regulatory activity of seven anti-FRP-1/CD98 MAbs were analysed using the U937/gp160 cell line, which is a CD4+ U937 cell line expressing HIV gp160. Two MAbs induced multinucleated giant cell formation in U937/gp160 cells and the other five MAbs showed no fusion-inducing ability. However, four of these MAbs suppressed multinucleated giant cell formation of U937/gp160 cells induced by the activating anti-FRP-1 MAbs. Interestingly, five of the MAbs induced multinucleated giant cells in peripheral blood monocytes and one MAb showing fusion-inducing ability in U937/gp160 cells suppressed multinucleated giant cell formation of monocytes induced by anti-FRP-1 MAbs. Furthermore, four of the anti-FRP-1 MAbs suppressed cell fusion of Jurkat/gp160 cells, which are Jurkat cells expressing HIV gp160. Thus, FRP-1/CD98 is capable of either activating or inhibiting HIV-mediated cell fusion depending on whether an enhancing or inhibiting antibody is used, indicating that FRP-1/CD98 is a multipotential molecule. Thus, HIV-mediated cell fusion can be regulated by modification of the FRP-1 system. Furthermore, the present study demonstrates that the FRP-1 and FRP-2 systems are interdependent.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/fisiologia , Proteínas de Transporte/fisiologia , Fusão Celular , Proteína gp160 do Envelope de HIV/fisiologia , Animais , Ligação Competitiva , Fibronectinas/farmacologia , Proteína-1 Reguladora de Fusão , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
Human CD4+ cell clones expressing either gp160 or gp120 of HIV-1 under the transcriptional control of an inducible promoter were used to examine the role of Ca2+ signalling in the induction of apoptosis by envelope glycoproteins. Singlecell killing with apoptosis was induced in the cells expressing gp 160 while no such apoptosis was found in the cells expressing gp 120. An increase of intracellular Ca2+ was found in the gp 160-expressing cells but not in the gp 120-expressing cells as determined by Intracellular Ca2+ imaging analysis before the appearance of DNA fragmentation. W7, a calmodulin antagonist, blocked the elevation of Ca2+ as well as the resultant DNA fragmentation, which thus implies that the calmodulin-dependent intracellular Ca2+ release system is first activated by gp 160 and thereafter apoptosis takes place. The above results thus indicate that Ca2+ signalling plays a crucial role in the apoptosis accompanying the single-cell death induced by gp 160 in CD4+ cells.