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1.
Nature ; 623(7989): 1026-1033, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37993716

RESUMO

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.


Assuntos
Antígenos CD4 , Membrana Celular , Proteína gp120 do Envelope de HIV , HIV-1 , Multimerização Proteica , Humanos , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Capsídeo/química , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Antígenos CD4/química , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , Infecções por HIV/virologia , HIV-1/química , HIV-1/ultraestrutura , Vírion/química , Vírion/metabolismo , Vírion/ultraestrutura
2.
Nature ; 623(7989): 1017-1025, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37993719

RESUMO

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.


Assuntos
Antígenos CD4 , Proteína gp120 do Envelope de HIV , HIV-1 , Conformação Proteica , Antígenos CD4/química , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Microscopia Crioeletrônica , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , HIV-1/química , HIV-1/ultraestrutura , Rotação , Reprodutibilidade dos Testes
3.
Nat Struct Mol Biol ; 26(12): 1167-1175, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792452

RESUMO

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5's tyrosine-sulfated amino (N) terminus, release of the gp41 fusion peptide and fusion. We present 3.3 Å and 3.5 Å cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral-host cell membrane fusion.


Assuntos
Anticorpos/química , Antígenos CD4/química , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Anticorpos/metabolismo , Reações Antígeno-Anticorpo , Sítios de Ligação , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Microscopia Crioeletrônica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Receptores CCR5/imunologia , Tirosina/análogos & derivados , Tirosina/química
4.
Nature ; 565(7739): 318-323, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30542158

RESUMO

HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.


Assuntos
Antígenos CD4/química , Antígenos CD4/ultraestrutura , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/ultraestrutura , Receptores CCR5/química , Receptores CCR5/ultraestrutura , Receptores de HIV/química , Receptores de HIV/ultraestrutura , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sítios de Ligação , Antígenos CD4/isolamento & purificação , Antígenos CD4/metabolismo , Linhagem Celular , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Proteína gp120 do Envelope de HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/ultraestrutura , Humanos , Ligantes , Maraviroc/química , Maraviroc/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores CCR5/isolamento & purificação , Receptores CCR5/metabolismo , Receptores de HIV/antagonistas & inibidores , Receptores de HIV/metabolismo
5.
Nature ; 547(7663): 360-363, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28700571

RESUMO

For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.


Assuntos
Regulação Alostérica , Microscopia Crioeletrônica , HIV-1/química , HIV-1/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Anticorpos/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Antígenos CD4/química , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/ultraestrutura , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Ligantes , Modelos Moleculares , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores de HIV/química , Receptores de HIV/metabolismo , Receptores de HIV/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
6.
Nat Struct Mol Biol ; 24(4): 370-378, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28218750

RESUMO

Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-Å resolution. A single CD4 molecule is embraced by a quaternary HIV-1-Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.


Assuntos
Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação , Antígenos CD4/ultraestrutura , Microscopia Crioeletrônica , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , Infecções por HIV/metabolismo , Humanos , Cinética , Mutagênese , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de Superfície
7.
Biophys J ; 95(1): 40-53, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18339756

RESUMO

We simulated the docking of human immunodeficiency virus (HIV) with a cell membrane using Brownian adhesive dynamics. The main advance in the current version of Brownian adhesive dynamics is that we use a simple bead-spring model to coarsely approximate the role of gp120 trimerization on HIV docking. We used our simulations to elucidate the effect of env spike density on the rate and probability of HIV binding, as well as the probability that each individual gp120 trimer is fully engaged. We found that for typical CD4 surface densities, viruses expressing as few as 8 env spikes will dock with binding rate constants comparable to viruses expressing 72 spikes. We investigated the role of cellular receptor diffusion on the degree of binding achieved by the virus on both short timescales (where binding has reached steady state but before substantial receptor accumulation in the viral-cell contact zone has occurred) and long timescales (where the system has reached steady state). On short timescales, viruses with 10-23 env trimers most efficiently form fully engaged trimers. On long timescales, all gp120 in the contact area will become bound to CD4. We found that it takes seconds for engaged trimers to cluster CD4 molecules in the contact zone, which partially explains the deleay in viral entry.


Assuntos
Antígenos CD4/química , Antígenos CD4/ultraestrutura , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/ultraestrutura , Modelos Químicos , Modelos Moleculares , Adesividade , Sítios de Ligação , Simulação por Computador , Difusão , Dimerização , Modelos Estatísticos , Ligação Proteica
8.
Biochim Biophys Acta ; 1768(9): 2107-19, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17560540

RESUMO

Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1(IIIB) gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric-electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.


Assuntos
Fármacos Anti-HIV/química , Arginina/química , Antígenos CD4/química , Modelos Químicos , Neomicina/química , Internalização do Vírus , Sítios de Ligação , Antígenos CD4/ultraestrutura , Simulação por Computador , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas
9.
Wound Repair Regen ; 10(4): 241-4, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12191006

RESUMO

Recently, we found CD3-CD4(bright) cells with comparative specificity for normal rat liver. In the current study, we investigated the type and form of both CD3-CD4(bright) cells and CD3-CD4(dull) cells in the rat liver. The surface phenotype of hepatic mononuclear cells in Lewis rats was identified by using monoclonal antibodies including anti-CD4, anti-CD3, and antimacrophage in conjunction with two- or three-color immunofluorescence analysis. CD3-CD4(bright) cells and CD3-CD4(dull) cells were examined morphologically using May-Giemsa staining and scanning electron microscopy. The distribution of CD3-CD4(bright) cells and CD3-CD4(dull) cells 48 hours after intravenous administration of liposome-encapsulated dichloromethylene diphosphate was also investigated. In comparison to CD3-CD4(dull) cells, CD3-CD4(bright) cells were slightly larger macrophages with abundant cytoplasmic granules, being present with comparative specificity for normal rat liver and showing negligible effects by intravenous liposome-encapsulated dichloromethylene diphosphate administration. These data suggest that in normal young rat liver these CD3-CD4(dull) and CD3-CD4(bright) cells may be dendritic cells and Kupffer cells that shift from the liver to the spleen or vice versa. These cells may also be able to locally proliferate in liver or spleen due to changes in the developing liver.


Assuntos
Complexo CD3/imunologia , Complexo CD3/ultraestrutura , Antígenos CD4/imunologia , Antígenos CD4/ultraestrutura , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/ultraestrutura , Regeneração Hepática/imunologia , Fígado/imunologia , Fígado/ultraestrutura , Animais , Complexo CD3/análise , Antígenos CD4/análise , Modelos Animais de Doenças , Contagem de Leucócitos , Masculino , Ratos , Ratos Endogâmicos Lew , Sensibilidade e Especificidade
11.
J Immunol ; 158(3): 1157-64, 1997 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9013955

RESUMO

The binding of the surface envelope glycoprotein gp120 to its receptor, CD4, has been well characterized and is the primary basis for the cell tropism of HIV. In this study, the interaction between recombinant soluble CD4 and native membrane-associated CD4 with gp120 is probed by the use of mAbs. Complexation of gp120 with both forms of CD4 induces conformational epitopes that can be defined with specific mAbs. CG1, CG7, and CG8 are three novel mAbs that have a distinct preference for CD4 complexed over noncomplexed with gp120. The epitopes of these unique mAbs were mapped by cross-inhibition with previously characterized mAbs to a region encompassing the CDR2 and CDR3 loops in domain 1 of CD4. Systematic analysis of CG mAbs binding to CD4 and CD4/gp120 complex delineates a region in the D1 domain of CD4 that undergoes conformational rearrangements upon gp120 binding to its receptor.


Assuntos
Antígenos CD4/ultraestrutura , Proteína gp120 do Envelope de HIV/ultraestrutura , Animais , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Antígenos CD4/química , Linfócitos T CD4-Positivos/imunologia , Mapeamento de Epitopos , Proteína gp120 do Envelope de HIV/química , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Conformação Proteica , Deleção de Sequência , Solubilidade
12.
Lik Sprava ; (7-8): 155-9, 1995.
Artigo em Russo | MEDLINE | ID: mdl-8846357

RESUMO

A model is proposed and well based of structure of the receptor apparatus of interacting HIV target cells: Langerhans cells (LC) and helper cells (HC)--designed to explain feasibility of realizing a double activation signal, together with some features of immune pathogenesis of HIV-infection (morphofunctional abnormalities in certain subpopulations of the immune system such as HC, suppressor cells, killer cells, natural killers, lymph cells, macrophages, LC), as well as immunogenetic predisposition to AIDS.


Assuntos
Modelos Biológicos , Modelos Estruturais , Receptores de HIV/ultraestrutura , Antígenos CD4/imunologia , Antígenos CD4/ultraestrutura , Antígenos CD8/imunologia , Antígenos CD8/ultraestrutura , Comunicação Celular , Humanos , Células de Langerhans/citologia , Células de Langerhans/imunologia , Receptores de HIV/imunologia , Relação Estrutura-Atividade , Propriedades de Superfície , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia
13.
Scand J Immunol ; 41(2): 148-56, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7863261

RESUMO

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down-modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down-modulation was ganglioside-dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down-modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3-treated cells, suggests intracellular degradation of the internalized CD4 molecules.


Assuntos
Antígenos CD4/sangue , Linfócitos T CD4-Positivos/imunologia , Gangliosídeo G(M3)/farmacologia , Antígenos CD4/ultraestrutura , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/ultraestrutura , Citometria de Fluxo , Humanos , Microscopia Imunoeletrônica
15.
J Exp Med ; 178(4): 1209-22, 1993 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-8376930

RESUMO

The phorbol ester phorbol myristate acetate (PMA) induces a rapid downregulation of CD4 from the surface of T cells and lymphocytic cell lines, as well as from CD4-transfected nonlymphoid cells. Here we have studied the mechanisms of this phorbol ester-induced CD4 modulation. Using HeLa-CD4 or NIH-3T3-CD4 cells, in which the endocytosis of CD4 is not influenced by the protein tyrosine kinase p56lck, we show that PMA enhanced the uptake of CD4, increasing the rate of CD4 endocytosis three to five-fold, and doubling the proportion of CD4 found inside the cells. Trafficking of a CD4 mutant lacking the major portion of the cytoplasmic domain, as well as fluid phase endocytosis were not affected by PMA treatment. Studies in which clathrin-coated pits were disrupted through the use of hypertonic media indicated that both the constitutive and PMA-induced CD4 uptake occurred through coated vesicles. Electron microscopy demonstrated directly that PMA increases the association of CD4 with coated pits. Immunofluorescent staining of internalized CD4 showed that PMA also diverted CD4 from the early endosome-plasma membrane recycling pathway to a mannose 6-phosphate receptor-containing late endosomal compartment. In lymphoid or p56lck-expressing transfected cells, these effects were preceded by the PMA-induced dissociation of CD4 and p56lck, which released CD4 and made possible increased endocytosis and altered intracellular trafficking. Together these results indicate that phorbol esters have multiple effects on the normal endocytosis and trafficking of CD4, and suggest that phosphorylation may influence the interaction of CD4 with coated pits.


Assuntos
Antígenos CD4/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células 3T3 , Animais , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/genética , Antígenos CD4/ultraestrutura , Clatrina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Microscopia Eletrônica , Transfecção
16.
Proc Natl Acad Sci U S A ; 90(2): 507-11, 1993 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-8093643

RESUMO

Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of human rhinovirus 14 and CD4, homologous to human rhinovirus 16 and intercellular adhesion molecule 1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.


Assuntos
Antígenos CD/ultraestrutura , Moléculas de Adesão Celular/ultraestrutura , Receptores Virais/ultraestrutura , Rhinovirus/ultraestrutura , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Criopreservação , Humanos , Processamento de Imagem Assistida por Computador , Molécula 1 de Adesão Intercelular , Microscopia Eletrônica , Modelos Moleculares , Receptores Virais/metabolismo , Rhinovirus/metabolismo
17.
J Virol ; 65(9): 4893-901, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1714523

RESUMO

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.


Assuntos
Antígenos CD4/fisiologia , Linfócitos T CD4-Positivos/microbiologia , HIV-1/crescimento & desenvolvimento , HIV-2/crescimento & desenvolvimento , Antígenos CD4/ultraestrutura , Fusão Celular , Análise Mutacional de DNA , Infecções por HIV/patologia , HIV-1/metabolismo , HIV-1/patogenicidade , HIV-2/metabolismo , HIV-2/patogenicidade , Humanos , Técnicas In Vitro , Hibridização de Ácido Nucleico , RNA Viral/genética , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Replicação Viral
18.
J Dermatol ; 18(7): 377-92, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1724250

RESUMO

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.


Assuntos
Antígenos CD4/imunologia , Epitopos , Produtos do Gene env/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Células de Langerhans/imunologia , Precursores de Proteínas/imunologia , Receptores de HIV/imunologia , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/imunologia , Antígenos de Superfície/ultraestrutura , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/ultraestrutura , Membrana Celular/ultraestrutura , Endocitose/imunologia , Células Epidérmicas , Produtos do Gene env/ultraestrutura , Proteína gp120 do Envelope de HIV/ultraestrutura , Proteína gp160 do Envelope de HIV , HIV-1/ultraestrutura , Humanos , Células de Langerhans/ultraestrutura , Microscopia Eletrônica , Precursores de Proteínas/ultraestrutura , Receptores de HIV/efeitos dos fármacos , Receptores de HIV/ultraestrutura , Tripsina/farmacologia
19.
J Immunol ; 145(12): 4072-8, 1990 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1701782

RESUMO

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.


Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/fisiopatologia , Fragmentos de Peptídeos/metabolismo , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Antígenos CD4/ultraestrutura , Fusão Celular , Epitopos , Humanos , Técnicas In Vitro , Fragmentos de Peptídeos/síntese química , Mapeamento de Peptídeos , Ligação Proteica , Relação Estrutura-Atividade
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