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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
J Biol Chem ; 295(46): 15576-15587, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-32883811

RESUMO

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

3.
Science ; 362(6421): 1423-1428, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30573630

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 and Vps4 puncta within nanotubes whose presence followed ATP release, correlated with force generation and nanotube constriction, and preceded scission. These observations directly verify long-standing predictions that ATP-hydrolyzing assemblies of ESCRT-III and Vps4 sever membranes.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Biocatálise , Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Hidrólise , Nanotubos , Proteínas de Saccharomyces cerevisiae/química , Lipossomas Unilamelares
4.
Cell Host Microbe ; 24(3): 417-428.e5, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30146390

RESUMO

Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Membrana Celular/virologia , Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Liberação de Vírus , Linhagem Celular , Membrana Celular/imunologia , Vírus Chikungunya/genética , Humanos , Replicação Viral
5.
PLoS Negl Trop Dis ; 12(7): e0006693, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063703

RESUMO

BACKGROUND: Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. CONCLUSIONS/SIGNIFICANCE: Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Proteínas Luminescentes/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Vírus Chikungunya/genética , Vírus Chikungunya/ultraestrutura , Microscopia Crioeletrônica , Feminino , Fluorescência , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
6.
Elife ; 52016 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-27343348

RESUMO

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5' untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.


Assuntos
HIV-1/fisiologia , RNA Viral/metabolismo , Lipossomas Unilamelares/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Humanos
7.
Proc Natl Acad Sci U S A ; 112(52): 15892-7, 2015 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-26668364

RESUMO

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Bicamadas Lipídicas/metabolismo , Algoritmos , Membrana Celular/virologia , Recuperação de Fluorescência Após Fotodegradação , HIV/fisiologia , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência/métodos , Modelos Biológicos , Transporte Proteico , Replicação Viral
8.
Dev Cell ; 35(4): 397-8, 2015 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-26609952

RESUMO

In a recent issue of Cell, Chiaruttini et al. (2015) reveal the mechanical properties of the mysterious spiral filaments formed by the yeast ESCRT-III protein Snf7. The spirals are shown to be springs whose bending drives membrane deformation and perhaps membrane scission.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Bicamadas Lipídicas/química , Modelos Moleculares , Leveduras/metabolismo
9.
Elife ; 32014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25490155

RESUMO

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.


Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Sequência de Aminoácidos , Animais , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/ultraestrutura , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
10.
J Virol ; 88(14): 7893-903, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24789788

RESUMO

Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. Importance: HIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds, often seemingly in contact with the Gag layer. Here, we show that this spatial association is observed independently of the proposed actin-binding domain of HIV-1. The absence of this domain also did not affect actin incorporation and had a minor effect on the viral assembly rate. Furthermore, actin was not enriched in the virus compared to the average levels in the respective producing cell. Our data argue against a specific recruitment of actin to HIV-1 budding sites by the nucleocapsid domain of Gag.


Assuntos
Actinas/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Montagem de Vírus , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , HIV-1/genética , Humanos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Estrutura Terciária de Proteína , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
11.
Proc Natl Acad Sci U S A ; 109(42): 16928-33, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-23027949

RESUMO

Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PI(4,5)P(2). These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYP(X)(n)L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/metabolismo , Subunidades Proteicas/metabolismo , Montagem de Vírus/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Oligonucleotídeos/metabolismo , Montagem de Vírus/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/isolamento & purificação
12.
J Biol Chem ; 287(33): 28144-51, 2012 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-22718754

RESUMO

The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Subunidades Proteicas/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corantes Fluorescentes/química , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
PLoS Pathog ; 6(11): e1001173, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21124872

RESUMO

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.


Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , HIV-1/química , HIV-1/ultraestrutura , RNA Viral/química , Vírion/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Células Cultivadas , Glioblastoma/metabolismo , HIV-1/fisiologia , Humanos , RNA Viral/metabolismo , Linfócitos T/virologia , Vírion/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
14.
Cell ; 143(6): 875-87, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145455

RESUMO

Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms.


Assuntos
Estruturas da Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Animais , Estruturas da Membrana Celular/química , Vesículas Citoplasmáticas/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos
15.
Mol Cell ; 39(4): 560-9, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20797628

RESUMO

Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding.


Assuntos
Neoplasias Encefálicas/ultraestrutura , Glioblastoma/ultraestrutura , Polirribossomos/ultraestrutura , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Tomografia com Microscopia Eletrônica , Glioblastoma/metabolismo , Humanos , Imageamento Tridimensional , Modelos Moleculares , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Relação Estrutura-Atividade
16.
Cell Host Microbe ; 4(6): 592-9, 2008 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19064259

RESUMO

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.


Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , HIV-1/fisiologia , HIV-1/ultraestrutura , Vírion/ultraestrutura , Montagem de Vírus , Linhagem Celular , Células HeLa , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura
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