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1.
Cell ; 187(4): 831-845.e19, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38301645

RESUMO

The paraneoplastic Ma antigen (PNMA) proteins are associated with cancer-induced paraneoplastic syndromes that present with an autoimmune response and neurological symptoms. Why PNMA proteins are associated with this severe autoimmune disease is unclear. PNMA genes are predominantly expressed in the central nervous system and are ectopically expressed in some tumors. We show that PNMA2, which has been co-opted from a Ty3 retrotransposon, encodes a protein that is released from cells as non-enveloped virus-like capsids. Recombinant PNMA2 capsids injected into mice induce autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, whereas a capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic disease show similar preferential binding to spike capsid epitopes. PNMA2 capsid-injected mice develop learning and memory deficits. These observations suggest that PNMA2 capsids act as an extracellular antigen, capable of generating an autoimmune response that results in neurological deficits.


Assuntos
Antígenos de Neoplasias , Neoplasias , Proteínas do Tecido Nervoso , Síndromes Paraneoplásicas do Sistema Nervoso , Animais , Humanos , Camundongos , Autoanticorpos , Capsídeo/metabolismo , Epitopos , Neoplasias/complicações , Síndromes Paraneoplásicas do Sistema Nervoso/metabolismo , Síndromes Paraneoplásicas do Sistema Nervoso/patologia , Antígenos de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo
2.
Cell ; 172(1-2): 275-288.e18, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29328916

RESUMO

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Exossomos/metabolismo , Produtos do Gene gag/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Endocitose , Feminino , Produtos do Gene gag/química , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia
4.
Nature ; 603(7902): 706-714, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104837

RESUMO

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Assuntos
COVID-19/patologia , COVID-19/virologia , Fusão de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Internalização do Vírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Convalescença , Feminino , Humanos , Soros Imunes/imunologia , Intestinos/patologia , Intestinos/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Mucosa Nasal/patologia , Mucosa Nasal/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Técnicas de Cultura de Tecidos , Virulência , Replicação Viral
5.
Cell ; 150(3): 508-20, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863005

RESUMO

Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.


Assuntos
Membrana Celular/ultraestrutura , Endocitose , Saccharomyces cerevisiae/ultraestrutura , Actinas/metabolismo , Tomografia com Microscopia Eletrônica , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo
6.
Nature ; 592(7853): 277-282, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545711

RESUMO

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.


Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/terapia , COVID-19/virologia , Evolução Molecular , Mutagênese/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Doença Crônica , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunização Passiva , Terapia de Imunossupressão , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Mutação , Filogenia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Eliminação de Partículas Virais , Soroterapia para COVID-19
7.
Nature ; 587(7834): 495-498, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32908308

RESUMO

Influenza A virus causes millions of severe cases of disease during annual epidemics. The most abundant protein in influenza virions is matrix protein 1 (M1), which mediates virus assembly by forming an endoskeleton beneath the virus membrane1. The structure of full-length M1, and how it oligomerizes to mediate the assembly of virions, is unknown. Here we determine the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We find that the C-terminal domain of M1 is disordered in solution but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerizing M1 into linear strands that coat the interior surface of the membrane of the assembling virion. In the M1 polymer, five histidine residues-contributed by three different monomers of M1-form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore reveal mechanisms of influenza virus assembly and disassembly.


Assuntos
Microscopia Crioeletrônica , Vírus da Influenza A Subtipo H3N2/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/ultraestrutura , Animais , Cães , Células HEK293 , Histidina , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A Subtipo H3N2/ultraestrutura , Células Madin Darby de Rim Canino , Modelos Moleculares , Proteínas da Matriz Viral/metabolismo , Vírion/química , Vírion/metabolismo , Vírion/ultraestrutura
8.
Nature ; 588(7838): 498-502, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32805734

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.


Assuntos
Microscopia Crioeletrônica , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Vírion/química , Vírion/ultraestrutura , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Maleabilidade , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Vírion/isolamento & purificação , Vírion/metabolismo
9.
Proc Natl Acad Sci U S A ; 120(18): e2220545120, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37094124

RESUMO

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but detailed structural information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryoelectron tomography and single particle analysis (SPA) cryoelectron microscopy to determine structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a method of liposome scaffold-based in vitro lattice assembly ("lattice templating") that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (lenacapavir). Our work reveals key structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and SPA as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.


Assuntos
Proteínas do Capsídeo , HIV-1 , Proteínas do Capsídeo/química , Capsídeo/metabolismo , HIV-1/metabolismo , Microscopia Crioeletrônica/métodos , Montagem de Vírus
10.
Proc Natl Acad Sci U S A ; 120(16): e2220557120, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37040417

RESUMO

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6, and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple cofactors to a single site is coordinated, all remain to be elucidated. Here, using single-particle cryoEM, we have determined the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to ~3 Å resolution. We also determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology.


Assuntos
Capsídeo , HIV-1 , Capsídeo/metabolismo , Proteínas do Capsídeo/química , HIV-1/genética , Ligação Proteica , Citoplasma/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(10): e2117781119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35238630

RESUMO

SignificanceThe mature capsids of HIV-1 are transiently stable complexes that self-assemble around the viral genome during maturation, and uncoat to release preintegration complexes that archive a double-stranded DNA copy of the virus in the host cell genome. However, a detailed view of how HIV cores rupture remains lacking. Here, we elucidate the physical properties involved in capsid rupture using a combination of large-scale all-atom molecular dynamics simulations and cryo-electron tomography. We find that intrinsic strain on the capsid forms highly correlated patterns along the capsid surface, along which cracks propagate. Capsid rigidity also increases with high strain. Our findings provide fundamental insight into viral capsid uncoating.


Assuntos
Capsídeo/fisiologia , HIV-1/fisiologia , Desenvelopamento do Vírus , Capsídeo/química , Proteínas do Capsídeo/química , Linhagem Celular , Tomografia com Microscopia Eletrônica/métodos , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica
12.
Biophys J ; 123(3): 389-406, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38196190

RESUMO

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2' acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.


Assuntos
HIV-1 , Simulação de Dinâmica Molecular , HIV-1/metabolismo , Ligação Proteica , Membranas/metabolismo , Lipídeos , Membrana Celular/metabolismo
13.
PLoS Pathog ; 18(7): e1010583, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35905112

RESUMO

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.


Assuntos
COVID-19 , SARS-CoV-2 , Dissulfetos , Humanos , Ligação Proteica , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
Nature ; 561(7724): 561-564, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30224749

RESUMO

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.


Assuntos
Chaetomium/química , Chaetomium/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/ultraestrutura , Chaetomium/metabolismo , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Transporte Proteico , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Nexinas de Classificação/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo
15.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33397805

RESUMO

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.


Assuntos
Proteínas do Capsídeo/ultraestrutura , Infecções por HIV/genética , HIV-1/genética , Fator de Transcrição Sp1/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Cristalografia por Raios X , Infecções por HIV/virologia , Soropositividade para HIV , HIV-1/patogenicidade , HIV-1/ultraestrutura , Humanos , Simulação de Dinâmica Molecular , Multimerização Proteica/genética , Proteólise , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/genética , Vírion/genética , Vírion/patogenicidade , Montagem de Vírus/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
16.
Nature ; 551(7680): 394-397, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-29144446

RESUMO

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.


Assuntos
Microscopia Crioeletrônica , Ebolavirus/química , Ebolavirus/ultraestrutura , Tomografia com Microscopia Eletrônica , Proteínas do Nucleocapsídeo/ultraestrutura , Nucleocapsídeo/química , Nucleocapsídeo/ultraestrutura , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Marburgvirus/química , Modelos Moleculares , Conformação Molecular , Proteínas do Nucleocapsídeo/química , RNA Viral/química , RNA Viral/ultraestrutura , Células Vero
18.
J Virol ; 95(15): e0020321, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33963055

RESUMO

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines.


Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Camundongos , Estabilidade Proteica , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
19.
PLoS Pathog ; 16(1): e1008277, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31986188

RESUMO

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.


Assuntos
Anemia Infecciosa Equina/metabolismo , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Anemia Infecciosa Equina/fisiologia , Ácido Fítico/metabolismo , Vírion/fisiologia , Sequência de Aminoácidos , Animais , Tomografia com Microscopia Eletrônica , Anemia Infecciosa Equina/virologia , Produtos do Gene gag/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , HIV-1/ultraestrutura , Cavalos , Interações Hospedeiro-Patógeno , Vírus da Anemia Infecciosa Equina/química , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/ultraestrutura , Alinhamento de Sequência , Vírion/genética , Vírion/ultraestrutura , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(20): 10048-10057, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31036670

RESUMO

Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation. They encode a Gag polypeptide, the capsid domains of which can oligomerize to form a virus-like particle. The structures of retroviral capsids have been extensively described. They assemble an immature viral particle through oligomerization of full-length Gag. Proteolytic cleavage of Gag results in a mature, infectious particle. In contrast, the absence of structural data on LTR retrotransposon capsids hinders our understanding of their function and evolutionary relationships. Here, we report the capsid morphology and structure of the archetypal Gypsy retrotransposon Ty3. We performed electron tomography (ET) of immature and mature Ty3 particles within cells. We found that, in contrast to retroviruses, these do not change size or shape upon maturation. Cryo-ET and cryo-electron microscopy of purified, immature Ty3 particles revealed an irregular fullerene geometry previously described for mature retrovirus core particles and a tertiary and quaternary arrangement of the capsid (CA) C-terminal domain within the assembled capsid that is conserved with mature HIV-1. These findings provide a structural basis for studying retrotransposon capsids, including those domesticated in higher organisms. They suggest that assembly via a structurally distinct immature capsid is a later retroviral adaptation, while the structure of mature assembled capsids is conserved between LTR retrotransposons and retroviruses.


Assuntos
Evolução Biológica , Capsídeo/ultraestrutura , Retroviridae/ultraestrutura , Microscopia Crioeletrônica , Retroviridae/genética
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