RESUMEN
A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.
Asunto(s)
VIH-1/metabolismo , Fosfatos de Inositol/metabolismo , Virión/metabolismo , Ensamble de Virus , Arginina/metabolismo , Cápside/química , Cápside/metabolismo , Cristalografía por Rayos X , VIH-1/química , VIH-1/genética , Técnicas In Vitro , Lisina/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Virión/química , Virión/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as '6BH5, 6BHT and 6BHS' instead of '6BHR, 6BHT and 6BHS'; this has been corrected online.
RESUMEN
With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.
Asunto(s)
Endosomas , Orthobunyavirus , Internalización del Virus , Animales , Microscopía por Crioelectrón , Endosomas/virología , Mamíferos , Orthobunyavirus/fisiologíaRESUMEN
The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
Asunto(s)
Electrones , Procesamiento de Imagen Asistido por Computador , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Tomografía Computarizada por Rayos XRESUMEN
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.
Asunto(s)
Anemia Infecciosa Equina/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus de la Anemia Infecciosa Equina/fisiología , Ácido Fítico/metabolismo , Virión/fisiología , Secuencia de Aminoácidos , Animales , Tomografía con Microscopio Electrónico , Anemia Infecciosa Equina/virología , Productos del Gen gag/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , VIH-1/ultraestructura , Caballos , Interacciones Huésped-Patógeno , Virus de la Anemia Infecciosa Equina/química , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/ultraestructura , Alineación de Secuencia , Virión/genética , Virión/ultraestructura , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.
Asunto(s)
Cápside/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , VIH-1/química , VIH-1/ultraestructura , Virión/química , Virión/ultraestructura , Cápside/química , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Células HEK293 , Humanos , Virus del Mono Mason-Pfizer/química , Virus del Mono Mason-Pfizer/ultraestructura , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Ensamble de VirusRESUMEN
Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
Asunto(s)
Cápside/química , Cápside/ultraestructura , Virus de la Leucemia Murina/química , Virus de la Leucemia Murina/ultraestructura , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Tomografía con Microscopio Electrónico , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/ultraestructura , Células HEK293 , VIH-1/química , VIH-1/genética , VIH-1/ultraestructura , Humanos , Virus de la Leucemia Murina/genética , Ratones , Modelos Moleculares , Dominios Proteicos , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Virión/química , Virión/genética , Virión/ultraestructuraRESUMEN
Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Microscopía por Crioelectrón/métodos , Manejo de Especímenes/métodos , Impresión Tridimensional , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
Cryo-electron tomography (cryo-ET) allows cellular ultrastructures and macromolecular complexes to be imaged in three-dimensions in their native environments. Cryo-electron tomograms are reconstructed from projection images taken at defined tilt-angles. In order to recover high-resolution information from cryo-electron tomograms, it is necessary to measure and correct for the contrast transfer function (CTF) of the microscope. Most commonly, this is performed using protocols that approximate the sample as a two-dimensional (2D) plane. This approximation accounts for differences in defocus and therefore CTF across the tilted sample. It does not account for differences in defocus of objects at different heights within the sample; instead, a 3D approach is required. Currently available approaches for 3D-CTF correction are computationally expensive and have not been widely implemented. Here we simulate the benefits of 3D-CTF correction for high-resolution subtomogram averaging, and present a user-friendly, computationally-efficient 3D-CTF correction tool, NovaCTF, that is compatible with standard tomogram reconstruction workflows in IMOD. We validate the approach on synthetic data and test it using subtomogram averaging of real data. Consistent with our simulations, we find that 3D-CTF correction allows high-resolution structures to be obtained with much smaller subtomogram averaging datasets than are required using 2D-CTF. We also show that using equivalent dataset sizes, 3D-CTF correction can be used to obtain higher-resolution structures. We present a 3.4Å resolution structure determined by subtomogram averaging.
Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Flujo de Trabajo , Proteínas de la Cápside/química , VIH-1/química , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
UNLABELLED: The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE: Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.
Asunto(s)
Proteínas de la Cápside/metabolismo , Genoma Viral , Virus del Mono Mason-Pfizer/fisiología , ARN Viral/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de la Cápside/genética , Línea Celular , Microscopía por Crioelectrón , Productos del Gen gag , Humanos , Virus del Mono Mason-Pfizer/ultraestructura , Mutación , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes , Ensamble de Virus/genéticaRESUMEN
The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
Asunto(s)
VIH-1/química , VIH-1/ultraestructura , Nanotubos/química , Nanotubos/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , VIH-1/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Virión/química , Virión/metabolismo , Virión/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
UNLABELLED: The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of â¼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCE: Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.
Asunto(s)
Cápside/ultraestructura , Productos del Gen gag/química , Virus del Sarcoma de Rous/ultraestructura , Cápside/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Tomografía con Microscopio Electrónico , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Productos del Gen gag/genética , VIH-1/química , VIH-1/ultraestructura , Virus del Mono Mason-Pfizer/química , Virus del Mono Mason-Pfizer/ultraestructura , Modelos Moleculares , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Virus del Sarcoma de Rous/química , Proteínas Virales/química , Proteínas Virales/aislamiento & purificación , Ensamble de Virus/fisiologíaRESUMEN
The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.
Asunto(s)
Tomografía con Microscopio Electrónico , Matriz Extracelular , Transporte Biológico , Movimiento Celular , Citosol , Tomografía con Microscopio Electrónico/métodos , Matriz Extracelular/ultraestructuraRESUMEN
Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Multimerización de Proteína , Virus Vaccinia/ultraestructura , Virus Vaccinia/química , Virus Vaccinia/metabolismo , Poxviridae/ultraestructura , Poxviridae/metabolismo , Poxviridae/química , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/ultraestructura , Proteínas del Núcleo Viral/metabolismo , HumanosRESUMEN
Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15 Å. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason-Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5 Å. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Productos del Gen gag/química , Procesamiento de Imagen Asistido por Computador/métodos , Virus del Mono Mason-Pfizer/química , Conformación ProteicaRESUMEN
Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall's mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed "meshing," which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is-at least in part-composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at -45° and +45° relative to the cell's long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.
Asunto(s)
Celulosa , Cebollas , Pared Celular/metabolismo , Tomografía con Microscopio Electrónico , Pectinas/metabolismoRESUMEN
The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses.
Asunto(s)
VIH-1/fisiología , Ácido Fítico/metabolismo , Retroviridae/fisiología , Ensamble de Virus , Sitios de Unión , Proteínas de la Cápside , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Mutación , Proteínas de los Retroviridae/metabolismo , Transcripción Reversa , Virus del Sarcoma de Rous/fisiología , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.
Asunto(s)
Proteínas de la Cápside/metabolismo , Cápside/ultraestructura , Ácido Fítico/metabolismo , Virus del Sarcoma de Rous/ultraestructura , Ensamble de Virus , Cápside/metabolismo , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Virus del Sarcoma de Rous/patogenicidad , Virus del Sarcoma de Rous/fisiología , Imagen Individual de Molécula , Transfección , Liberación del VirusRESUMEN
De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.