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2.
Cells ; 11(11)2022 05 27.
Article in English | MEDLINE | ID: mdl-35681454

ABSTRACT

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.


Subject(s)
COVID-19 , Transmissible gastroenteritis virus , Detergents/pharmacology , Humans , SARS-CoV-2 , Transmissible gastroenteritis virus/metabolism , Virion/metabolism
3.
Mol Microbiol ; 117(4): 837-850, 2022 04.
Article in English | MEDLINE | ID: mdl-34967475

ABSTRACT

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes. We previously identified double membrane vesicles (DMVs) in the cytoplasm of cells infected with Berne virus (BEV), the prototype member of the Torovirus genus (Nidovirales Order). Our previous analysis by transmission electron microscopy suggested that the DMVs form a reticulovesicular network (RVN) analogous those described for the related severe acute respiratory syndrome coronavirus (SARS-CoV-1). Here, we used serial sectioning and electron tomography to characterize the architecture of torovirus replication organelles, and to learn about their biogenesis and dynamics during the infection. The formation of a RVN in BEV infected cells was confirmed, where the outer membranes of the DMVs are interconnected with each other and with the ER. Paired or zippered ER membranes connected with the DMVs were also observed, and likely represent early structures that evolve to give rise to DMVs. Also, paired membranes forming small spherule-like invaginations were observed at late time post-infection. Although resembling in size, the tomographic analysis show that these structures are clearly different from the true spherules described previously for coronaviruses. Hence, BEV shows important similarities, but also some differences, in the architecture of the replication organelles with other nidoviruses.


Subject(s)
Torovirus , Electron Microscope Tomography , Endoplasmic Reticulum , Virus Replication
4.
Viruses ; 13(11)2021 10 20.
Article in English | MEDLINE | ID: mdl-34834916

ABSTRACT

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.


Subject(s)
Tomography, X-Ray/methods , Virus Diseases/virology , Virus Physiological Phenomena , Animals , Antiviral Agents/pharmacology , Humans , Tomography, X-Ray/instrumentation , Virus Diseases/diagnostic imaging , Virus Diseases/drug therapy , Viruses/chemistry , Viruses/drug effects
5.
mSphere ; 5(5)2020 10 14.
Article in English | MEDLINE | ID: mdl-33055261

ABSTRACT

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.


Subject(s)
Babesia/growth & development , Erythrocytes/parasitology , Host-Pathogen Interactions , Trophozoites/growth & development , Animals , Babesia/pathogenicity , Babesia/ultrastructure , Babesiosis/parasitology , Cattle , Cattle Diseases/parasitology , Erythrocytes/ultrastructure , Humans , Microscopy, Electron, Transmission , Microscopy, Video , Reproduction, Asexual , Time-Lapse Imaging , Tomography, X-Ray , Trophozoites/ultrastructure
6.
Sci Rep ; 9(1): 7187, 2019 05 10.
Article in English | MEDLINE | ID: mdl-31076588

ABSTRACT

We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the proteome of thymocyte centrosomes to data published for KE37 cells, focusing on proteins associated with centriole disengagement and centrosome separation. The data obtained enhances our understanding of the protein system joining the centrioles, a system comprised of a branched network of fibers linked to an apparently amorphous density that was partially characterized here. A number of proteins were localized to the amorphous density by immunolabeling (C-NAP1, cohesin SMC1, condensin SMC4 and NCAPD2), yet not DNA. In conjuction, these data not only extend our understanding of centrosomes but they will help refine the model that focus on the protein system associated with the centriolar junction.


Subject(s)
Centrosome/metabolism , Proteomics/methods , Thymocytes/cytology , Animals , Cell Line , Cryoelectron Microscopy , Electron Microscope Tomography , Gene Regulatory Networks , Sheep , Thymocytes/metabolism
7.
J Cell Sci ; 130(1): 83-89, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27505890

ABSTRACT

Macroautophagy is morphologically characterized by autophagosome formation. Autophagosomes are double-membraned vesicles that sequester cytoplasmic components for further degradation in the lysosome. Basal autophagy is paramount for intracellular quality control in post-mitotic cells but, surprisingly, the number of autophagosomes in post-mitotic neurons is very low, suggesting that alternative degradative structures could exist in neurons. To explore this possibility, we have examined neuronal subcellular architecture by performing three-dimensional (3D) electron tomography analysis of mouse brain tissue that had been preserved through high-pressure freezing. Here, we report that sequestration of neuronal cytoplasmic contents occurs at the Golgi complex in distinct and dynamic structures that coexist with autophagosomes in the brain. These structures are composed of several concentric double-membraned layers that appear to be formed simultaneously by the direct bending and sealing of discrete Golgi stacks. These structures are labelled for proteolytic enzymes, and lysosomes and late endosomes are found in contact with them, leading to the possibility that the sequestered material could be degraded inside them. Our findings highlight the key role that 3D electron tomography, together with tissue rapid-freezing techniques, will have in gaining new knowledge about subcellular architecture.


Subject(s)
Brain/ultrastructure , Electron Microscope Tomography/methods , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Imaging, Three-Dimensional , Neurons/metabolism , Neurons/ultrastructure , Animals , Cryopreservation , Mice, Inbred C57BL
8.
Eur J Immunol ; 46(10): 2376-2387, 2016 10.
Article in English | MEDLINE | ID: mdl-27405273

ABSTRACT

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.


Subject(s)
Actins/metabolism , Cell Movement , Clathrin/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Phosphoproteins/metabolism , T-Lymphocytes/physiology , Cell Movement/genetics , Cell Polarity , Clathrin/genetics , Humans , Immunological Synapses , Jurkat Cells , Protein Transport , RNA, Small Interfering/genetics , Transport Vesicles/metabolism
9.
ACS Nano ; 10(7): 6597-611, 2016 07 26.
Article in English | MEDLINE | ID: mdl-27328170

ABSTRACT

Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.


Subject(s)
Hepacivirus , Hepatitis C/virology , Tomography, X-Ray , Animals , Antiviral Agents , Humans , Mitochondria , Virus Replication , X-Rays
10.
Sci Rep ; 6: 22354, 2016 Mar 10.
Article in English | MEDLINE | ID: mdl-26960695

ABSTRACT

We used soft X-ray three-dimensional imaging to quantify the mass of superparamagnetic iron oxide nanoparticles (SPION) within whole cells, by exploiting the iron oxide differential absorption contrast. Near-edge absorption soft X-ray nanotomography (NEASXT) combines whole-cell 3D structure determination at 50 nm resolution, with 3D elemental mapping and high throughput. We detected three-dimensional distribution of SPIONs within cells with 0.3 g/cm(3) sensitivity, sufficient for detecting the density corresponding to a single nanoparticle.


Subject(s)
Metal Nanoparticles , X-Ray Microtomography/methods , Humans , MCF-7 Cells
11.
J Nanobiotechnology ; 14: 15, 2016 Mar 03.
Article in English | MEDLINE | ID: mdl-26939942

ABSTRACT

BACKGROUND: Recent advances in nanoparticle design have generated new possibilities for nano-biotechnology and nano-medicine. Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line. RESULTS: We incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach. After vitrification, samples tested by correlative cryo-epifluorescent microscopy showed SPION accumulation in acidic vesicles related to the endocytic pathway. Microscopy grids bearing MCF-7 cells were then analysed by cryo-SXT to generate whole cell volume 3D maps. Cryo-SXT is an emerging technique that benefits from high X-ray penetration into the biological material to image close-to-native vitrified cells at nanometric resolution with no chemical fixation or staining agents. This unique possibility of obtaining 3D information from whole cells allows quantitative statistical analysis of SPION-containing vesicle (SCV) accumulation inside cells, including vesicle number and size, distances between vesicles, and their distance from the nucleus. CONCLUSIONS: Correlation between fluorescent microscopy, cryo-SXT and transmission electron microscopy allowed us to identify SCV and to generate 3D data for statistical analysis of SPION:cell interaction. This study supports continuous transfer of the internalized SPION from the plasma membrane to an accumulation area near the cell nucleus. Statistical analysis showed SCV increase in number and size concomitant with longer incubation times, and therefore an increase in their accumulated volume within the cell. This cumulative effect expands the accumulation area and cell organelles such as mitochondria are consequently displaced to the periphery. Our 3D cryo-SXT approach demonstrates that a comprehensive quantitative description of SPION:cell interaction is possible, which will serve as a basis for metal-based nanoparticle design and for selection of those best suited for hyperthermia treatment, drug delivery and image diagnosis in nanobiomedicine.


Subject(s)
Cell Membrane/metabolism , Cell Nucleus/metabolism , Cells/metabolism , Nanoparticles/metabolism , Cell Line, Tumor , Cryopreservation/methods , Humans , Imaging, Three-Dimensional/methods , MCF-7 Cells , Microscopy, Fluorescence/methods , Tomography, X-Ray/methods
12.
J Synchrotron Radiat ; 22(4): 1112-7, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26134819

ABSTRACT

The performance of MISTRAL is reported, the soft X-ray transmission microscopy beamline at the ALBA light source (Barcelona, Spain) which is primarily dedicated to cryo soft X-ray tomography (cryo-SXT) for three-dimensional visualization of whole unstained cells at spatial resolutions down to 30 nm (half pitch). Short acquisition times allowing for high-throughput and correlative microscopy studies have promoted cryo-SXT as an emerging cellular imaging tool for structural cell biologists bridging the gap between optical and electron microscopy. In addition, the beamline offers the possibility of imaging magnetic domains in thin magnetic films that are illustrated here with an example.


Subject(s)
Magnetics , Microscopy/methods , Tomography/methods , X-Rays
13.
PLoS One ; 10(7): e0133595, 2015.
Article in English | MEDLINE | ID: mdl-26208356

ABSTRACT

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1ß, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.


Subject(s)
Carrier Proteins/immunology , HIV Antigens/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Recombinant Fusion Proteins/immunology , Viral Fusion Proteins/immunology , AIDS Vaccines/genetics , AIDS Vaccines/immunology , Adaptive Immunity , Animals , Antibodies, Neutralizing/immunology , CD4 Antigens/metabolism , Carrier Proteins/genetics , Cell Line , Dendritic Cells/immunology , Dendritic Cells/metabolism , HIV Antibodies/immunology , HIV Antigens/genetics , HIV Envelope Protein gp120/genetics , HIV-1/genetics , Humans , Immunity, Innate , Immunoglobulin G/immunology , Immunologic Memory , Membrane Proteins , Polysaccharides/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Viral Fusion Proteins/genetics
14.
J Nanobiotechnology ; 13: 16, 2015 Feb 26.
Article in English | MEDLINE | ID: mdl-25880445

ABSTRACT

BACKGROUND: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells. RESULTS: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells. CONCLUSIONS: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cells.


Subject(s)
Breast Neoplasms/metabolism , Coated Materials, Biocompatible/metabolism , Ferric Compounds/metabolism , Magnetite Nanoparticles/chemistry , Succimer/metabolism , Breast/cytology , Breast/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Coated Materials, Biocompatible/chemistry , Cytoskeleton/drug effects , Endocytosis , Endosomes/metabolism , Female , Ferric Compounds/chemistry , Humans , Pinocytosis , Succimer/chemistry
15.
Cell Host Microbe ; 15(5): 611-22, 2014 May 14.
Article in English | MEDLINE | ID: mdl-24832455

ABSTRACT

Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.


Subject(s)
Bacterial Infections/microbiology , Dendritic Cells/immunology , Listeria monocytogenes/immunology , Salmonella enterica/immunology , Staphylococcus aureus/immunology , T-Lymphocytes/immunology , Animals , Cells, Cultured , Cytokines/immunology , Dendritic Cells/microbiology , Female , Humans , Immunity, Innate , Male , Mice , Mice, Inbred C57BL , Phagocytosis , T-Lymphocytes/microbiology
16.
Science ; 338(6114): 1634-7, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23180776

ABSTRACT

The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.


Subject(s)
Influenza A Virus, H1N1 Subtype/chemistry , RNA, Viral/chemistry , Ribonucleoproteins/chemistry , Viral Proteins/chemistry , Virion/chemistry , Animals , Cell Nucleus/metabolism , Cell Nucleus/virology , Cryoelectron Microscopy , Electron Microscope Tomography , Image Processing, Computer-Assisted , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H1N1 Subtype/ultrastructure , Madin Darby Canine Kidney Cells , Microscopy, Electron , Models, Molecular , Nucleocapsid Proteins , Protein Conformation , Protein Structure, Secondary , RNA, Viral/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/ultrastructure , Ribonucleoproteins/metabolism , Ribonucleoproteins/ultrastructure , Transcription, Genetic , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Viral Core Proteins/ultrastructure , Viral Proteins/metabolism , Viral Proteins/ultrastructure , Virion/ultrastructure
17.
J Struct Biol ; 177(2): 202-11, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22178221

ABSTRACT

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.


Subject(s)
Host-Pathogen Interactions , Microscopy/methods , Tomography, X-Ray/methods , Vaccinia virus/physiology , Animals , Cell Line , Cell Shape , Chick Embryo , Cryopreservation/methods , Cytoplasmic Structures/ultrastructure , Cytoplasmic Structures/virology , Single-Cell Analysis , Vaccinia virus/ultrastructure , Virion/ultrastructure
18.
J Struct Biol ; 168(2): 234-9, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19616103

ABSTRACT

Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250x270x360nm(3). The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.


Subject(s)
Vaccinia virus/ultrastructure , Cryoelectron Microscopy , Tomography, X-Ray , Vaccinia virus/physiology
19.
Microsc Res Tech ; 72(1): 42-8, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18837435

ABSTRACT

Centrioles are components of the centrosome, which is present in most eukaryotic cells (from protozoa to mammals). They organize the microtubule skeleton during interphase and the mitotic spindle during cell division. In ciliate cells, centrioles form basal bodies that are involved in cellular motility. Despite their important roles in biology, the detailed structure of centrioles remains obscure. This work contributes to a more complete model of centriole structure. The authors used electron tomography of isolated centrosomes from the human lymphoblast KE37 to explore the details of subdistal appendages and centriole lumen organization in mother centrioles. Their results reveal that each of the nine subdistal appendages is composed of two halves (20 nm diameter each) fused in a 40 nm tip that extends 100 nm from where it anchors to microtubules. The centriole lumen is filled at the distal domain by a 45 nm periodic stack of rings. Each ring has a 30 nm diameter, is 15 nm thick, and appears to be tilted at 53 degrees perpendicular to the centriole axis. The rings are anchored to microtubules by arms. Based on their results, the authors propose a model of the mother centriole distal structure.


Subject(s)
Centrioles/ultrastructure , Electron Microscope Tomography , Imaging, Three-Dimensional , Models, Molecular , Cell Cycle , Cell Line, Tumor , Humans
20.
Biol Cell ; 101(7): 401-14, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19076058

ABSTRACT

BACKGROUND INFORMATION: VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. RESULTS: We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent 'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. CONCLUSIONS: The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.


Subject(s)
Cell Membrane/chemistry , Vaccinia virus/chemistry , Vaccinia virus/physiology , Vaccinia/virology , Virus Assembly , Cell Membrane/virology , Electron Microscope Tomography , HeLa Cells , Humans , Vaccinia virus/ultrastructure , Viral Structural Proteins/metabolism
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