Posts Tagged ‘Microscopy’

Single-Cell Imaging of HIV-1 Proviruses

Thursday, March 28th, 2013

HIV proviruses at the periphery of the nucleus Technical developments in imaging-based techniques have greatly improved our understanding of HIV–host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus–host interplay during all steps of HIV-1 replication cycle. Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment, which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA.

A new paper describes a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. A 3D topological analysis demonstrated that integrated viral DNA localizes at the periphery of the nuclei revealing important insights in the nuclear biology of HIV-1.

 

Single-Cell Imaging of HIV-1 Provirus (SCIP). PNAS USA 19 March 2013, doi: 10.1073/pnas.1216254110
Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.

Detection of HIV-1 in Living Cells

Friday, December 7th, 2012

HIV-infected cell In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it was previously impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus.

This paper describes a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. A single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity was fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems.

Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. This new reporter system was used for automated image analysis, providing a new tool for cell-based HIV research. Generation of additional chromobodies with different binding sites should enable kinetic and functional studies to specifically probe the HIV assembly process.

 

Direct and Dynamic Detection of HIV-1 in Living Cells. (2012) PLoS ONE 7(11): e50026. doi:10.1371/journal.pone.0050026

Structure of Ebola virus [video]

Thursday, March 1st, 2012

Recently some of my final year virology students commented to me that it was a shame I was not still making regular MicrobiologyBytes podcasts. But as I said when I stopped posting weekly podcasts, I don’t feel that audio adds value to the content of what I like to share here. But video may be a different matter. Not that you’re going to see videos of me talking, because you won’t learn much microbiology that way, and I’ve only ever seen one good talking head video. But some papers lend themselves to a more visual treatment, and this is a very good example:

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. PNAS USA 27 February 2012 doi: 10.1073/pnas.1120453109
Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.


Native HIV-1 Budding Sites

Monday, November 29th, 2010

HIV-1 Gag The production of new HIV-1 particles is initiated at the plasma membrane where the virus polyprotein Gag assembles into a budding site, and proceeds through release of an immature virion which is subsequently transformed to the infectious virion by proteolytic cleavage of Gag. Recently HIV-1 budding sites have been studied by cryo electron tomography. This technique allows three-dimensional structure determination of single objects at macromolecular resolution, and is uniquely suited to study variable structures such as HIV-1 particles and budding sites.

Using cryo electron tomography, researchers obtained three-dimensional images with unprecedented detail of the formation of HIV-1 particles. By analyzing these images they showed that the organization of released immature HIV-1 is determined at its intracellular assembly without major subsequent rearrangements. They were able to identify a lattice structure of the viral protein Gag present in budding sites that seem to lack the viral genome and thus cannot be precursors of infectious viruses. Some HIV-1 infected T-cells preferentially carry these budding sites, suggesting that they have lost a crucial control of the proteolytic maturation of the virus.

Cryo Electron Tomography of Native HIV-1 Budding Sites. (2010) PLoS Pathog 6(11): e1001173. doi:10.1371/journal.ppat.1001173
The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

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Microbial nanoscopy

Tuesday, July 20th, 2010

atomic force microscopy With its ability to observe single microbial cells at nanometre resolution, to monitor structural dynamics in response to environmental changes or drugs, and to detect and manipulate single-cell surface constituents, atomic force microscopy (AFM) provides new insight into the structure–function relationships of cell envelopes. This emerging field of microbial nanoscopy should have an important impact on many disciplines of microbiology, including cellular and molecular microbiology, pathogenesis, diagnosis, antimicrobial therapy and environmental microbiology.

How cell envelope constituents are organised and how they interact with the environment are key questions in microbiology. Unlike other bioimaging tools, AFM provides information about the nanoscale surface architecture of living cells and about the localization and interactions of their individual constituents. These past years have witnessed remarkable advances in our use of the AFM molecular toolbox to observe and force probe microbial cells. Recent milestones include the real-time imaging of the nanoscale organization of cell walls, the quantification of subcellular chemical heterogeneities, the mapping and functional analysis of individual cell wall constituents and the analysis of the mechanical properties of single receptors and sensors.

Microbial nanoscopy: a closer look at microbial cell surfaces. Trends Microbiol. Jul 12 2010

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Giant microscope meets giant virus

Friday, June 25th, 2010

Mimivirus Mimivirus appears most closely related structurally to large algal viruses such as PBCV-1 and other irridoviruses, though it possesses additional features not present in those viruses. The icosahedral capsid consists of 20 large triangular plates joined at their edges to produce the required 12 fivefold vertices. The capsid surface is composed of trimeric major capsid proteins, each subunit of which consists of two jelly roll beta barrels, arranged in a very open hexagonal lattice, with the appearance of a honeycomb. The center-to-center distance of the capsomeres is 14 nm. Because there is some ambiguity as to what lies exactly on the fivefold vertex, there is an uncertainty in the triangulation number T, which could have any one of nine possibilities lying between 972 and 1200.

The fibres that coat particles are reported to have lengths of about 125 nm and are probably anchored to the major capsid proteins or perhaps to an integument layer of protein disposed immediately above the capsid. The most striking and unique feature of mimivirus is a prominent five armed, star-shaped apparatus that occupies one vertex of every virus. While somewhat obscured on the intact virus, it is prominently displayed on particles which lack the coating of fibers. This star shaped assembly, termed a “stargate,” opens up once the virus is inside the host cell to produce a wide opening. The DNA of the virus, which is enclosed inside a membrane sac, then emerges from the interior, fuses with a cellular membrane, and delivers its nucleic acid contents to the cell. Another remarkable is that the DNA does not enter the capsid through the stargate, but through a separate portal, and by a completely different mechanism, in the center of a distal icosahedral face, i.e. at a threefold axis.

Atomic force microscopy investigation of the giant mimivirus. Virology. 2010 404(1): 127-137
Mimivirus was investigated by atomic force microscopy in its native state following serial degradation by lysozyme and bromelain. The 750-nm diameter virus is coated with a forest of glycosylated protein fibers of lengths about 140 nm with diameters 1.4 nm. Fibers are capped with distinctive ellipsoidal protein heads of estimated Mr=25 kDa. The surface fibers are attached to the particle through a layer of protein covering the capsid, which is in turn composed of the major capsid protein (MCP). The latter is organized as an open network of hexagonal rings with central depressions separated by 14 nm. The virion exhibits an elaborate apparatus at a unique vertex, visible as a star shaped depression on native particles, but on defibered virions as five arms of 50 nm width and 250 nm length rising above the capsid by 20 nm. The apparatus is integrated into the capsid and not applied atop the icosahedral lattice. Prior to DNA release, the arms of the star disengage from the virion and it opens by folding back five adjacent triangular faces. A membrane sac containing the DNA emerges from the capsid in preparation for fusion with a membrane of the host cell. Also observed from disrupted virions were masses of distinctive fibers of diameter about 1 nm, and having a 7-nm periodicity. These are probably contained within the capsid along with the DNA bearing sac. The fibers were occasionally observed associated with toroidal protein clusters interpreted as processive enzymes modifying the fibers.

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Shedding light on the inner workings of the immune response

Monday, March 15th, 2010

Leishmania Leishmania donovani is a protozoan parasite that causes severe disease in humans with associated pathology in the spleen and liver. In experimental models of L. donovani infection, the hepatic response to infection is characterised by the presence of a focal mononuclear cell-rich inflammatory response (a granuloma) surrounding cells infected with intracellular amastigotes. Granulomas provide focus to the ensuing immune response, helping to contain parasite dissemination and providing the major effector site responsible for parasites elimination from the liver. Although granulomas are believed to form around infected resident liver macrophages (Kupffer cells), the role of these cells in intra-granuloma antigen presentation is currently unknown. Researchers used sophisticated microscopy to identify how killer T lymphocytes behaved when they enter sites of inflammation caused by L. donovani, and which infected cells they were able to recognise.

Leishmaniasis is a globally important but neglected disease, affecting approximately two million people every year. For most people, infection results in a slow-to-heal skin ulcer. In others, however, the parasite targets the liver, spleen and bone marrow, leading to over 70,000 deaths annually. The Leishmania parasite is eventually contained by a characteristic type of inflammatory response that forms cellular structures called granulomas. Little is known about the inner workings of these granulomas, in spite of their occurrence in other human diseases, from tuberculosis to rheumatoid arthritis. The scientists used an advanced laser-based microscopy technique, called “2-photon imaging”, to view the inner workings of the granuloma in mice infected with Leishmania. This enabled them to study how killer lymphocytes, such as those that can be induced by vaccination, are able to enter into the granulomas, penetrate deep into the core of the structure and seek out specific types of parasite-infected cells. Although this technique can not be used currently for the study of inflammatory disease in humans, the insights provided into the biology of granulomas and the hidden world of inflammation should help to improve vaccines and drugs, and allow researchers to now construct in silico models for this type of inflammatory process. These data have important implications for the understanding of how granulomas function to limit infection and may have important implications for the development of vaccines to Leishmania.

Dynamic Imaging of Experimental Leishmania donovani-Induced Hepatic Granulomas Detects Kupffer Cell-Restricted Antigen Presentation to Antigen-Specific CD8+ T Cells. PLoS Pathog 6(3): e1000805. doi:10.1371/journal.ppat.1000805
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intravital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

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HIV-1 Assembly and Release

Tuesday, November 24th, 2009

HIV assembly Human immunodeficiency virus (HIV) particles are formed and released at the plasma membrane of the infected cell. Researchers analyzed the dynamics of HIV assembly and release making use of fluorescently labeled HIV structural proteins. They determined that assembly of the virus protein shell occurs within ~8–9 min after nucleation of an assembly site and virus particles are formed individually and not from large patches. Virion release was observed ~25 min after nucleation of the assembly site. Assembly of the Gag shell thus appears to constitute only a minor part of the period required for particle formation indicating that traversing the membrane and fission are the rate-limiting stages in virion formation. Using a photoconvertible label in the viral Gag protein, they established that the Gag molecules driving nucleation of a new assembly site and in bud growth are recruited preferentially from the cytosolic pool of Gag molecules and from recently membrane-attached Gag. No intracellular assembly or vesicular trafficking of Gag was observed. The described results add essential dynamic information to our picture of virus release and provide an experimental basis for interfering with this stage of virus replication.

Dynamics of HIV-1 Assembly and Release. PLoS Pathog 5(11): e1000652 doi: 10.1371/journal.ppat.1000652

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Cell-to-cell transmission of retroviruses

Tuesday, July 28th, 2009

Retroviruses, such as HIV, that are already within cells are more easily transmitted when they spread through direct contact between cells than if they are floating free in the bloodstream. Researchers have recorded video of virus activity within cells that helps explains why cell-to-cell transmission is so efficient and may in turn provide insights into potential targets for a new generation of antiretroviral drugs.

Cell-to-cell transmission of retroviruses is a thousand times more efficient than extracellular infection, and because retroviruses spread through the tight cell-cell interface, they are out of reach of the immune system. Using imaging technology that can track individual virus particles in real time, the research team discovered that retrovirus-infected cells can specifically assemble daughter viruses at the point of contact between cells. Ten times more of these particles are found at these cellular connection points than elsewhere at the surface of cells. The ability of infected cells to specifically produce viruses only at cell interfaces offers an explanation of how viruses spread so efficiently. The team identified a clue to how virus assembly is targeted to these points of contact: it involves a retrovirus protein called Env that docks with uninfected cells and then attracts the viral particles to these sites. If this adhesion molecule lacked a cytoplasmic tail, then the virus particles did not assemble at the patches of contact between cells.

Somewhere down the road it is possible that we may be able to develop completely new antiviral strategies based on targeting cell-to-cell transmission.

Assembly of the Murine Leukemia Virus Is Directed towards Sites of Cell–Cell Contact. 2009 PLoS Biol 7(7): e1000163 doi:10.1371/journal.pbio.1000163
We have investigated the underlying mechanism by which direct cell–cell contact enhances the efficiency of cell-to-cell transmission of retroviruses. Applying 4D imaging to a model retrovirus, the murine leukemia virus, we directly monitor and quantify sequential assembly, release, and transmission events for individual viral particles as they happen in living cells. We demonstrate that de novo assembly is highly polarized towards zones of cell–cell contact. Viruses assembled approximately 10-fold more frequently at zones of cell contact with no change in assembly kinetics. Gag proteins were drawn to adhesive zones formed by viral Env glycoprotein and its cognate receptor to promote virus assembly at cell–cell contact. This process was dependent on the cytoplasmic tail of viral Env. Env lacking the cytoplasmic tail while still allowing for contact formation, failed to direct virus assembly towards contact sites. Our data describe a novel role for the viral Env glycoprotein in establishing cell–cell adhesion and polarization of assembly prior to becoming a fusion protein to allow virus entry into cells.

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