MicrobiologyBytes

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.

Related:

• HIV-1 Assembly and Release
• Studying the structure of HIV
• Giant microscope meets giant virus

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

Related:

• The architecture of peptidoglycan
• Giant microscope meets giant virus

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.

Related:

• Atomic force microscopy
• Marvellous mimivirus
• Mimivirus replicates in the cytoplasm

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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• Novel application for an old drug: tamoxifen in the treatment of Leishmania infection
• Foamy macrophages allow TB agent to survive in infected individuals

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

Related:

• Studying the structure of HIV
• HIV spreading from cell to cell
• Cell-to-cell transmission of retroviruses

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.

Related:

• Retroviruses
• Retrovirus infection of resting cells
• HIV infection is due to the sins of the fathers

Cells arrange their components – proteins, lipids, and nucleic acids – in organized and reproducible ways to optimize their activities and to improve cell efficiency and survival. Eukaryotic cells have a complex arrangement of subcellular structures such as membrane-bound organelles and cytoskeletal transport systems. However, subcellular organization is also important in prokaryotic cells, including rod-shaped bacteria such as E. coli, most of which lack such well-developed systems of organelles and motor proteins for transporting cellular cargoes. In fact, it has remained somewhat mysterious how bacteria are able to organize and spatially segregate their interiors.

The E. coli chemotaxis network, a system important for the bacterial response to environmental cues, is one of the best-understood biological signal transduction pathways and serves as a useful model for studying bacterial spatial organization because its components display a nonrandom, periodic distribution in mature cells. Chemotaxis receptors aggregate and cluster into large sensory complexes that localize to the poles of bacteria. To understand how these clusters form and what controls their size and density, a recent study used ultrahigh-resolution light microscopy, called photoactivated localization microscopy, to visualize individual chemoreceptors in single E. coli cells. From these high-resolution images, the authors were able to determine that receptors are not actively distributed or attached to specific locations in cells. Instead, it appears that random receptor diffusion and receptor–receptor interactions are sufficient to generate the observed complex, ordered pattern. This simple mechanism, termed stochastic self-assembly, may prove to be widespread in both prokaryotic and eukaryotic cells.

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Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy. 2009 PLoS Biol 7(6): e1000137 doi:10.1371/journal.pbio.1000137
The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

Related:

• Real-time imaging of bacterial infections in vivo
• Tracking Lyme Disease in Living Hosts
• How smart are bacteria?

Source: CDC (Public domain)

Mimivirus is the largest virus known to scientists, about half of a micrometre (0.0005 millimeter) in diameter. It is more than 10 times larger than the virus that causes the common cold and – unlike other viruses – is large enough to be seen with a light microscope. An international team of researchers have now determined key structural features of Mimivirus, findings that could help scientists study how the simplest life forms evolved and whether this unusual virus causes any human diseases. Mimivirus infects amoebae, but it is also thought that it may act as a human pathogen, because antibodies to the virus have been discovered in people with pneumonia. However, many details about the virus remain unknown. Now researchers have determined the basic design of the virus’s outer shell, or capsid, and also of the hundreds of smaller units – called capsomeres – making up this outer shell. Their findings confirmed the existence of a starfish-shaped structure that covers a ‘special vertex’ – an opening in the capsid where the genetic material leaves the virus to infect its host; an indentation in the virus’s genetic material itself is positioned opposite this opening.

The findings are important in terms of studying the evolution of cells, bacteria and viruses. Mimivirus is like an intermediate between a cell and a virus. We usually think of cells as being alive and a virus is thought of as being non-living because it needs a host cell to complete its life cycle. Mimivirus straddles a middle ground between viruses and living cells, perhaps redefining what a virus is. Scientists had previously been unable to determine the virus’s structure because they had assumed that, like many other viruses, it’s capsid had a design known as icosahedral symmetry. These authors discovered the true structure when they tried reconstructing the virus, assuming it had not the standard icosahedral symmetry but another configuration called five-fold symmetry. If you start out thinking the object has icosahedral symmetry, then you assume there are 60 identical pieces, and that influences how you reconstruct the virus’s structure.

The researchers took images of the virus using an atomic force microscope, revealing a pattern of holes regularly spaced throughout the virus’s outer shell. The capsids of most other large, pseudo-icosahedral viruses do not contain such holes, and their function is unknown. The researchers used cryo-electron microscopy reconstruction to determine the structural details. This reconstruction method enabled them to reassemble three-dimensional images from two-dimensional pictures, much as a complete architectural drawing of a house can be assembled with two-dimensional drawings of the sides, the roof and other elements. An icosahedron has a roughly spherical shape containing 20 triangular facets and 60 identical subunits. Like an icosahedron, the mimivirus capsid also has 20 facets. However, unlike an icosohedron, five facets of the capsid are slightly different than the others and surround the special vertex. Icosohedra contain 12 similar vertices, whereas the mimivirus contains eleven such vertices, with the 12th being different than the others.

Structural studies of the giant Mimivirus. 2009 PLoS Biol 7(4): e1000092
Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral doublestranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 608. The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 A°.

Related:

• Marvellous mimivirus
• Mimivirus and the Stargate
• Marine mimivirus relatives are large algal viruses