2012. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J Struct Biol, 177 (2), pp. 193-201. Read abstract | Read more
Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the 'water-window' wavelength region (2.34-4.37nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach - the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only. Hide abstract
2011. Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons. PLoS Pathog, 7 (12), pp. e1002406. Read abstract | Read more
During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the 'married' model or as non-enveloped capsids suggested by the 'separate' model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the 'separate model' for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles. Hide abstract
2011. Eisosome proteins assemble into a membrane scaffold. J Cell Biol, 195 (5), pp. 889-902. Read abstract | Read more
Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity. Hide abstract
2011. Conserved eukaryotic fusogens can fuse viral envelopes to cells. Science, 332 (6029), pp. 589-592. Read abstract | Read more
Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle. Hide abstract
2010. Cryo Electron Tomography of Native HIV-1 Budding Sites PLOS PATHOG, 6 (11), | Read more
2010. The three-dimensional organization of polyribosomes in intact human cells. Mol Cell, 39 (4), pp. 560-569. Read abstract | Read more
Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding. Hide abstract
2008. Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe, 4 (6), pp. 592-599. Read abstract | Read more
Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether. Hide abstract
2008. Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry. Proc Natl Acad Sci U S A, 105 (30), pp. 10559-10564. Read abstract | Read more
The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional "poles" of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly. Hide abstract
2008. Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus. Proc Natl Acad Sci U S A, 105 (7), pp. 2375-2379. Read abstract | Read more
Bunyaviridae is a large family of viruses that have gained attention as "emerging viruses" because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or "spikes," were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry. Hide abstract
2007. Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells. Cell, 131 (3), pp. 516-529. Read abstract | Read more
Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol. Hide abstract
2006. Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog, 2 (8), pp. e83. Read abstract | Read more
The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike. Hide abstract
2006. Structure of complex viruses and virus-infected cells by electron cryo tomography. Curr Opin Microbiol, 9 (4), pp. 437-442. Read abstract | Read more
In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail. Hide abstract
2006. Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ PLoS Pathogens, 2 (8), pp. 0790-0797.
2006. The mechanism of HIV-1 core assembly: insights from three-dimensional reconstructions of authentic virions. Structure, 14 (1), pp. 15-20. Read abstract | Read more
Infectious HIV particles contain a characteristic cone-shaped core encasing the viral RNA and replication proteins. The core exhibits significant heterogeneity in size and shape, yet consistently forms a well-defined structure. The mechanism by which the core is assembled in the maturing virion remains poorly understood. Using cryo-electron tomography, we have produced three-dimensional reconstructions of authentic, unstained HIV-1. These reveal the viral morphology with unprecedented clarity and suggest the following mechanism for core formation inside the extracellular virion: core growth initiates at the narrow end of the cone and proceeds toward the distal side of the virion until limited by the viral membrane. Curvature and closure of the broad end of the core are then directed by the inner surface of the viral membrane. This mechanism accommodates significant flexibility in lattice growth while ensuring the closure of cores of variable size and shape. Hide abstract
2003. Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science, 302 (5649), pp. 1396-1398. Read abstract | Read more
Herpes simplex virus, a DNA virus of high complexity, consists of a nucleocapsid surrounded by the tegument-a protein compartment-and the envelope. The latter components, essential for infectivity, are pleiomorphic. Visualized in cryo-electron tomograms of isolated virions, the tegument was seen to form an asymmetric cap: On one side, the capsid closely approached the envelope; on the other side, they were separated by approximately 35 nanometers of tegument. The tegument substructure was particulate, with some short actin-like filaments. The envelope contained 600 to 750 glycoprotein spikes that varied in length, spacing, and in the angles at which they emerge from the membrane. Their distribution was nonrandom, suggesting functional clustering. Hide abstract
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