-
[show abstract]
[hide abstract]
ABSTRACT: The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions.
Methods in cell biology 01/2012; 111:235-57. · 2.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
PLoS Biology 11/2011; 9(11):e1001196. · 11.45 Impact Factor
-
Rainer Beck,
Simone Prinz,
Petra Diestelkötter-Bachert,
Simone Röhling,
Frank Adolf,
Kathrin Hoehner, Sonja Welsch,
Paolo Ronchi,
Britta Brügger,
John A G Briggs,
Felix Wieland
[show abstract]
[hide abstract]
ABSTRACT: Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle. Using a reconstituted system for COPI vesicle formation from purified components, we find that a dimerization-deficient Arf1 mutant, which does not display the ability to modulate membrane curvature in vitro or to drive formation of coated vesicles, is able to recruit coatomer to allow formation of COPI-coated buds but does not support scission. Chemical cross-linking of this Arf1 mutant restores vesicle release. These experiments show that initial curvature of the bud is defined primarily by coatomer, whereas the membrane curvature modulating activity of dimeric Arf1 is required for membrane scission.
The Journal of Cell Biology 09/2011; 194(5):765-77. · 10.26 Impact Factor
-
Rainer Beck,
Simone Prinz,
Petra Diestelkötter-Bachert,
Simone Röhling,
Frank Adolf,
Kathrin Hoehner, Sonja Welsch,
Paolo Ronchi,
Britta Brügger,
John A.G. Briggs,
Felix Wieland
[show abstract]
[hide abstract]
ABSTRACT: Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced
to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle. Using a reconstituted system for
COPI vesicle formation from purified components, we find that a dimerization-deficient Arf1 mutant, which does not display
the ability to modulate membrane curvature in vitro or to drive formation of coated vesicles, is able to recruit coatomer
to allow formation of COPI-coated buds but does not support scission. Chemical cross-linking of this Arf1 mutant restores
vesicle release. These experiments show that initial curvature of the bud is defined primarily by coatomer, whereas the membrane
curvature modulating activity of dimeric Arf1 is required for membrane scission.
The Journal of Cell Biology 09/2011; 194(5):765-777. · 10.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1) at the virological synapse (VS) is an efficient mode of dissemination between CD4(+) T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+) T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS). Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC) and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL) and we show that the HIV-1 envelope glycoprotein (Env) localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+) T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+) T cells to enhance its dissemination.
PLoS Pathogens 09/2011; 7(9):e1002226. · 9.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Productive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little is known about the structure of this compartment and its potential regulation by infection. Here we characterized this compartment in macrophages using electron tomography and electron microscopy with stereology. We found an intricate, interconnected membrane network that constitutes a preexisting physiologic structure in macrophages but which expands in size upon HIV-1 infection. Membranes required for this expansion were apparently derived from preexisting pools of plasma membrane. Physical connections between this compartment and the extracellular milieu were frequently made by tube-like structures of insufficient diameter for virion passage. We conclude that HIV-1 induces the expansion of a complex membranous labyrinth in macrophages in which the virus buds and can be retained, with potential consequences for transmission and immune evasion.
Journal of Virology 05/2011; 85(15):7922-7. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and
rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of
signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm.
We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also
apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule
plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway
through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for
direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.
The Journal of Cell Biology 01/2011; 192(1):111-119. · 10.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.
The Journal of Cell Biology 01/2011; 192(1):111-9. · 10.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Viruses are perfect opportunists that have evolved to modify numerous cellular processes in order to complete their replication cycle in the host cell. An article by Reggiori and coworkers in this issue of Cell Host & Microbe reveals how coronaviruses can divert a cellular quality control pathway that normally functions in degradation of mis-folded proteins to replicate the viral genome.
Cell host & microbe 06/2010; 7(6):424-6. · 13.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4(+) T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.
Journal of Virology 04/2010; 84(7):3516-27. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a "submarine-like" budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular "rocket-like" protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.
PLoS Pathogens 01/2010; 6(4):e1000875. · 9.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations. To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.
Journal of Biological Chemistry 09/2009; 284(43):29692-703. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.
Cell host & microbe 05/2009; 5(4):365-75. · 13.02 Impact Factor
-
Christine Goffinet,
Ina Allespach,
Stefanie Homann,
Hanna-Mari Tervo,
Anja Habermann,
Daniel Rupp,
Lena Oberbremer,
Christian Kern,
Nadine Tibroni, Sonja Welsch,
Jacomine Krijnse-Locker,
George Banting,
Hans-Georg Kräusslich,
Oliver T Fackler,
Oliver T Keppler
[show abstract]
[hide abstract]
ABSTRACT: Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.
Cell host & microbe 04/2009; 5(3):285-97. · 13.02 Impact Factor
-
Christine Goffinet,
Ina Allespach,
Stefanie Homann,
Hanna-Mari Tervo,
Anja Habermann,
Daniel Rupp,
Lena Oberbremer,
Christian Kern,
Nadine Tibroni, Sonja Welsch,
Jacomine Krijnse Locker,
George Banting,
Hans-Georg Kräusslich,
Oliver Fackler,
Oliver Keppler
Retrovirology. 01/2009;
-
[show abstract]
[hide abstract]
ABSTRACT: Macrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4(+) T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4(+) T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1-infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.
Blood 06/2008; 111(9):4660-3. · 9.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.
FEBS Letters 06/2007; 581(11):2089-97. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: HIV-1 assembly and release are believed to occur at the plasma membrane in most host cells with the exception of primary macrophages, for which exclusive budding at late endosomes has been reported. Here, we applied a novel ultrastructural approach to assess HIV-1 budding in primary macrophages in an immunomarker-independent manner. Infected macrophages were fed with BSA-gold and stained with the membrane-impermeant dye ruthenium red to identify endosomes and the plasma membrane, respectively. Virus-filled vacuolar structures with a seemingly intracellular localization displayed intense staining with ruthenium red, but lacked endocytosed BSA-gold, defining them as plasma membrane. Moreover, HIV budding profiles were virtually excluded from gold-filled endosomes while frequently being detected on ruthenium red-positive membranes. The composition of cellular marker proteins incorporated into HIV-1 supported a plasma membrane-derived origin of the viral envelope. Thus, contrary to current opinion, the plasma membrane is the primary site of HIV-1 budding also in infected macrophages.
PLoS Pathogens 04/2007; 3(3):e36. · 9.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular-vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells.
Traffic 12/2006; 7(11):1551-66. · 4.92 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.
Molecular Biology of the Cell 07/2005; 16(6):2822-35. · 4.94 Impact Factor
