The nucleocapsid domain of Gag is dispensable for actin incorporation into HIV-1 and for association of viral budding sites with cortical F-actin.
Stauffer, S., Rahman, S.A., de Marco, A., Carlson, L.A., Glass, B., Oberwinkler, H., Herold, N., Briggs, J.A., Muller, B., Grunewald, K. & Krausslich, H.G.
J Virol. 2014 Jul;88(14):7893-903. doi: 10.1128/JVI.00428-14. Epub 2014 Apr 30.
Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. Importance: HIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds, often seemingly in contact with the Gag layer. Here, we show that this spatial association is observed independently of the proposed actin-binding domain of HIV-1. The absence of this domain also did not affect actin incorporation and had a minor effect on the viral assembly rate. Furthermore, actin was not enriched in the virus compared to the average levels in the respective producing cell. Our data argue against a specific recruitment of actin to HIV-1 budding sites by the nucleocapsid domain of Gag.
Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.
Bharat, T.A., Castillo Menendez, L.R., Hagen, W.J., Lux, V., Igonet, S., Schorb, M., Schur, F.K., Krausslich, H.G. & Briggs, J.A.
Proc Natl Acad Sci U S A. 2014 May 19. pii: 201401455.
The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-A resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion.
Bharat, T.A., Malsam, J., Hagen, W.J., Scheutzow, A., Sollner, T.H. & Briggs, J.A.
EMBO Rep. 2014 Mar;15(3):308-14. doi: 10.1002/embr.201337807. Epub 2014 Feb 3.
Synaptic vesicles fuse with the plasma membrane in response to Ca(2+) influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca(2)(+)-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.
Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy.
Nikic, I., Plass, T., Schraidt, O., Szymanski, J., Briggs, J.A., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2014;53(8):2245?2249. doi: 10.1002/anie.201309847.
The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.
Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation.
Daum, S., Kruger, D., Meister, A., Auerswald, J., Prinz, S., Briggs, J.A. & Bacia, K.
Biol Chem. 2014 Jul 1;395(7-8):801-12. doi: 10.1515/hsz-2014-0117.
Abstract As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.
Determination of protein structure at 8.5A resolution using cryo-electron tomography and sub-tomogram averaging.
Schur, F.K., Hagen, W.J., de Marco, A. & Briggs, J.A.
J Struct Biol. 2013 Dec;184(3):394-400. doi: 10.1016/j.jsb.2013.10.015. Epub 2013Oct 30.
Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15 A. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason-Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5 A. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions.
Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity.
Schorb, M. & Briggs, J.A.
Ultramicroscopy. 2013 Nov 11. pii: S0304-3991(13)00291-X. doi:10.1016/j.ultramic.2013.10.015.
Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals.
The structure of the COPII transport-vesicle coat assembled on membranes.
Zanetti, G., Prinz, S., Daum, S., Meister, A., Schekman, R., Bacia, K. & Briggs, J.A.
Elife. 2013 Sep 17;2:e00951. doi: 10.7554/eLife.00951.
Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.
Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.
Szymborska, A., de Marco, A., Daigle, N., Cordes, V.C., Briggs, J.A. & Ellenberg, J.
Science. 2013 Aug 9;341(6146):655-8. doi: 10.1126/science.1240672. Epub 2013 Jul11.
Much of life's essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.
Variable Internal Flexibility Characterizes the Helical Capsid Formed by Agrobacterium VirE2 Protein on Single-Stranded DNA.
Bharat, T.A., Zbaida, D., Eisenstein, M., Frankenstein, Z., Mehlman, T., Weiner, L., Sorzano, C.O., Barak, Y., Albeck, S., Briggs, J.A., Wolf, S.G. & Elbaum, M.
Structure. 2013 Jul 2;21(7):1158-67. doi: 10.1016/j.str.2013.04.027. Epub 2013Jun 13.
Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.
Vesicle coats: structure, function, and general principles of assembly.
Faini, M., Beck, R., Wieland, F.T. & Briggs, J.A.
Trends Cell Biol. 2013 Jun;23(6):279-88. doi: 10.1016/j.tcb.2013.01.005. Epub2013 Feb 13.
The transport of proteins and lipids between distinct cellular compartments is conducted by coated vesicles. These vesicles are formed by the self-assembly of coat proteins on a membrane, leading to collection of the vesicle cargo and membrane bending to form a bud. Scission at the bud neck releases the vesicle. X-ray crystallography and electron microscopy (EM) have recently generated models of isolated coat components and assembled coats. Here, we review these data to present a structural overview of the three main coats: clathrin, COPII, and COPI. The three coats have similar function, common ancestry, and structural similarities, but exhibit fundamental differences in structure and assembly. We describe the implications of structural similarities and differences for understanding the function, assembly principles, and evolution of vesicle coats.
Structural biology in situ--the potential of subtomogram averaging.
Curr Opin Struct Biol. 2013 Apr;23(2):261-7. doi: 10.1016/j.sbi.2013.02.003. Epub2013 Mar 4.
Cryo-electron tomography provides low-resolution 3D views of cells, organelles, or viruses. Macromolecular complexes present in multiple copies can be subsequently identified within the 3D reconstruction (the tomogram), computationally extracted, and averaged to obtain higher resolution 3D structures, as well as a map of their spatial distribution. This method, called subtomogram averaging or subvolume averaging, allows structures of macromolecular complexes to be resolved in situ. Recent applications have provided in situ structural data at resolutions of 2-4 nm on samples including polysomes, nuclear pores, vesicle coats, and viral surface proteins. Here I describe the method and discuss limitations, advances and recent applications. I speculate how the method will solve more structures at higher resolution, allowing in situ structural biology.
Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.
Fabrowski, P., Necakov, A.S., Mumbauer, S., Loeser, E., Reversi, A., Streichan, S., Briggs, J.A. & De Renzis, S.
Nat Commun. 2013;4:2244. doi: 10.1038/ncomms3244.
During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development.
Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity.
de Marco, A., Heuser, A.M., Glass, B., Krausslich, H.G., Muller, B. & Briggs, J.A.
J Virol. 2012 Dec;86(24):13708-16. doi: 10.1128/JVI.01704-12. Epub 2012 Oct 10.
HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two C-terminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.
Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography.
Kukulski, W., Schorb, M., Kaksonen, M. & Briggs, J.A.
Cell. 2012 Aug 3;150(3):508-20. doi: 10.1016/j.cell.2012.05.046.
Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are approximately 100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.
Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion.
Steringer, J.P., Bleicken, S., Andreas, H., Zacherl, S., Laussmann, M., Temmerman, K., Contreras, F.X., Bharat, T.A., Lechner, J., Muller, H.M., Briggs, J.A., Garcia-Saez, A.J. & Nickel, W.
J Biol Chem. 2012 Aug 10;287(33):27659-69. Epub 2012 Jun 23.
Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.
Structure of the immature retroviral capsid at 8 A resolution by cryo-electron microscopy.
Bharat, T.A., Davey, N.E., Ulbrich, P., Riches, J.D., de Marco, A., Rumlova, M., Sachse, C., Ruml, T. & Briggs, J.A.
Nature. 2012 Jul 19;487(7407):385-9.
The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid). Assembly of an infectious virion proceeds in two stages. In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation. However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-A resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.
Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release.
Malsam, J., Parisotto, D., Bharat, T.A., Scheutzow, A., Krause, J.M., Briggs, J.A. & Sollner, T.H.
EMBO J. 2012 Jun 15. doi: 10.1038/emboj.2012.164.
Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.
The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions.
Faini, M., Prinz, S., Beck, R., Schorb, M., Riches, J.D., Bacia, K., Brugger, B., Wieland, F.T. & Briggs, J.A.
Science. 2012 Jun 15;336(6087):1451-4. Epub 2012 May 24.
Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.
Imaging cellular structure across scales with correlated light, superresolution, and electron microscopy.
Briggs, J.; Lakadamyali Melike
Molecular Biology of the cell, Volume: 23, Issue: 6, Pages: 979-980, DOI: 10.1091/mbc.E11-12-0971, Published: MAR 15 2012
Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.
Bharat, T.A., Noda, T., Riches, J.D., Kraehling, V., Kolesnikova, L., Becker, S., Kawaoka, Y. & Briggs, J.A.
Proc Natl Acad Sci U S A. 2012 Mar 13;109(11):4275-80. doi:10.1073/pnas.1120453109. Epub 2012 Feb 27.
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.
In vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus XMRV.
Hadravova, R., de Marco, A., Ulbrich, P., Stokrova, J., Dolezal, M., Pichova, I., Ruml, T., Briggs, J.A. & Rumlova, M.
J Virol. 2012 Feb;86(3):1297-306. Epub 2011 Nov 16.
Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a beta-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of beta-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of DeltaProCANC or Delta10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.
Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers.
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A.
Methods Cell Biol. 2012;111:235-57. doi: 10.1016/B978-0-12-416026-2.00013-3.
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.
Computational Identification of Novel Amino-Acid Interactions in HIV Gag via Correlated Evolution.
Kalinina, O.V., Oberwinkler, H., Glass, B., Krausslich, H.G., Russell, R.B. & Briggs, J.A.
PLoS One. 2012;7(8):e42468. Epub 2012 Aug 3.
Pairs of amino acid positions that evolve in a correlated manner are proposed to play important roles in protein structure or function. Methods to detect them might fare better with families for which sequences of thousands of closely related homologs are available than families with only a few distant relatives. We applied co-evolution analysis to thousands of sequences of HIV Gag, finding that the most significantly co-evolving positions are proximal in the quaternary structures of the viral capsid. A reduction in infectivity caused by mutating one member of a significant pair could be rescued by a compensatory mutation of the other.
Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells.
Bharat, T.A., Riches, J.D., Kolesnikova, L., Welsch, S., Krahling, V., Davey, N., Parsy, M.L., Becker, S. & Briggs, J.A.
PLoS Biol. 2011 Nov;9(11):e1001196. Epub 2011 Nov 15.
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.
Coatomer and dimeric ADP ribosylation factor 1 promote distinct steps in membrane scission.
Beck, R., Prinz, S., Diestelkotter-Bachert, P., Rohling, S., Adolf, F., Hoehner, K., Welsch, S., Ronchi, P., Brugger, B., Briggs, J.A. & Wieland, F.
J Cell Biol. 2011 Sep 5;194(5):765-77.
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 molecular architecture of HIV.
Briggs, J.A. & Krausslich, H.G.
J Mol Biol. 2011 Jul 22;410(4):491-500. doi: 10.1016/j.jmb.2011.04.021.
Assembly of human immunodeficiency virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an immature virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the virus assembly site, the immature virus, the maturation intermediates and the mature virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.
Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A.
J Cell Biol. 2011 Jan 10;192(1):111-9. doi: 10.1083/jcb.201009037. Epub 2011 Jan3.
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 approximately 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.
Multibudded tubules formed by COPII on artificial liposomes.
Bacia, K., Futai, E., Prinz, S., Meister, A., Daum, S., Glatte, D., Briggs, J.A. & Schekman, R.
Sci Rep. 2011;1:17. doi: 10.1038/srep00017. Epub 2011 Jun 17.
COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission.
Conserved and variable features of Gag structure and arrangement in immature retrovirus particles.
de Marco, A., Davey, N.E., Ulbrich, P., Phillips, J.M., Lux, V., Riches, J.D., Fuzik, T., Ruml, T., Krausslich, H.G., Vogt, V.M. & Briggs, J.A.
J Virol. 2010 Nov;84(22):11729-36. doi: 10.1128/JVI.01423-10. Epub 2010 Sep 1.
The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.
Structural Analysis of HIV-1 Maturation Using Cryo-Electron Tomography.
de Marco, A., Muller, B., Glass, B., Riches, J.D., Krausslich, H.G. & Briggs, J.A.
PLoS Pathog. 2010 Nov 24;6(11):e1001215.
HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.
Cryo Electron Tomography of Native HIV-1 Budding Sites.
Carlson, L.A., de Marco, A., Oberwinkler, H., Habermann, A., Briggs, J.A., Krausslich, H.G. & Grunewald, K.
PLoS Pathog. 2010 Nov 24;6(11):e1001173.
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.
Electron tomography reveals the steps in filovirus budding.
Welsch, S., Kolesnikova, L., Krahling, V., Riches, J.D., Becker, S. & Briggs, J.A.
PLoS Pathog. 2010 Apr 29;6(4):e1000875.
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.
Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition.
Martin, N., Welsch, S., Jolly, C., Briggs, J.A., Vaux, D. & Sattentau, Q.J.
J Virol. 2010 Apr;84(7):3516-27. doi: 10.1128/JVI.02651-09. Epub 2010 Jan 20.
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.
Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging.
Zanetti, G., Riches, J.D., Fuller, S.D. & Briggs, J.A.
J Struct Biol. 2009 Nov;168(2):305-12. doi: 10.1016/j.jsb.2009.08.002. Epub 2009Aug 8.
Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.
Computational model of membrane fission catalyzed by ESCRT-III.
Fabrikant, G., Lata, S., Riches, J.D., Briggs, J.A., Weissenhorn, W. & Kozlov, M.M.
PLoS Comput Biol. 2009 Nov;5(11):e1000575. Epub 2009 Nov 20.
ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.
Structure and assembly of immature HIV.
Briggs, J.A., Riches, J.D., Glass, B., Bartonova, V., Zanetti, G. & Krausslich, H.G.
Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11090-5. doi:10.1073/pnas.0903535106. Epub 2009 Jun 22.
The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximately 17-A resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.
Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis.
Carlson, L.A., Briggs, J.A., Glass, B., Riches, J.D., Simon, M.N., Johnson, M.C., Muller, B., Grunewald, K. & Krausslich, H.G.
Cell Host Microbe. 2008 Dec 11;4(6):592-9.
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.
HIV-1-cellular interactions analyzed by single virus tracing.
Endress, T., Lampe, M., Briggs, J.A., Krausslich, H.G., Brauchle, C., Muller, B. & Lamb, D.C.
Eur Biophys J. 2008 Oct;37(8):1291-301. Epub 2008 Apr 10.
Single virus tracing (SVT) allows the direct investigation of the entry pathway of viruses into living cells. Using fluorescently labeled virus-like particles (VLPs) and SVT, we have studied the interaction between human immunodeficiency virus type 1 (HIV-1) and the plasma membrane of living cells. From the trajectories of freely diffusing VLPs in solution, we established that the particle preparation was homogeneous and the particles had a hydrodynamic radius of 86 +/- 5 nm, consistent with the size of single HI viruses. The VLPs that come in contact with the cell surface either become immobilized or rapidly dissociate from the cell surface. The fraction of virions that become immobilized on the plasma membrane correlates with the surface heparan sulfate linked proteoglycans (HSPG) concentration of the cell line tested. The particles that are not immobilized make an average of 1.5 contacts with the cell surface before diffusing away. For most cell lines investigated, the contact duration follows an exponential distribution with a lifetime between 20 and 50 ms depending on the cell type.
Structure of a hexameric RNA packaging motor in a viral polymerase complex.
Huiskonen, J.T., Jaalinoja, H.T., Briggs, J.A., Fuller, S.D. & Butcher, S.J.
J Struct Biol. 2007 May;158(2):156-64. Epub 2006 Oct 7.
Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.
Double-labelled HIV-1 particles for study of virus-cell interaction.
Lampe, M., Briggs, J.A., Endress, T., Glass, B., Riegelsberger, S., Krausslich, H.G., Lamb, D.C., Brauchle, C. & Muller, B.
Virology. 2007 Mar 30;360(1):92-104. Epub 2006 Nov 9.
Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the viral envelope with a cellular membrane. While the viral and cellular proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the viral matrix protein combined with a red label fused to the viral Vpr protein designed to distinguish between complete virions and subviral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.
Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ.
Zanetti, G., Briggs, J.A., Grunewald, K., Sattentau, Q.J. & Fuller, S.D.
PLoS Pathog. 2006 Aug;2(8):e83.
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.
Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus.
Briggs, J.A., Johnson, M.C., Simon, M.N., Fuller, S.D. & Vogt, V.M.
J Mol Biol. 2006 Jan 6;355(1):157-68. Epub 2005 Nov 2.
Retrovirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency virus type 1 (HIV-1) Gag CA (capsid) domain in the immature virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroviruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in immature retrovirus particles.
The mechanism of HIV-1 core assembly: insights from three-dimensional reconstructions of authentic virions.
Briggs, J.A., Grunewald, K., Glass, B., Forster, F., Krausslich, H.G. & Fuller, S.D.
Structure. 2006 Jan;14(1):15-20.
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.
Classification and three-dimensional reconstruction of unevenly distributed or symmetry mismatched features of icosahedral particles.
Briggs, J.A., Huiskonen, J.T., Fernando, K.V., Gilbert, R.J., Scotti, P., Butcher, S.J. & Fuller, S.D.
J Struct Biol. 2005 Jun;150(3):332-9. Epub 2005 Apr 19.
Methods for the three-dimensional reconstruction of icosahedral particles, such as spherical viruses, from electron micrographs are well established. These methods take advantage of the 60-fold symmetry of the icosahedral group. Several features within these particles, however, may deviate from icosahedral symmetry. Examples include viral genomes, symmetry mismatched vertex proteins, unique DNA packaging vertices, flexible proteins, and proteins that are present at less than 100% occupancy. Such asymmetrically distributed features are smeared in the final density map when icosahedral symmetry is applied. Here, we describe a novel approach to classifying, analysing, and obtaining three-dimensional reconstructions of such features. The approach uses the orientation information derived from the icosahedral orientation search to facilitate multivariate statistical analysis and to limit the orientational degrees of freedom for reconstruction. We demonstrate the application of this approach to images of Kelp fly Virus. In this case, each virion may have two different types of fivefold vertex. We use our approach to produce independent reconstructions of the two types of vertex.
The stoichiometry of Gag protein in HIV-1.
Briggs, J.A., Simon, M.N., Gross, I., Krausslich, H.G., Fuller, S.D., Vogt, V.M. & Johnson, M.C.
Nat Struct Mol Biol. 2004 Jul;11(7):672-5. Epub 2004 Jun 20.
The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
Cryoelectron microscopy of mouse mammary tumor virus.
Briggs, J.A., Watson, B.E., Gowen, B.E. & Fuller, S.D.
J Virol. 2004 Mar;78(5):2606-8.
Cryoelectron microscopy of Mouse mammary tumor virus, a Betaretrovirus, provided information about glycoprotein structure and core formation. The virions showed the broad range of diameters typical of retroviruses. Betaretroviruses assemble cytoplasmically, so the broad size range cannot reflect the use of the plasma membrane as a platform for assembly.
Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper.
Schwarz-Linek, U., Werner, J.M., Pickford, A.R., Gurusiddappa, S., Kim, J.H., Pilka, E.S., Briggs, J.A., Gough, T.S., Hook, M., Campbell, I.D. & Potts, J.R.
Nature. 2003 May 8;423(6936):177-81.
Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats in an unfolded region of the protein. The bacterium-binding site in the amino-terminal domain (1-5F1) of Fn contains five sequential Fn type 1 (F1) modules. Here we show the structure of a streptococcal (S. dysgalactiae) FnBP peptide (B3) in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel beta-strands on sequential F1 modules-the first example of a tandem beta-zipper. Sequence analyses of larger regions of FnBPs from S. pyogenes and S. aureus reveal a repeating pattern of F1-binding motifs that match the pattern of F1 modules in 1-5F1 of Fn. In the process of Fn-mediated invasion of host cells, therefore, the bacterial proteins seem to exploit the modular structure of Fn by forming extended tandem beta-zippers. This work is a vital step forward in explaining the full mechanism of the integrin-dependent FnBP-mediated invasion of host cells.
Structural organization of authentic, mature HIV-1 virions and cores.
Briggs, J.A., Wilk, T., Welker, R., Krausslich, H.G. & Fuller, S.D.
EMBO J. 2003 Apr 1;22(7):1707-15.
Mature, infectious HIV-1 particles contain a characteristic cone-shaped core that encases the viral RNA and replication proteins. The architectures of mature virions and isolated cores were studied using cryo-electron microscopy. The average size ( approximately 145 nm) of the virion was unchanged during maturation. Most virions contained a single core but roughly one-third contained two or more cores. Consideration of the capsid protein concentration during core assembly indicated that core formation in vivo is template-mediated rather than concentration-driven. Although most cores were conical, 7% were tubular. These displayed a stacked-disc arrangement with 7-, 8-, 9- or 10-fold axial symmetry. Layer line filtration of these images showed that the capsid subunit arrangement is consistent with a 9.6 nm hexamer resembling that previously seen in the helical tubes assembled from purified capsid protein. A common reflection (1/3.2 nm) shared between the tubular and conical cores suggested they share a similar organization. The extraordinary flexibility observed in the assembly of the mature core appears to be well suited to accommodating variation and hence there may be no single structure for the infectious virion.
Do lipid rafts mediate virus assembly and pseudotyping?
Briggs, J.A., Wilk, T. & Fuller, S.D.
J Gen Virol. 2003 Apr;84(Pt 4):757-68.
Co-infection of a host cell by two unrelated enveloped viruses can lead to the production of pseudotypes: virions containing the genome of one virus but the envelope proteins of both viruses. The selection of components during virus assembly must therefore be flexible enough to allow the incorporation of unrelated viral membrane proteins, yet specific enough to exclude the bulk of host proteins. This apparent contradiction has been termed the pseudotypic paradox. There is mounting evidence that lipid rafts play a role in the assembly pathway of non-icosahedral, enveloped viruses. Viral components are concentrated initially in localized regions of the plasma membrane via their interaction with lipid raft domains. Lateral interactions of viral structural proteins amplify the changes in local lipid composition which in turn enhance the concentration of viral proteins in the rafts. An affinity for lipid rafts may be the common feature of enveloped virus proteins that leads to the formation of pseudotypes.
Contribution of energy values to the analysis of global searching molecular dynamics simulations of transmembrane helical bundles.
Torres, J., Briggs, J.A. & Arkin, I.T.
Biophys J. 2002 Jun;82(6):3063-71.
Molecular interactions between transmembrane alpha-helices can be explored using global searching molecular dynamics simulations (GSMDS), a method that produces a group of probable low energy structures. We have shown previously that the correct model in various homooligomers is always located at the bottom of one of various possible energy basins. Unfortunately, the correct model is not necessarily the one with the lowest energy according to the computational protocol, which has resulted in overlooking of this parameter in favor of experimental data. In an attempt to use energetic considerations in the aforementioned analysis, we used global searching molecular dynamics simulations on three homooligomers of different sizes, the structures of which are known. As expected, our results show that even when the conformational space searched includes the correct structure, taking together simulations using both left and right handedness, the correct model does not necessarily have the lowest energy. However, for the models derived from the simulation that uses the correct handedness, the lowest energy model is always at, or very close to, the correct orientation. We hypothesize that this should also be true when simulations are performed using homologous sequences, and consequently lowest energy models with the right handedness should produce a cluster around a certain orientation. In contrast, using the wrong handedness the lowest energy structures for each sequence should appear at many different orientations. The rationale behind this is that, although more than one energy basin may exist, basins that do not contain the correct model will shift or disappear because they will be destabilized by at least one conservative (i.e. silent) mutation, whereas the basin containing the correct model will remain. This not only allows one to point to the possible handedness of the bundle, but can be used to overcome ambiguities arising from the use of homologous sequences in the analysis of global searching molecular dynamics simulations. In addition, because clustering of lowest energy models arising from homologous sequences only happens when the estimation of the helix tilt is correct, it may provide a validation for the helix tilt estimate.
Role of adaptor complex AP-3 in targeting wild-type and mutated CD63 to lysosomes.
Rous, B.A., Reaves, B.J., Ihrke, G., Briggs, J.A., Gray, S.R., Stephens, D.J., Banting, G. & Luzio, J.P.
Mol Biol Cell. 2002 Mar;13(3):1071-82.
CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit mu 3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with mu 3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells. Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with mu 3 but not at all with mu 2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells. In contrast, it localized to the cell surface in transfected cells of pearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the delta subunit of AP-3. Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes. Using this AP-3-dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from the trans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes.
Multiple site-specific infrared dichroism of CD3-zeta, a transmembrane helix bundle.
Torres, J., Briggs, J.A. & Arkin, I.T.
J Mol Biol. 2002 Feb 15;316(2):365-74.
The structure of the transmembrane domain of CD3-zeta a component of the T-cell receptor involved in signal transduction, has been studied in its native state (a lipid bilayer) by multiple site-specific infrared dichroism. For the first time, the transmembrane domain has been labelled at multiple positions along the sequence, representing a total of 11 samples, each labelled at a different residue with an isotopically modified carbonyl group, (13)C [double bond] (18)O. A strategy is outlined that, based on the above data, can yield the rotational orientation and the local helix tilt for each labelled residue, giving a detailed description of helix geometry. The results obtained indicate that the transmembrane segment is in an alpha-helical conformation throughout, with an average helix tilt of 12 degrees. The N-terminal side of the helix is more tilted than the C-terminal.In an accompanying paper we describe the implementation of the infrared data in a model-building study of the CD3-zeta transmembrane complex. The model obtained is entirely consistent with results based on evolutionary conservation data. Taken together, this study represents the first step towards elucidation of the backbone structure of a transmembrane alpha-helical bundle by infrared spectroscopy.
Convergence of experimental, computational and evolutionary approaches predicts the presence of a tetrameric form for CD3-zeta.
Torres, J., Briggs, J.A. & Arkin, I.T.
J Mol Biol. 2002 Feb 15;316(2):375-84.
Experimental results using multiple site-specific infrared dichroism have shown that, when reconstituted into lipid bilayers, the orientation of the transmembrane domain of CD3-zeta is not compatible with a dimeric right-handed model reported previously. This model, obtained using a computational approach that uses evolutionary data, is in agreement with mutagenesis data and homology modelling. This suggested that, in our experimental conditions, the oligomeric state of CD3-zeta may not be dimeric. We have explored this possibility by performing global searching molecular dynamics simulations assuming different homo-oligomeric sizes (from 2 to 6). In these simulations, the helix tilt was restrained to the average helix tilt obtained experimentally, 12 degrees. Only a left-handed tetrameric model was compatible with the experimentally observed tilt and rotational orientation of the helix, and was also the lowest-energy model amongst the candidate structures obtained. Furthermore, simulations performed using close homologues demonstrate that this model is compatible with evolutionary conservation data. Finally, the pattern of residue conservation in the zeta family of proteins strongly argues in favour of the presence of a left-handed hetero-oligomer with an orientation compatible with the tetramer we present. These results show that both the known dimeric and the so far undetected tetrameric form may be of functional importance in the cell.
A new method to model membrane protein structure based on silent amino acid substitutions.
Briggs, J.A., Torres, J. & Arkin, I.T.
Proteins. 2001 Aug 15;44(3):370-5.
The importance of accurately modeling membrane proteins cannot be overstated, in lieu of the difficulties in solving their structures experimentally. Often, however, modeling procedures (e.g., global searching molecular dynamics) generate several possible candidates rather then pointing to a single model. Herein we present a new approach to select among candidate models based on the general hypothesis that silent amino acid substitutions, present in variants identified from evolutionary conservation data or mutagenesis analysis, do not affect the stability of a native structure but may destabilize the non-native structures also found. The proof of this hypothesis has been tested on the alpha-helical transmembrane domains of two homodimers, human glycophorin A and human CD3-zeta, a component of the T-cell receptor. For both proteins, only one structure was identified using all the variants. For glycophorin A, this structure is virtually identical to the structure determined experimentally by NMR. We present a model for the transmembrane domain of CD3-zeta that is consistent with predictions based on mutagenesis, homology modeling, and the presence of a disulfide bond. Our experiments suggest that this method allows the prediction of transmembrane domain structure based only on widely available evolutionary conservation data.