165 results on '"Naiqian, Cheng"'
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2. Biphasic Packing of DNA and Internal Proteins in Bacteriophage T4 Heads Revealed by Bubblegram Imaging
- Author
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Weimin Wu, Naiqian Cheng, Lindsay W. Black, Hendrik Dietz, and Alasdair C. Steven
- Subjects
cryo-electron microscopy ,DNA packaging ,coaxial spool ,DNA origami ,radiation damage ,bubblegram ,Microbiology ,QR1-502 - Abstract
The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.
- Published
- 2020
- Full Text
- View/download PDF
3. The Mottled Capsid of the Salmonella Giant Phage SPN3US, a Likely Maturation Intermediate with a Novel Internal Shell
- Author
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J. Bernard Heymann, Bing Wang, William W. Newcomb, Weimin Wu, Dennis C. Winkler, Naiqian Cheng, Erin R. Reilly, Ru-Ching Hsia, Julie A. Thomas, and Alasdair C. Steven
- Subjects
cryoEM (cryo-electron microscopy) ,bacteriophage ,virus ,single particle analysis ,3D reconstruction ,scaffold ,Microbiology ,QR1-502 - Abstract
“Giant” phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them “mottled capsids”. Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a “twisted hook” conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.
- Published
- 2020
- Full Text
- View/download PDF
4. The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress
- Author
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William W. Newcomb, Juan Fontana, Dennis C. Winkler, Naiqian Cheng, J. Bernard Heymann, and Alasdair C. Steven
- Subjects
HSV capsid ,cryo-electron microscopy ,cryo-electron tomography ,nuclear egress ,nuclear egress complex ,nuclear envelope ,Microbiology ,QR1-502 - Abstract
ABSTRACT Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions. IMPORTANCE On its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate—the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.
- Published
- 2017
- Full Text
- View/download PDF
5. Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging
- Author
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Weimin Wu, Justin C. Leavitt, Naiqian Cheng, Eddie B. Gilcrease, Tina Motwani, Carolyn M. Teschke, Sherwood R. Casjens, and Alasdair C. Steven
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. IMPORTANCE While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.
- Published
- 2016
- Full Text
- View/download PDF
6. Subassemblies and Asymmetry in Assembly of Herpes Simplex Virus Procapsid
- Author
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Anastasia A. Aksyuk, William W. Newcomb, Naiqian Cheng, Dennis C. Winkler, Juan Fontana, J. Bernard Heymann, and Alasdair C. Steven
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation. IMPORTANCE This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit “protomers” are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.
- Published
- 2015
- Full Text
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7. Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids
- Author
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Giovanni Cardone, Robert L. Duda, Naiqian Cheng, Lili You, James F. Conway, Roger W. Hendrix, and Alasdair C. Steven
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local minima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross-linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in maturation. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable intermediates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conformations of its precursor and mature states. IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in capsid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of principle, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.
- Published
- 2014
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8. Maturation of the Human Papillomavirus 16 Capsid
- Author
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Giovanni Cardone, Adam L. Moyer, Naiqian Cheng, Cynthia D. Thompson, Israel Dvoretzky, Douglas R. Lowy, John T. Schiller, Alasdair C. Steven, Christopher B. Buck, and Benes L. Trus
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. IMPORTANCE Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.
- Published
- 2014
- Full Text
- View/download PDF
9. Biphasic Packing of DNA and Internal Proteins in Bacteriophage T4 Heads Revealed by Bubblegram Imaging
- Author
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Naiqian Cheng, Hendrik Dietz, Lindsay W. Black, Weimin Wu, and Alasdair C. Steven
- Subjects
0301 basic medicine ,Models, Molecular ,Cryo-electron microscopy ,viruses ,Shell (structure) ,lcsh:QR1-502 ,cryo-electron microscopy ,02 engineering and technology ,Article ,lcsh:Microbiology ,Bacteriophage ,03 medical and health sciences ,chemistry.chemical_compound ,Capsid ,Virology ,Microscopy ,DNA origami ,Bacteriophage T4 ,biology ,DNA packaging ,Virus Assembly ,Cryoelectron Microscopy ,Nucleocapsid Proteins ,021001 nanoscience & nanotechnology ,biology.organism_classification ,bubblegram ,Nucleoprotein ,030104 developmental biology ,Infectious Diseases ,chemistry ,radiation damage ,DNA, Viral ,Biophysics ,coaxial spool ,0210 nano-technology ,DNA - Abstract
The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions, it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.
- Published
- 2020
10. The Mottled Capsid of the Salmonella Giant Phage SPN3US, a Likely Maturation Intermediate with a Novel Internal Shell
- Author
-
Bing Wang, Erin R Reilly, J. Bernard Heymann, Dennis C. Winkler, William W. Newcomb, Ru-Ching Hsia, Weimin Wu, Julie A. Thomas, Alasdair C. Steven, and Naiqian Cheng
- Subjects
0301 basic medicine ,cryoEM (cryo-electron microscopy) ,Icosahedral symmetry ,viruses ,Population ,lcsh:QR1-502 ,Genome, Viral ,virus ,scaffold ,Cleavage (embryo) ,Article ,lcsh:Microbiology ,Bacteriophage ,03 medical and health sciences ,chemistry.chemical_compound ,Capsid ,0302 clinical medicine ,bacteriophage ,Salmonella ,Virology ,DNA Packaging ,Giant Virus ,030212 general & internal medicine ,3D reconstruction ,education ,single particle analysis ,education.field_of_study ,biology ,Chemistry ,Virus Assembly ,Cryoelectron Microscopy ,Virion ,ejection proteins ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,030104 developmental biology ,Infectious Diseases ,Virion assembly ,Giant Viruses ,Biophysics ,Capsid Proteins ,Salmonella Phages ,DNA - Abstract
&ldquo, Giant&rdquo, phages have genomes of >, 200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them &ldquo, mottled capsids&rdquo, Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a &ldquo, twisted hook&rdquo, conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å, repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.
- Published
- 2020
11. Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae
- Author
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Naiqian Cheng, Alasdair C. Steven, Weimin Wu, Paul T. Wingfield, Rick Huang, and Norman R. Watts
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0301 basic medicine ,Protein Conformation ,Dimer ,viruses ,Viral Packaging ,Database and Informatics Methods ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Potential Energy ,Electron Microscopy ,Biology (General) ,Materials ,Peptide sequence ,Free Energy ,Microscopy ,Crystallography ,Ecology ,biology ,Physics ,Classical Mechanics ,Alanine scanning ,Condensed Matter Physics ,Negative stain ,Chemistry ,Computational Theory and Mathematics ,Capsid ,Modeling and Simulation ,Physical Sciences ,Crystal Structure ,Thermodynamics ,Sequence Analysis ,Research Article ,Hepatitis B virus ,Bioinformatics ,Icosahedral symmetry ,QH301-705.5 ,Materials Science ,Viral Structure ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Sequence Motif Analysis ,Virology ,Genetics ,Solid State Physics ,Amino Acid Sequence ,Dimers ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Binding Sites ,Computational Biology ,Biology and Life Sciences ,Polymer Chemistry ,biology.organism_classification ,Viral Replication ,Protein Subunits ,030104 developmental biology ,chemistry ,Hepadnaviridae ,Oligomers ,Capsid Proteins ,Protein Multimerization ,030217 neurology & neurosurgery - Abstract
Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 Å resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism., Author summary Hepatitis B virus has infected approximately one third of the human population and causes almost 1 million deaths from liver disease annually. The capsid is a defining feature of a virus, distinct from host components, and therefore a target for intervention. Unusually for a virus, Hepatitis B assembles two capsids, with different geometries, from the same dimeric protein. Geometric principles dictate that the subunits in this system occupy seven different environments. From comparing the two capsids by cryo-electron microscopy at high resolution under the exact same conditions we find that the polypeptide chains adopt seven different conformations. We use these structures to calculate potential energies (analogous to elastic deformation or strain) for the individual chains, dimers, and several higher-order groupings discernible in the two lattices. We also calculate the binding energies between chains. We find that some groupings have substantially lower energy and are therefore potentially more stable, allowing us to predict likely intermediates on the two assembly pathways. We also observe such intermediates by electron microscopy of in vitro capsid assembly reactions. This is the first structural characterization of the early assembly intermediates of this important human pathogen.
- Published
- 2020
12. Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids
- Author
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Oliver W, Bayfield, Evgeny, Klimuk, Dennis C, Winkler, Emma L, Hesketh, Maria, Chechik, Naiqian, Cheng, Eric C, Dykeman, Leonid, Minakhin, Neil A, Ranson, Konstantin, Severinov, Alasdair C, Steven, and Alfred A, Antson
- Subjects
DNA packaging ,viruses ,Virus Assembly ,Cryoelectron Microscopy ,DNA Viruses ,Virion ,Biological Sciences ,portal protein ,Biophysics and Computational Biology ,Capsid ,DNA, Viral ,Physical Sciences ,cryo-EM ,Bacteriophages - Abstract
Significance Understanding molecular events during virus assembly and genome packaging is important for understanding viral life cycles, and the functioning of other protein–nucleic acid machines. The model system developed for the thermophilic bacteriophage P23-45 offers advantages over other systems. Cryo-EM reconstructions reveal modifications to a canonical capsid protein fold, resulting in capsids that are abnormally large for this virus class. Structural information on the portal protein, through which the genome is packaged, demonstrates that the capsid influences the portal’s conformation. This has implications for understanding how processes inside and outside the capsid can be coordinated., Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
- Published
- 2019
13. The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly
- Author
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Jonathan M. Grimes, Michael A. DiMattia, Naiqian Cheng, David I. Stuart, A.C. Steven, Rick Huang, Paul T. Wingfield, Norman R. Watts, and J B Heymann
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0301 basic medicine ,Physics ,Hiv 1 rev ,Quantitative Biology::Biomolecules ,viruses ,Dimer ,RNA ,rev Gene Products, Human Immunodeficiency Virus ,Nanotechnology ,Rev Protein ,Article ,Quantitative Biology::Subcellular Processes ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Structural Biology ,HIV-1 ,Biophysics ,Viral rna ,Protein Multimerization ,Nuclear export signal ,Molecular Biology ,Protein Binding - Abstract
HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev-response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing-angles of 90° to 140° . In parallel, we performed cryo-EM of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing-angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs which offers an explanation for how the arrangement of Rev subunits adapts to the ‘A’-shaped architecture of the RRE in export-active complexes.
- Published
- 2016
14. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging
- Author
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William W. Newcomb, Dennis C. Winkler, Anastasia A. Aksyuk, Naiqian Cheng, Alasdair C. Steven, and Weimin Wu
- Subjects
0301 basic medicine ,Scaffold protein ,Icosahedral symmetry ,viruses ,Proteolysis ,Protein subunit ,Immunology ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Capsid ,0302 clinical medicine ,Virology ,DNA Packaging ,medicine ,030212 general & internal medicine ,medicine.diagnostic_test ,Virus Assembly ,Structure and Assembly ,Cryoelectron Microscopy ,Capsomere ,Virion ,biochemical phenomena, metabolism, and nutrition ,Cell biology ,030104 developmental biology ,Herpes simplex virus ,chemistry ,Insect Science ,Capsid Proteins ,DNA - Abstract
The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids—which develop into infectious virions—are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation damage was used to localize internal proteins of HSV-1, yielding insights into how capsid maturation is regulated. The scaffolding protein, which forms inner shells in the procapsid and B capsid, is exceptionally bubbling-prone. In the mature DNA-filled C capsid, a previously undetected protein was found to underlie the icosahedral vertices: this is tentatively assigned as a storage form of the viral protease. We also observed a capsid species that appears to contain substantial amounts of scaffolding protein as well as DNA, suggesting that DNA packaging and expulsion of the scaffolding protein are coupled processes.
- Published
- 2016
15. Exploiting radiation damage to map proteins in nucleoprotein complexes: The internal structure of bacteriophage T7
- Author
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Naiqian Cheng, Alasdair C. Steven, Weimin Wu, and Norman R. Watts
- Subjects
Cryo-electron microscopy ,Bubble ,Cryoelectron Microscopy ,Biology ,biology.organism_classification ,Article ,Nucleoprotein ,Core (optical fiber) ,Bacteriophage ,chemistry.chemical_compound ,Crystallography ,Nucleoproteins ,chemistry ,Capsid ,Structural Biology ,Bacteriophage T7 ,Microscopy ,Biophysics ,Capsid Proteins ,DNA - Abstract
In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique (“bubblegram imaging”) by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 s) intervals between exposures of 17 electrons/A 2 each, bubbling starts in the third exposure, with 1–4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.
- Published
- 2014
16. Conformational Shift of a Major Poliovirus Antigen Confirmed by Immuno-Cryogenic Electron Microscopy
- Author
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Alasdair C. Steven, Naiqian Cheng, David M. Belnap, Jun Lin, and James M. Hogle
- Subjects
viruses ,Poliovirus ,Immunoglobulin Fab Fragments ,Immunology ,Biology ,medicine.disease_cause ,Molecular biology ,law.invention ,Protein structure ,Capsid ,Antigen ,law ,medicine ,Biophysics ,Immunology and Allergy ,Particle ,Electron microscope ,Eukaryotic Ribosome - Abstract
Small, interfacial conformational changes occur in some Ag–Ab interactions. Using cryogenic electron microscopy (cryo-EM), we have demonstrated such changes in a major antigenic site of a poliovirus capsid protein. During cell entry, native human poliovirus (160S particle) converts to a cell entry intermediate (135S particle) and later to an RNA-released (80S) particle. By mixing particles with Fabs of the neutralizing C3 mAb, we labeled the external loop connecting the B and C β-strands (BC loop) of the capsid protein VP1 (residues 95–105) in the 160S and 135S states. We then determined three-dimensional structures by cryo-EM and enhanced their interpretability by fitting high-resolution coordinates of C3 Fab and the capsid proteins into the density maps. Binding of C3 to either 160S or 135S particles caused residues of the BC loop, located on the tip of a prominent peak known as the “mesa,” to move by an estimated 5 Å. C3 Abs are neutralizing and can bind bivalently. The orientation of the bound Fabs in our reconstructions suggests that C3 neutralizes poliovirus by binding two adjacent BC loops on the same mesa and inhibiting conformational changes in the viral capsid.
- Published
- 2013
17. α-Synuclein Oligomers with Broken Helical Conformation Form Lipoprotein Nanoparticles
- Author
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Alasdair C. Steven, Jobin Varkey, Balachandra G. Hegde, Naoko Mizuno, Ralf Langen, and Naiqian Cheng
- Subjects
Circular dichroism ,animal diseases ,Lipoproteins ,Phosphatidylserines ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,mental disorders ,Fluorescence Resonance Energy Transfer ,Humans ,Particle Size ,Protein Structure, Quaternary ,Molecular Biology ,Protein secondary structure ,Phosphatidylglycerol ,Vesicle ,Cryoelectron Microscopy ,Membranes, Artificial ,Phosphatidylglycerols ,Cell Biology ,nervous system diseases ,Crystallography ,Cholesterol ,Förster resonance energy transfer ,Tubule ,Membrane ,nervous system ,Membrane protein ,chemistry ,Mitochondrial Membranes ,Chromatography, Gel ,Phosphatidylcholines ,alpha-Synuclein ,Nanoparticles ,lipids (amino acids, peptides, and proteins) ,Molecular Biophysics - Abstract
α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.
- Published
- 2013
18. Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure
- Author
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Stephen J. Stahl, Zhaochun Chen, Weimin Wu, Alasdair C. Steven, Paul T. Wingfield, Naiqian Cheng, Robert H. Purcell, Norman R. Watts, and Patrizia Farci
- Subjects
Models, Molecular ,Hepatitis B virus ,medicine.drug_class ,Biology ,medicine.disease_cause ,Monoclonal antibody ,Chromatography, Affinity ,Protein Structure, Secondary ,Article ,Epitope ,Antigen ,Antibody Specificity ,Structural Biology ,medicine ,Humans ,Hepatitis B Antibodies ,Protein Structure, Quaternary ,Viral Core Proteins ,Cryoelectron Microscopy ,Liver Failure, Acute ,Hepatitis B ,Virology ,Molecular biology ,HBcAg ,Epitope mapping ,Capsid ,Epitope Mapping ,Conformational epitope - Abstract
Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25Å-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous “floor”. Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis.
- Published
- 2013
19. Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging
- Author
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Eddie B. Gilcrease, Carolyn M. Teschke, Justin C. Leavitt, Alasdair C. Steven, Tina Motwani, Naiqian Cheng, Sherwood R. Casjens, and Weimin Wu
- Subjects
0301 basic medicine ,biology ,Protein subunit ,Cryoelectron Microscopy ,Mutant ,Virion ,biology.organism_classification ,Models, Biological ,Microbiology ,Virology ,QR1-502 ,3. Good health ,Bacteriophage ,Viral Proteins ,03 medical and health sciences ,Barrel ,030104 developmental biology ,Dodecameric protein ,Capsid ,Biophysics ,Binding site ,Bacteriophage P22 ,Bubblegram ,Research Article - Abstract
The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection., IMPORTANCE While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.
- Published
- 2016
20. α-Synuclein Amyloid Fibrils with Two Entwined, Asymmetrically Associated Protofibrils
- Author
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Joseph S. Wall, Andrey V. Kajava, Naiqian Cheng, Alasdair C. Steven, J. Bernard Heymann, Jobin Varkey, Altaira D. Dearborn, Ralf Langen, Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Biology [York, UK], University of York [York, UK], Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), and University of Southern California (USC)
- Subjects
0301 basic medicine ,Microscopy, Electron, Scanning Transmission ,Amyloid ,Cryo-electron microscopy ,Protein subunit ,Molecular Sequence Data ,macromolecular substances ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Fibril ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Microscopy ,Scanning transmission electron microscopy ,Image Processing, Computer-Assisted ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,Alpha-synuclein ,Ions ,Binding Sites ,Sequence Homology, Amino Acid ,Chemistry ,Cryoelectron Microscopy ,Cell Biology ,Amyloid fibril ,Crystallography ,030104 developmental biology ,Biophysics ,alpha-Synuclein ,Lewy Bodies ,Molecular Biophysics - Abstract
Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of α-synuclein. We used cryo-electron microscopy and scanning transmission electron microscopy (STEM) to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass per length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated β-structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77-nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other, but not all, fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils that are associated differently.
- Published
- 2016
21. Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation
- Author
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Balachandra G. Hegde, Jobin Varkey, Alasdair C. Steven, Naiqian Cheng, Natalie C. Kegulian, Naoko Mizuno, and Ralf Langen
- Subjects
Tube formation ,Protein Folding ,Chemistry ,Bilayer ,Vesicle ,Lipid Bilayers ,Electron Spin Resonance Spectroscopy ,Membrane structure ,Parkinson Disease ,Phosphatidylglycerols ,Cell Biology ,Biochemistry ,Micelle ,Protein Structure, Secondary ,Crystallography ,Membrane Biology ,alpha-Synuclein ,Humans ,lipids (amino acids, peptides, and proteins) ,Protein folding ,Lipid bilayer ,Molecular Biology ,Membrane biophysics ,Micelles - Abstract
α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.
- Published
- 2012
22. Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giantPseudomonas aeruginosaphage φKZ
- Author
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Lindsay W. Black, Alasdair C. Steven, Dennis C. Winkler, Julie A. Thomas, Susan T. Weintraub, Naiqian Cheng, and Weimin Wu
- Subjects
Protease ,medicine.diagnostic_test ,biology ,Molecular mass ,Proteolysis ,medicine.medical_treatment ,Prohead ,Myoviridae ,Cleavage (embryo) ,biology.organism_classification ,Microbiology ,Capsid ,Biochemistry ,medicine ,Molecular Biology ,Function (biology) - Abstract
Encased within the 280 kb genome in the capsid of the giant myovirus φKZ is an unusual cylindrical proteinaceous “inner body” of highly ordered structure. We present here mass spectrometry, bioinformatic, and biochemical studies that reveal novel information about the φKZ head and the complex inner body. The identification of 39 cleavage sites in 19 φKZ head proteins indicates cleavage of many prohead proteins forms a major morphogenetic step in φKZ head maturation. The φKZ head protease, gp175, is newly identified here by a bioinformatics approach, as confirmed by a protein expression assay. Gp175 is distantly related to T4 gp21 and recognizes and cleaves head precursors at related but distinct S/A/G-X-E recognition sites. Within the φKZ head there are six high copy number proteins that are probable major components of the inner body. The molecular weights of five of these proteins are reduced 35–65% by cleavages making their mature form similar (26–31 kDa), while their precursors are dissimilar (36–88 kDa). Together the six abundant proteins sum to the estimated mass of the inner body (15–20 MDa). The identification of these proteins is important for future studies on the composition and function of the inner body.
- Published
- 2012
23. Subassemblies and Asymmetry in Assembly of Herpes Simplex Virus Procapsid
- Author
-
Juan Fontana, Naiqian Cheng, William W. Newcomb, Dennis C. Winkler, J. Bernard Heymann, Alasdair C. Steven, Anastasia A. Aksyuk, and Dermody, TS
- Subjects
Models, Molecular ,Scaffold protein ,Icosahedral symmetry ,Virus Assembly ,viruses ,Protein subunit ,Cryoelectron Microscopy ,Capsomere ,Protomer ,biochemical phenomena, metabolism, and nutrition ,Biology ,Microbiology ,Virology ,QR1-502 ,chemistry.chemical_compound ,Capsid ,chemistry ,Biophysics ,Simplexvirus ,Protein Multimerization ,DNA ,Research Article ,Macromolecule - Abstract
The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation., IMPORTANCE This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit “protomers” are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.
- Published
- 2015
24. Primary Envelopment of the Herpes Simplex 1 Virion
- Author
-
Alasdair C. Steven, William W. Newcomb, J. Bernard Heymann, Dennis C. Winkler, Naiqian Cheng, and Juan Fontana
- Subjects
0301 basic medicine ,03 medical and health sciences ,0302 clinical medicine ,Primary (chemistry) ,Simplex ,Chemistry ,030212 general & internal medicine ,Envelopment ,030112 virology ,Instrumentation ,Virology - Published
- 2017
25. Applications of Bubblegram Imaging
- Author
-
Juan Fontana, Weimin Wu, Naiqian Cheng, and Alasdair C. Steven
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Chemistry ,Instrumentation ,Biomedical engineering - Published
- 2017
26. Stepwise Expansion of the Bacteriophage ϕ6 Procapsid: Possible Packaging Intermediates
- Author
-
Naiqian Cheng, Leonard Mindich, Jian Qiao, Alasdair C. Steven, Daniel Nemecek, and J. Bernard Heymann
- Subjects
Calorimetry, Differential Scanning ,biology ,Cryo-electron microscopy ,Virus Assembly ,viruses ,Cryoelectron Microscopy ,Mutant ,RNA ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Article ,Bacteriophage ,RNA silencing ,Crystallography ,Capsid ,Structural Biology ,RNA, Viral ,Intermediate state ,Bacteriophages ,Binding site ,Molecular Biology - Abstract
The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.
- Published
- 2011
27. In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone
- Author
-
Ulrich Baxa, Paul W. Keller, Alasdair C. Steven, Joseph S. Wall, and Naiqian Cheng
- Subjects
Amyloid ,Protein subunit ,Peptide Termination Factors ,macromolecular substances ,Transfection ,Biology ,Fibril ,Microbiology ,Ribosome ,Negative stain ,In vitro ,Biochemistry ,Biophysics ,Molecular Biology - Abstract
In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter (∼8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield ∼1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.
- Published
- 2010
28. Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy
- Author
-
Brian Firek, Philip D. Ross, Roger W. Hendrix, Robert L. Duda, Naiqian Cheng, James F. Conway, and Alasdair C. Steven
- Subjects
Calorimetry, Differential Scanning ,Protein Conformation ,Cryo-electron microscopy ,Chemistry ,viruses ,Cryoelectron Microscopy ,Calorimetry ,DNA condensation ,Article ,Nucleic acid thermodynamics ,Crystallography ,chemistry.chemical_compound ,Capsid ,Structural Biology ,Ionic strength ,DNA, Viral ,Nucleic Acid Conformation ,Thermodynamics ,Bacteriophages ,Capsid Proteins ,Denaturation (biochemistry) ,Molecular Biology ,DNA - Abstract
Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg(2+), DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 degrees C and 96 degrees C. In other conditions (absence of Mg(2+) and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit-probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.
- Published
- 2009
29. Visualization of a missing link in retrovirus capsid assembly
- Author
-
Rebecca C. Craven, John G. Purdy, Giovanni Cardone, Alasdair C. Steven, and Naiqian Cheng
- Subjects
Models, Molecular ,Pentamer ,viruses ,Protein subunit ,Static Electricity ,Random hexamer ,010402 general chemistry ,01 natural sciences ,Article ,03 medical and health sciences ,Capsid ,Retrovirus ,Static electricity ,Protein Structure, Quaternary ,030304 developmental biology ,0303 health sciences ,Rous sarcoma virus ,Polymorphism, Genetic ,Multidisciplinary ,biology ,Virus Assembly ,Cryoelectron Microscopy ,HIV ,Link (geometry) ,biology.organism_classification ,3. Good health ,0104 chemical sciences ,Protein Subunits ,Crystallography ,Mutation ,Capsid Proteins ,Mutant Proteins ,Protein Multimerization - Abstract
For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers. Retrovirus capsids are polymorphic, consisting of variable hexamer and pentamer aggregates with structures thought to resemble fullerenes. A cryo-electon microscopy/molecular modelling study of capsids assembled from the full-length Rous sarcoma virus capsid protein now confirms the existence of fullerene-like capsid architecture. Two different forms were observed, one composed of 12 pentamers and the other of 12 pentamers and 20 hexamers. Retrovirus capsids are polymorphic, consisting of variable hexamer and pentamer aggregates that are thought to reflect fullerenes. This paper reports the cryo-electron microscopy analysis of RSV capsid protein visualizing pentamers, and confirms that retrovirus capsid has a fullerene-based architecture.
- Published
- 2009
30. Non-canonical Binding of an Antibody Resembling a Naïve B Cell Receptor Immunoglobulin to Hepatitis B Virus Capsids
- Author
-
Stephen J. Stahl, Joe G. Vethanayagam, Naiqian Cheng, Matti Sällberg, Giovanni Cardone, Catharina Hultgren, Paul T. Wingfield, Norman R. Watts, and Alasdair C. Steven
- Subjects
Models, Molecular ,Hepatitis B virus ,Molecular Sequence Data ,Naive B cell ,B-cell receptor ,Antibody Affinity ,Immunoglobulin Variable Region ,Receptors, Antigen, B-Cell ,Priming (immunology) ,Antigen-Antibody Complex ,Article ,Antigen-Antibody Reactions ,Immunoglobulin Fab Fragments ,Mice ,Capsid ,Antigen ,Structural Biology ,Image Processing, Computer-Assisted ,Animals ,Amino Acid Sequence ,Hepatitis B Antibodies ,Molecular Biology ,B-Lymphocytes ,Sequence Homology, Amino Acid ,biology ,Models, Immunological ,Surface Plasmon Resonance ,Molecular biology ,Kinetics ,Antigen-antibody interaction ,Multiprotein Complexes ,biology.protein ,Antibody - Abstract
The hepatitis B virus capsid (core antigen) is able to bind to and activate naïve B cells and these become efficient primary antigen-presenting cells for the priming of T cells. We have investigated this interaction by using cryo-electron microscopy, three-dimensional image reconstruction, and molecular modeling to visualize capsids decorated with Fab fragments of a receptor immunoglobulin, and surface plasmon resonance to measure the binding affinity. By both criteria, the mode of binding differs from those of the six monoclonal anti-core antigen antibodies previously characterized. The Fab interacts with two sites approximately 30 A apart. One interaction is canonical, whereby the CDR loops engage the tip of one of the 25 A spikes that protrude from the capsid surface. The second interaction is non-canonical; in it, the Fab framework contacts the tip of an adjacent spike. The binding affinity of this Fab for capsids, K(D) approximately 4 x 10(-7) M, is relatively low for an antibody-antigen interaction, but is approximately 150-fold lower still ( approximately 2.5 x 10(-5) M) for unassembled capsid protein dimers. The latter observation indicates that both of the observed interactions are required to achieve stable binding of capsids by this receptor immunoglobulin. Considerations of conserved sequence motifs in other such molecules suggest that other naïve B cells may interact with HBV capsids in much the same way.
- Published
- 2008
31. Allosteric Signaling and a Nuclear Exit Strategy: Binding of UL25/UL17 Heterodimers to DNA-Filled HSV-1 Capsids
- Author
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Giovanni Cardone, Lyuben N. Marekov, Fred L. Homa, Naiqian Cheng, Jay C. Brown, William W. Newcomb, Benes L. Trus, and Alasdair C. Steven
- Subjects
viruses ,Allosteric regulation ,Population ,Herpesvirus 1, Human ,Plasma protein binding ,Biology ,Article ,Mass Spectrometry ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Capsid ,Allosteric Regulation ,Image Processing, Computer-Assisted ,medicine ,education ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,education.field_of_study ,Cryoelectron Microscopy ,030302 biochemistry & molecular biology ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,Transport protein ,Protein Transport ,Cell nucleus ,medicine.anatomical_structure ,chemistry ,Biophysics ,Capsid Proteins ,Dimerization ,Nucleus ,DNA ,Protein Binding - Abstract
UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T=16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent, site that is not seen at other quasi-equivalent sites nor on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25-null capsids, and denaturant-extracted capsids, we conclude that (i) the C-capsid-specific component is a heterodimer of UL25 and UL17; and (ii) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.
- Published
- 2007
32. Visualization of the herpes simplex virus portal in situ by cryo-electron tomography
- Author
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Dennis C. Winkler, John E. Heuser, William W. Newcomb, Jay C. Brown, Giovanni Cardone, Naiqian Cheng, Benes L. Trus, and Alasdair C. Steven
- Subjects
Cryo-electron microscopy ,Icosahedral symmetry ,Pentamer ,viruses ,Context (language use) ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Article ,Viral Proteins ,03 medical and health sciences ,Virology ,Chlorocebus aethiops ,Image Processing, Computer-Assisted ,medicine ,Animals ,Three-dimentional image reconsruction ,A-DNA ,Microscopy, Immunoelectron ,Vero Cells ,030304 developmental biology ,0303 health sciences ,Portal protein ,Immuno-gold labelling ,Cryoelectron Microscopy ,030302 biochemistry & molecular biology ,Immunohistochemistry ,3. Good health ,Herpes simplex virus ,Capsid assemby ,Capsid ,Biophysics ,Cryo-electron tomography ,Capsid Proteins - Abstract
Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the twelve vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid. From a cryo-EM reconstruction of capsids immuno-gold-labeled with anti-UL6 antibodies, we confirmed that UL6 resides at a vertex. To visualize the portal in the context of the assembled capsid, we used cryo-electron tomography to determine the three-dimensional structures of individual A-capsids (empty, mature capsids). The similarity in size and overall shape of the portal and a UL19 pentamer - both are cylinders of ~ 800 kDa - combined with residual noise in the tomograms, prevented us from identifying the portal vertices directly; however, this was accomplished by a computational classification procedure. Averaging the portal-containing subtomograms produced a structure that tallies with the isolated portal, as previously reconstructed by cryo-EM. The portal is mounted on the outer surface of the capsid floor layer, with its narrow end pointing outwards. This disposition differs from that of known phage portals in that the bulk of its mass lies outside, not inside, the floor. This distinction may be indicative of functional divergence at the level of portal-related functions other than its role as a DNA channel.
- Published
- 2007
33. Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry
- Author
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James M. Hogle, Mike Strauss, David M. Belnap, Naiqian Cheng, Roane T. Noel, and David J. Filman
- Subjects
Models, Molecular ,viruses ,Immunology ,Nectins ,Biology ,medicine.disease_cause ,Microbiology ,Cell membrane ,Nectin ,Virology ,medicine ,Humans ,CD155 ,Receptor ,Poliovirus ,Cryoelectron Microscopy ,Virus Internalization ,Ligand (biochemistry) ,Molecular biology ,Virus-Cell Interactions ,medicine.anatomical_structure ,Capsid ,Ectodomain ,Insect Science ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Receptors, Virus ,Capsid Proteins ,Cell Adhesion Molecules ,HeLa Cells - Abstract
Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called “pocket factor”), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. IMPORTANCE The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.
- Published
- 2015
34. A Free Energy Cascade with Locks Drives Assembly and Maturation of Bacteriophage HK97 Capsid
- Author
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Naiqian Cheng, Alasdair C. Steven, James F. Conway, Philip D. Ross, Brian Firek, Lindsay Dierkes, Roger W. Hendrix, and Robert L. Duda
- Subjects
Models, Molecular ,Steric effects ,Conformational change ,Calorimetry, Differential Scanning ,medicine.diagnostic_test ,Chemistry ,Cryo-electron microscopy ,Proteolysis ,Protein subunit ,Cryoelectron Microscopy ,Capsomere ,Article ,Protein Structure, Tertiary ,Crystallography ,Capsid ,Structural Biology ,Covalent bond ,Mutation ,medicine ,Thermodynamics ,Bacteriophages ,Capsid Proteins ,Molecular Biology - Abstract
We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.
- Published
- 2006
35. Capsid Conformational Sampling in HK97 Maturation Visualized by X-Ray Crystallography and Cryo-EM
- Author
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James F. Conway, John E. Johnson, Robert L. Duda, Roger W. Hendrix, Lu Gan, Gabriel C. Lander, Lars Liljas, Jeffrey A. Speir, Brian Firek, and Naiqian Cheng
- Subjects
Models, Molecular ,Protein Folding ,Cryo-electron microscopy ,Protein Conformation ,Protein subunit ,Molecular Conformation ,Electrons ,Biology ,Crystallography, X-Ray ,03 medical and health sciences ,Protein structure ,Capsid ,Structural Biology ,Molecule ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Molecular Structure ,Virus Assembly ,030302 biochemistry & molecular biology ,Cryoelectron Microscopy ,Prohead ,Hydrogen-Ion Concentration ,Bacteriophage lambda ,Crystallography ,Cross-Linking Reagents ,Covalent bond ,Protein folding ,Crystallization - Abstract
Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.
- Published
- 2006
- Full Text
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36. Epitope Diversity of Hepatitis B Virus Capsids: Quasi-equivalent Variations in Spike Epitopes and Binding of Different Antibodies to the same Epitope
- Author
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Stephen J. Stahl, Joe G. Vethanayagam, A.C. Steven, Audray K. Harris, Norman R. Watts, Paul T. Wingfield, James F. Conway, Naiqian Cheng, and David M. Belnap
- Subjects
Models, Molecular ,Hepatitis B virus ,biology ,Linear epitope ,medicine.drug_class ,Cryoelectron Microscopy ,Antibodies, Monoclonal ,Monoclonal antibody ,Major histocompatibility complex ,Hepatitis B Core Antigens ,Molecular biology ,Epitope ,Epitopes ,Capsid ,Epitope mapping ,Structural Biology ,medicine ,biology.protein ,Antigenic variation ,Binding Sites, Antibody ,Binding site ,Molecular Biology ,Epitope Mapping ,Conformational epitope - Abstract
To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.
- Published
- 2006
37. Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids
- Author
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Alasdair C. Steven, James F. Conway, Giovanni Cardone, Naiqian Cheng, Lili You, Roger W. Hendrix, and Robert L. Duda
- Subjects
0303 health sciences ,Chemistry ,Protein subunit ,Virus Assembly ,030302 biochemistry & molecular biology ,Capsomere ,Mutant ,Cryoelectron Microscopy ,Energy landscape ,Random hexamer ,Calorimetry ,Virology ,Microbiology ,QR1-502 ,Folding (chemistry) ,03 medical and health sciences ,Capsid ,Biophysics ,Virus maturation ,Bacteriophages ,030304 developmental biology ,Research Article - Abstract
As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local minima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross-linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in maturation. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable intermediates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conformations of its precursor and mature states., IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in capsid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of principle, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.
- Published
- 2014
38. Virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity
- Author
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Naiqian Cheng, James F. Conway, Benes L. Trus, Alasdair C. Steven, and J. Bernard Heymann
- Subjects
Models, Molecular ,Conformational change ,Proteases ,Protein Conformation ,medicine.medical_treatment ,Context (language use) ,Biology ,Virus Replication ,Models, Biological ,Article ,Structure-Activity Relationship ,Viral Proteins ,Protein structure ,Structural Biology ,Virus maturation ,medicine ,Animals ,Humans ,Computer Simulation ,Molecular Biology ,Protease ,Virus Assembly ,Molecular biology ,Protease inhibitor (biology) ,Kinetics ,Models, Chemical ,Capsid ,Virus Diseases ,Viruses ,Biophysics ,Capsid Proteins ,Virus Activation ,medicine.drug - Abstract
For many viruses, the final stage of assembly involves structural transitions that convert an innocuous precursor particle into an infectious agent. This process -- maturation -- is controlled by proteases that trigger large-scale conformational changes. In this context, protease inhibitor antiviral drugs act by blocking maturation. Recent work has succeeded in determining the folds of representative examples of the five major proteins -- major capsid protein, scaffolding protein, portal, protease and accessory protein -- that are typically involved in capsid assembly. These data provide a framework for detailed mechanistic investigations and elucidation of mutations that affect assembly in various ways. The nature of the conformational change has been elucidated: it entails rigid-body rotations and translations of the arrayed subunits that transfer the interactions between them to different molecular surfaces, accompanied by refolding and redeployment of local motifs. Moreover, it has been possible to visualize maturation at the submolecular level in movies based on time-resolved cryo-electron microscopy.
- Published
- 2005
39. Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization
- Author
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Philip D. Ross, Alasdair C. Steven, Roger W. Hendrix, Robert L. Duda, Brian Firek, James F. Conway, and Naiqian Cheng
- Subjects
Models, Molecular ,Conformational change ,Protein Conformation ,Cryo-electron microscopy ,Protein subunit ,macromolecular substances ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Protein structure ,Bacteriophages ,Thermal stability ,Denaturation (biochemistry) ,Molecular Biology ,Calorimetry, Differential Scanning ,General Immunology and Microbiology ,Viral Core Proteins ,Virus Assembly ,General Neuroscience ,Cryoelectron Microscopy ,Genetic Complementation Test ,Brownian ratchet ,technology, industry, and agriculture ,Virology ,Protein Subunits ,Capsid ,Mutation ,Biophysics ,Capsid Proteins - Abstract
In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.
- Published
- 2005
40. Visualization of the Binding of Hsc70 ATPase to Clathrin Baskets
- Author
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David M. Belnap, Lois E. Greene, Kenji Iwasaki, Alasdair C. Steven, Naiqian Cheng, J. Bernard Heymann, Evan Eisenberg, and Yang-In Yim
- Subjects
animal structures ,biology ,Vesicle ,Cell Biology ,Auxilin ,Biochemistry ,Clathrin ,Clathrin Heavy Chains ,Cell biology ,A-site ,embryonic structures ,biology.protein ,Ap180 ,Clathrin adaptor proteins ,Binding site ,Molecular Biology - Abstract
Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.
- Published
- 2005
41. Control of Crosslinking by Quaternary Structure Changes during Bacteriophage HK97 Maturation
- Author
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Lu Gan, Alasdair C. Steven, Roger W. Hendrix, Naiqian Cheng, Brian Firek, John E. Johnson, James F. Conway, and Robert L. Duda
- Subjects
Protein Conformation ,viruses ,Bacillus Phages ,macromolecular substances ,medicine.disease_cause ,Bacteriophage ,Capsid ,medicine ,Bacteriophage HK97 ,Protein Structure, Quaternary ,Escherichia coli ,Molecular Biology ,Binding Sites ,biology ,Lysine ,Cryoelectron Microscopy ,Capsomere ,technology, industry, and agriculture ,Cell Biology ,biology.organism_classification ,Protein Subunits ,Structural change ,Biochemistry ,Covalent bond ,Biophysics ,Capsid Proteins ,Protein quaternary structure ,Asparagine - Abstract
Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli. These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. Analogous transformations also occur in unrelated viruses and protein complexes. We find that expansion and crosslinking happen concurrently during maturation at low pH. Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.
- Published
- 2004
- Full Text
- View/download PDF
42. A Polymerase-Activating Host Factor, YajQ, Bound to the Bacteriophage ϕ6 Capsid
- Author
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Leonard Mindich, Naiqian Cheng, Daniel Nemecek, Jian Qiao, Rick Huang, Alasdair C. Steven, and J. Bernard Heymann
- Subjects
0301 basic medicine ,Bacteriophage ,03 medical and health sciences ,030104 developmental biology ,biology ,Capsid ,Chemistry ,biology.protein ,biology.organism_classification ,Instrumentation ,Virology ,Polymerase ,Host factor - Published
- 2016
43. Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase
- Author
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Judith G. Levin, Tsutomu Murakami, Naiqian Cheng, Eric O. Freed, Shixing Tang, and Alasdair C. Steven
- Subjects
Models, Molecular ,Immunology ,Mutant ,Biology ,medicine.disease_cause ,Membrane Fusion ,Microbiology ,Virus ,Viral entry ,Virology ,medicine ,Infectivity ,Mutation ,Virulence ,Structure and Assembly ,Viral Core Proteins ,Lipid bilayer fusion ,Molecular biology ,HIV Reverse Transcriptase ,Reverse transcriptase ,Microscopy, Electron ,Capsid ,Insect Science ,DNA, Viral ,HIV-1 ,Capsid Proteins - Abstract
We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (
- Published
- 2003
44. Architecture of Ure2p Prion Filaments
- Author
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Kimberly L. Taylor, Reed B. Wickner, Ulrich Baxa, Martha N. Simon, Naiqian Cheng, Alasdair C. Steven, and Joseph S. Wall
- Subjects
chemistry.chemical_classification ,Conformational change ,Amyloid ,Globular protein ,macromolecular substances ,Cell Biology ,Biology ,Biochemistry ,Fusion protein ,Protein filament ,chemistry.chemical_compound ,Crystallography ,Protein structure ,Monomer ,chemistry ,Scanning transmission electron microscopy ,Molecular Biology - Abstract
The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.
- Published
- 2003
45. The Cryptophycin−Tubulin Ring Structure Indicates Two Points of Curvature in the Tubulin Dimer
- Author
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Naiqian Cheng, Wendy West, Dan L. Sackett, A.C. Steven, and Norman R. Watts
- Subjects
Light ,Protein Conformation ,Stereochemistry ,Protein subunit ,Dimer ,Peptides, Cyclic ,Biochemistry ,chemistry.chemical_compound ,Biopolymers ,Tubulin ,Depsipeptides ,medicine ,Animals ,Scattering, Radiation ,Trypsin ,chemistry.chemical_classification ,biology ,Hydrolysis ,Computational Biology ,Image Enhancement ,Cyclic peptide ,Rats ,Spindle apparatus ,Protein Subunits ,chemistry ,Cryptophycin ,Microscopy, Electron, Scanning ,biology.protein ,Dimerization ,Ultracentrifugation ,Cryptophycins ,medicine.drug - Abstract
Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.
- Published
- 2002
46. Structural Characterization of the Dilute Aqueous Surfactant Solution of Cetylpyridinium Bromide/Hexanol/Sodium Bromide
- Author
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Laura G. Barrientos, Naiqian Cheng, A.C. Steven, Klaus Gawrisch, and Angela M. Gronenborn
- Subjects
Aqueous solution ,Liquid crystalline ,Surfactant system ,Surfaces and Interfaces ,Condensed Matter Physics ,Sodium bromide ,chemistry.chemical_compound ,chemistry ,Pulmonary surfactant ,CETYLPYRIDINIUM BROMIDE ,Electrochemistry ,Organic chemistry ,General Materials Science ,Spectroscopy ,Nuclear chemistry ,Hexanol - Abstract
The liquid crystalline alignment medium based on a quasi-ternary surfactant system comprising cetylpyridinium bromide (CPBr)/hexanol/sodium bromide [6.5% CPBr/hexanol (1/1.33, w/w), 30 mM NaBr in 1...
- Published
- 2002
47. The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface
- Author
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James F. Conway, Naiqian Cheng, Stephen J. Stahl, David M. Belnap, Paul T. Wingfield, Norman R. Watts, and Alasdair C. Steven
- Subjects
Models, Molecular ,Hepatitis B virus ,Protein Folding ,Cellobiose dehydrogenase ,Protein Conformation ,Recombinant Fusion Proteins ,viruses ,Sequence (biology) ,Peptide ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,Capsid ,Protein structure ,Escherichia coli ,Image Processing, Computer-Assisted ,Morphogenesis ,Hepatitis B e Antigens ,Molecular Biology ,chemistry.chemical_classification ,General Immunology and Microbiology ,Circular Dichroism ,General Neuroscience ,Cryoelectron Microscopy ,Hepatitis B Core Antigens ,Molecular biology ,Peptide Fragments ,Protein Structure, Tertiary ,Amino Acid Substitution ,chemistry ,Subtraction Technique ,Chromatography, Gel ,Nucleic acid ,Biophysics ,Carbohydrate Dehydrogenases ,Protein folding ,Crystallization ,Linker - Abstract
Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell-forming ‘core’ domain and the nucleic acid-binding ‘protamine’ domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain-containing capsids, although its size-determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo-EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence-similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.
- Published
- 2002
48. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress
- Author
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Naiqian Cheng, Juan Fontana, Egbert Hoiczyk, Daniel Nemecek, Colleen A. McHugh, Joseph S. Wall, Anastasia A. Aksyuk, Alasdair C. Steven, Dennis C. Winkler, J. Bernard Heymann, and Alan S Lam
- Subjects
Models, Molecular ,Myxococcus xanthus ,Macromolecular Substances ,Iron ,Bacterial Physiological Phenomena ,General Biochemistry, Genetics and Molecular Biology ,Virus-like particle ,Bacterial Proteins ,Bacterial microcompartment ,Organelle ,Molecular Biology ,General Immunology and Microbiology ,biology ,General Neuroscience ,Cryoelectron Microscopy ,Articles ,biology.organism_classification ,Cell biology ,Ferritin ,Oxidative Stress ,Capsid ,biology.protein ,Nanoparticles ,Protein Multimerization ,Bacteria ,Archaea - Abstract
Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
- Published
- 2014
49. A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition
- Author
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Naiqian Cheng, Eric O. Freed, Kayoko Waki, J. Bernard Heymann, Rick Huang, Alasdair C. Steven, Rebecca C. Craven, Paul W. Keller, and Matthew R. England
- Subjects
Models, Molecular ,Icosahedral symmetry ,medicine.medical_treatment ,viruses ,Immunology ,Gene Products, gag ,Peptide ,Biology ,Cleavage (embryo) ,Microbiology ,gag Gene Products, Human Immunodeficiency Virus ,Retrovirus ,Capsid ,Virology ,medicine ,Humans ,chemistry.chemical_classification ,Rous sarcoma virus ,Protease ,Maturation inhibitor ,Structure and Assembly ,biology.organism_classification ,Molecular biology ,chemistry ,Insect Science ,Mutation ,Biophysics ,HIV-1 ,Capsid Proteins - Abstract
Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.
- Published
- 2013
50. [Untitled]
- Author
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Andreas Plückthun, Alexander Wlodawer, James F. Conway, Alasdair C. Steven, Fan Yang, Zbigniew Dauter, Mario E. Cerritelli, Patrik Forrer, and Naiqian Cheng
- Subjects
Viral protein ,Chemistry ,Trimer ,medicine.disease_cause ,Biochemistry ,Crystallography ,Protein structure ,Capsid ,Structural Biology ,Icosahedral viral capsid ,Genetics ,medicine ,Protein folding ,Peptide sequence ,Protein secondary structure - Abstract
The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.
- Published
- 2000
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