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Sample records for gadolinium hydroxypyridonate-viral capsid

  1. High Relaxivity Gadolinium Hydroxypyridonate-Viral Capsid Conjugates: Nano-sized MRI Contrast Agents

    SciTech Connect

    Meux, Susan C.; Datta, Ankona; Hooker, Jacob M.; Botta, Mauro; Francis, Matthew B.; Aime, Silvio; Raymond, Kenneth N.

    2007-08-29

    High relaxivity macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids are assessed. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a five-fold increase in relaxivity, leading to a peak relaxivity (per Gd{sup 3+} ion) of 41.6 mM{sup -1}s{sup -1} at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation to flexible lysines, while internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that (1) there is facile diffusion of water to the interior of capsids and (2) the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules (q = 2) and the NMRD fittings highlight the differences in the local motion for the internal ({tau}{sub RI} = 440 ps) and external ({tau}{sub RI} = 310 ps) conjugates. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

  2. Chirality of Viral Capsids

    NASA Astrophysics Data System (ADS)

    Dharmavaram, Sanjay; Xie, Fangming; Bruinsma, Robijn; Klug, William; Rudnick, Joseph

    Most icosahedral viruses are classified by their T-number which identifies their capsid in terms of the number of capsomers and their relative arrangement. Certain T-numbers (T = 7 for instance) are inherently chiral (with no reflection planes) while others (e.g. T = 1) are achiral. We present a Landau-Brazovskii (LB) theory for weak crystallization in which a scalar order parameter that measures density of capsid proteins successfully predicts the various observed T-numbers and their respective chiralities. We find that chiral capsids gain stability by spontaneously breaking symmetry from an unstable chiral state. The inherently achiral LB-free energy does not preferentially select a particular chiral state from its mirror reflection. Based on the physical observation that proteins are inherently chiral molecules with directional interactions, we propose a new chiral term to the LB energy as a possible selection mechanism for chirality.

  3. Continuum Theory of Retroviral Capsids

    NASA Astrophysics Data System (ADS)

    Nguyen, T. T.; Bruinsma, R. F.; Gelbart, W. M.

    2006-02-01

    We present a self-assembly phase diagram for the shape of retroviral capsids, based on continuum elasticity theory. The spontaneous curvature of the capsid proteins drives a weakly first-order transition from spherical to spherocylindrical shapes. The conical capsid shape which characterizes the HIV-1 retrovirus is never stable under unconstrained energy minimization. Only under conditions of fixed volume and/or fixed spanning length can the conical shape be a minimum energy structure. Our results indicate that, unlike the capsids of small viruses, retrovirus capsids are not uniquely determined by the molecular structure of the constituent proteins but depend in an essential way on physical constraints present during assembly.

  4. Gadolinium photoionization process

    DOEpatents

    Paisner, J.A.; Comaskey, B.J.; Haynam, C.A.; Eggert, J.H.

    1993-04-13

    A method is provided for selective photoionization of the odd-numbered atomic mass gadolinium isotopes 155 and 157. The selective photoionization is accomplished by circular or linear parallel polarized laser beam energy effecting a three-step photoionization pathway.

  5. Gadolinium photoionization process

    DOEpatents

    Paisner, Jeffrey A.; Comaskey, Brian J.; Haynam, Christopher A.; Eggert, Jon H.

    1993-01-01

    A method is provided for selective photoionization of the odd-numbered atomic mass gadolinium isotopes 155 and 157. The selective photoionization is accomplished by circular or linear parallel polarized laser beam energy effecting a three-step photoionization pathway.

  6. Modeling Viral Capsid Assembly

    PubMed Central

    2014-01-01

    I present a review of the theoretical and computational methodologies that have been used to model the assembly of viral capsids. I discuss the capabilities and limitations of approaches ranging from equilibrium continuum theories to molecular dynamics simulations, and I give an overview of some of the important conclusions about virus assembly that have resulted from these modeling efforts. Topics include the assembly of empty viral shells, assembly around single-stranded nucleic acids to form viral particles, and assembly around synthetic polymers or charged nanoparticles for nanotechnology or biomedical applications. I present some examples in which modeling efforts have promoted experimental breakthroughs, as well as directions in which the connection between modeling and experiment can be strengthened. PMID:25663722

  7. Gadolinium-Induced Fibrosis.

    PubMed

    Todd, Derrick J; Kay, Jonathan

    2016-01-01

    Gadolinium-based contrast agents (GBCAs), once believed to be safe for patients with renal disease, have been strongly associated with nephrogenic systemic fibrosis (NSF), a severe systemic fibrosing disorder that predominantly afflicts individuals with advanced renal dysfunction. We provide a historical perspective on the appearance and disappearance of NSF, including its initial recognition as a discrete clinical entity, its association with GBCA exposure, and the data supporting a causative relationship between GBCA exposure and NSF. On the basis of this body of evidence, we propose that the name gadolinium-induced fibrosis (GIF) more accurately reflects the totality of knowledge regarding this disease. Use of high-risk GBCAs, such as formulated gadodiamide, should be avoided in patients with renal disease. Restriction of GBCA use in this population has almost completely eradicated new cases of this debilitating condition. Emerging antifibrotic therapies may be useful for patients who suffer from GIF. PMID:26768242

  8. HIV Capsid Assembly, Mechanism, and Structure.

    PubMed

    Chen, Bo

    2016-05-10

    The HIV genome materials are encaged by a proteinaceous shell called the capsid, constructed from ∼1000-1500 copies of the capsid proteins. Because its stability and integrity are critical to the normal life cycle and infectivity of the virus, the HIV capsid is a promising antiviral drug target. In this paper, we review the studies shaping our understanding of the structure and dynamics of the capsid proteins and various forms of their assemblies, as well as the assembly mechanism. PMID:27074418

  9. Nonicosahedral pathways for capsid expansion

    NASA Astrophysics Data System (ADS)

    Cermelli, Paolo; Indelicato, Giuliana; Twarock, Reidun

    2013-09-01

    For a significant number of viruses a structural transition of the protein container that encapsulates the viral genome forms an important part of the life cycle and is a prerequisite for the particle becoming infectious. Despite many recent efforts the mechanism of this process is still not fully understood, and a complete characterization of the expansion pathways is still lacking. We present here a coarse-grained model that captures the essential features of the expansion process and allows us to investigate the conditions under which a viral capsid becomes unstable. Based on this model we demonstrate that the structural transitions in icosahedral viral capsids are likely to occur through a low-symmetry cascade of local expansion events spreading in a wavelike manner over the capsid surface.

  10. A primer on gadolinium chemistry

    PubMed Central

    Sherry, A. Dean; Caravan, Peter; Lenkinski, Robert E.

    2010-01-01

    Gadolinium is widely known by all practitioners of MRI but few appreciate the basic solution chemistry of this trivalent lanthanide ion. Given the recent linkage between gadolinium contrast agents and nephrogenic systemic fibrosis, some basic chemistry of this ion must be more widely understood. This short primer on gadolinium chemistry is intended to provide the reader the background principles necessary to understand the basics of chelation chemistry, water hydration numbers, and the differences between thermodynamic stability and kinetic stability or inertness. We illustrate the fundamental importance of kinetic dissociation rates in determining gadolinium toxicity in vivo by presenting new data for a novel europium DOTA-tetraamide complex that is relatively unstable thermodynamically yet extraordinarily inert kinetically and also quite non-toxic. This, plus other literature evidence forms the basis of the fundamental axiom that it is the kinetic stability of a gadolinium complex, not its thermodynamic stability, that determines its in vivo toxicity. PMID:19938036

  11. Biophysical chemistry: Unravelling capsid transformations

    NASA Astrophysics Data System (ADS)

    Uchida, Masaki; Douglas, Trevor

    2013-06-01

    The interactions between a virus capsid and its cargo are essential for viral infection as well as in the design of synthetic virus-like particles. Now a combination of analytical techniques has unravelled key steps in the transformation of a model virus and the release of its RNA cargo.

  12. The A, B, Cs of Herpesvirus Capsids

    PubMed Central

    Tandon, Ritesh; Mocarski, Edward S.; Conway, James F.

    2015-01-01

    Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle. PMID:25730559

  13. Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

    PubMed Central

    Wong, S; Momoeda, M; Field, A; Kajigaya, S; Young, N S

    1994-01-01

    We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened. Images PMID:8207846

  14. Irreversible growth model for virus capsid assembly

    NASA Astrophysics Data System (ADS)

    Hicks, Stephen D.; Henley, C. L.

    2006-09-01

    We model the spontaneous assembly of a capsid (a virus’ closed outer shell) from many copies of identical units, using entirely irreversible steps and only information local to the growing edge. Our model is formulated in terms of (i) an elastic Hamiltonian with stretching and bending stiffness and a spontaneous curvature, and (ii) a set of rate constants for the addition of new units or bonds. An ensemble of highly irregular capsids is generated, unlike the well-known icosahedrally symmetric viruses, but (we argue) plausible as a way to model the irregular capsids of retroviruses such as HIV. We found that (i) the probability of successful capsid completion decays exponentially with capsid size; (ii) capsid size depends strongly on spontaneous curvature and weakly on the ratio of the bending and stretching elastic stiffnesses of the shell; (iii) the degree of localization of Gaussian curvature (a measure of facetedness) depends heavily on the ratio of elastic stiffnesses.

  15. Molecular Architecture of the Retroviral Capsid.

    PubMed

    Perilla, Juan R; Gronenborn, Angela M

    2016-05-01

    Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores. PMID:27039020

  16. Laserspektroskopie Gadoliniums Fingerabdruck

    SciTech Connect

    Blaum, Klaus; Bushaw, Bruce A.; Nortershauser, Wilfried

    2003-05-31

    The minimum energy that is required to remove the first electron from e neutral atom, one call ionization energy E1. It is not only for every element of the periodic table but also even for every nuclide characteristic and therefore belongs to the most basic atomic properties. The ionization energy is important for the determination of a ‘row’ of properties, from the chemical reactivity to the color light absorbed and emitted from an atom. The value of E1 depends on the stationary state that the farthest electron originally occupies. If the electron finds itself, however, in the first excited stat, only 3.4 eV is required. Previously the ionization energy could be determined with high accuracy in the range of 10-8 by means of laser spectroscopy only for atoms with one or two valence electrons, whereby the most accurate to date result from measurements on Hydrogen. We could now, for the first time in a rare earth element, reach that kind of accuracy. Motivation for these investigations was the development of an analysis method for the ultra-trace detection of Gadolinium in tissue- and meteorite samples.

  17. Controlling Viral Capsid Assembly with Templating

    PubMed Central

    Hagan, Michael F.

    2009-01-01

    We develop coarse-grained models that describe the dynamic encapsidation of functionalized nanoparticles by viral capsid proteins. We find that some forms of cooperative interactions between protein subunits and nanoparticles can dramatically enhance rates and robustness of assembly, as compared to the spontaneous assembly of subunits into empty capsids. For large core-subunit interactions, subunits adsorb onto core surfaces en masse in a disordered manner, and then undergo a cooperative rearrangement into an ordered capsid structure. These assembly pathways are unlike any identified for empty capsid formation. Our models can be directly applied to recent experiments in which viral capsid proteins assemble around the functionalized inorganic nanoparticles [Sun et al., Proc. Natl. Acad. Sci (2007) 104, 1354]. In addition, we discuss broader implications for understanding the dynamic encapsidation of single-stranded genomic molecules during viral replication and for developing multicomponent nanostructured materials. PMID:18643099

  18. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  19. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-01-01

    Lambda-like dsDNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm. of pressure during genome packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and Hydrogen/Deuterium exchange data presented here demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

  20. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  1. Thermodynamic properties of gadolinium disilicide

    SciTech Connect

    Lukashenko, G.M.; Polotskaya, R.I.

    1986-11-01

    The authors determine the Gibbs energy, enthalpy, formation heat, and other thermodynamic properties of gadolinium disilicide by measuring the electromotive force in the 830-960 K temperature range in electrolytes consisting of molten tin and various chlorides. The relationship of these properties to crystal structure is briefly discussed.

  2. Theoretical studies of viral capsid proteins.

    PubMed

    Phelps, D K; Speelman, B; Post, C B

    2000-04-01

    Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus. PMID:10753813

  3. Tumor growth suppression by gadolinium-neutron capture therapy using gadolinium-entrapped liposome as gadolinium delivery agent.

    PubMed

    Dewi, Novriana; Yanagie, Hironobu; Zhu, Haito; Demachi, Kazuyuki; Shinohara, Atsuko; Yokoyama, Kazuhito; Sekino, Masaki; Sakurai, Yuriko; Morishita, Yasuyuki; Iyomoto, Naoko; Nagasaki, Takeshi; Horiguchi, Yukichi; Nagasaki, Yukio; Nakajima, Jun; Ono, Minoru; Kakimi, Kazuhiro; Takahashi, Hiroyuki

    2013-07-01

    Neutron capture therapy (NCT) is a promising non-invasive cancer therapy approach and some recent NCT research has focused on using compounds containing gadolinium as an alternative to currently used boron-10 considering several advantages that gadolinium offers compared to those of boron. In this study, we evaluated gadolinium-entrapped liposome compound as neutron capture therapy agent by in vivo experiment on colon-26 tumor-bearing mice. Gadolinium compound were injected intravenously via tail vein and allowed to accumulate into tumor site. Tumor samples were taken for quantitative analysis by ICP-MS at 2, 12, and 24 h after gadolinium compound injection. Highest gadolinium concentration was observed at about 2 h after gadolinium compound injection with an average of 40.3 μg/g of wet tumor tissue. We performed neutron irradiation at JRR-4 reactor facility of Japan Atomic Energy Research Institute in Tokaimura with average neutron fluence of 2×10¹² n/cm². The experimental results showed that the tumor growth suppression of gadolinium-injected irradiated group was revealed until about four times higher compared to the control group, and no significant weight loss were observed after treatment suggesting low systemic toxicity of this compound. The gadolinium-entrapped liposome will become one of the candidates for Gd delivery system on NCT. PMID:23743325

  4. Prediction of stability changes upon mutation in an icosahedral capsid.

    PubMed

    Hickman, Samuel J; Ross, James F; Paci, Emanuele

    2015-09-01

    Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. PMID:26178267

  5. Stabilising the Herpes Simplex Virus capsid by DNA packaging

    NASA Astrophysics Data System (ADS)

    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  6. A Kinase Chaperones Hepatitis B Virus Capsid Assembly and Captures Capsid Dynamics in vitro

    PubMed Central

    Chen, Chao; Wang, Joseph Che-Yen; Zlotnick, Adam

    2011-01-01

    The C-terminal domain (CTD) of Hepatitis B virus (HBV) core protein is involved in regulating multiple stages of the HBV lifecycle. CTD phosphorylation correlates with pregenomic-RNA encapsidation during capsid assembly, reverse transcription, and viral transport, although the mechanisms remain unknown. In vitro, purified HBV core protein (Cp183) binds any RNA and assembles aggressively, independent of phosphorylation, to form empty and RNA-filled capsids. We hypothesize that there must be a chaperone that binds the CTD to prevent self-assembly and nonspecific RNA packaging. Here, we show that HBV capsid assembly is stalled by the Serine Arginine protein kinase (SRPK) binding to the CTD, and reactivated by subsequent phosphorylation. Using the SRPK to probe capsids, solution and structural studies showed that SRPK bound to capsid, though the CTD is sequestered on the capsid interior. This result indicates transient CTD externalization and suggests that capsid dynamics could be crucial for directing HBV intracellular trafficking. Our studies illustrate the stochastic nature of virus capsids and demonstrate the appropriation of a host protein by a virus for a non-canonical function. PMID:22114561

  7. Modeling virus capsids and their protein binding -- the search for weak regions within the HIV capsid

    NASA Astrophysics Data System (ADS)

    Sankey, Otto; Benson, Daryn

    2010-10-01

    Viruses remain a threat to the health of humans worldwide with 33 million infected with AIDS. Viruses are ubiquitous infecting animals, plants, and bacteria. Each virus infects in its own unique manner making the problem seem intractable. However, some general physical steps apply to many viruses and the application of basic physical modeling can potentially have great impact. The aim of this theoretical study is to investigate the stability of the HIV viral capsid (protein shell). The structural shell can be compromised by physical probes such as pulsed laser light. But what are the weakest regions of the capsid so that we can begin to understand vulnerabilities of these deadly materials? The atomic structure of HIV capsids is not precisely known and we begin by describing our work to model the capsid structure. Next we describe a course grained model to investigate protein interactions within the capsid.

  8. GADOLINIUM SOLUBILITY AND VOLATILITY DURING DWPF PROCESSING

    SciTech Connect

    Reboul, S

    2008-01-30

    Understanding of gadolinium behavior, as it relates to potential neutron poisoning applications at the DWPF, has increased over the past several years as process specific data have been generated. Of primary importance are phenomena related to gadolinium solubility and volatility, which introduce the potential for gadolinium to be separated from fissile materials during Chemical Process Cell (CPC) and Melter operations. Existing data indicate that gadolinium solubilities under moderately low pH conditions can vary over several orders of magnitude, depending on the quantities of other constituents that are present. With respect to sludge batching processes, the gadolinium solubility appears to be highly affected by iron. In cases where the mass ratio of Fe:Gd is 300 or more, the gadolinium solubility has been observed to be low, one milligram per liter or less. In contrast, when the ratio of Fe:Gd is 20 or less, the gadolinium solubility has been found to be relatively high, several thousands of milligrams per liter. For gadolinium to serve as an effective neutron poison in CPC operations, the solubility needs to be limited to approximately 100 mg/L. Unfortunately, the Fe:Gd ratio that corresponds to this solubility limit has not been identified. Existing data suggest gadolinium and plutonium are not volatile during melter operations. However, the data are subject to inherent uncertainties preventing definitive conclusions on this matter. In order to determine if gadolinium offers a practical means of poisoning waste in DWPF operations, generation of additional data is recommended. This includes: Gd solubility testing under conditions where the Fe:Gd ratio varies from 50 to 150; and Gd and Pu volatility studies tailored to quantifying high temperature partitioning. Additional tests focusing on crystal aging of Gd/Pu precipitates should be pursued if receipt of gadolinium-poisoned waste into the Tank Farm becomes routine.

  9. Membrane-mediated interaction between retroviral capsids

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan

    2012-02-01

    A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

  10. Stochastic modeling of virus capsid assembly pathways

    NASA Astrophysics Data System (ADS)

    Schwartz, Russell

    2009-03-01

    Virus capsids have become a key model system for understanding self-assembly due to their high complexity, robust and efficient assembly processes, and experimental tractability. Our ability to directly examine and manipulate capsid assembly kinetics in detail nonetheless remains limited, creating a need for computer models that can infer experimentally inaccessible features of the assembly process and explore the effects of hypothetical manipulations on assembly trajectories. We have developed novel algorithms for stochastic simulation of capsid assembly [1,2] that allow us to model capsid assembly over broad parameter spaces [3]. We apply these methods to study the nature of assembly pathway control in virus capsids as well as their sensitivity to assembly conditions and possible experimental interventions. [4pt] [1] F. Jamalyaria, R. Rohlfs, and R. Schwartz. J Comp Phys 204, 100 (2005). [0pt] [2] N. Misra and R. Schwartz. J Chem Phys 129, in press (2008). [0pt] [3] B. Sweeney, T. Zhang, and R. Schwartz. Biophys J 94, 772 (2008).

  11. Dynamic Models for Templated Viral Capsid Assembly

    NASA Astrophysics Data System (ADS)

    Hagan, Michael

    2008-03-01

    The replication of many viruses with single-stranded genomes requires the simultaneous assembly of an ordered protein shell, or capsid, and encapsidation of the genome. In this talk, I will present coarse-grained computational and theoretical models that describe the assembly of viral capsid proteins around interior cores, such as polymers and rigid spheres. These models are motivated by two recently developed experimental model systems in which viral proteins dynamically encapsidate inorganic nanoparticles and polyelectrolytes. Model predictions suggest that some forms of cooperative interactions between subunits and cores can dramatically enhance rates and robustness of assembly, as compared to the spontaneous assembly of subunits into empty capsids. For large core-subunit interactions, subunits adsorb onto a core en masse in a disordered manner, and then undergo a cooperative rearrangement into an ordered capsid structure. These assembly pathways are unlike any seen for empty capsids formation. While model predictions suggest that cooperative interactions between disparate assembling components can overcome some limitations of spontaneous assembly, the complexity of multicomponent assembly introduces new forms of kinetic traps that can frustrate assembly, and hence introduces new limitations. These findings have implications for a mechanism in which viruses use interactions between proteins and genomic molecules to promote and control assembly, and thereby control the replication process.

  12. Metals Fact Sheet: Gadolinium GD

    SciTech Connect

    1992-10-01

    Gadolinium is a silvery-white, malleable, ductile metallic element used to improve the high-temperature characteristics of iron, chromium, and related metallic alloys. It was named after the French chemist, Gadolin, discoverer of yttrium. This article discusses sources of the element, the world supply and demand, and also a number of applications. With the largest thermal neutron absorption cross section of any element, one of these applications is as a burnable poison in reactors and as neutron absorbers in other nuclear devices.

  13. The semipermeability of simple spherical virus capsids.

    PubMed

    Durham, A C; Witz, J; Bancroft, J B

    1984-02-01

    Hydrogen-ion titration curves are reported for tomato bushy stunt virus, two strains of cowpea chlorotic mottle virus, and turnip crinkle virus, with particular attention to the hysteresis loops associated with the swelling and contraction of virions. There appears to be an archetypal shape of hysteresis loops, which is shared by viruses in several groups, suggestive of many intermediate states in the swelling of any one particle. In contrast, eggplant mosaic virus behaves as if its protein capsid is impermeable to small ions in mild conditions; its cation-binding sites were revealed by treatment with high concentrations of salt or urea, or at raised temperatures. Putting these observations together with the fact that a spherical virus capsid is a closed, holey, charged surface leads to a theory of titration hysteresis: its key feature is that the protein capsids of simple viruses are inherently semipermeable, with many of the ion-handling properties usually attributed only to complex lipid membranes. PMID:18639804

  14. Dynamic pathways for viral capsid assembly

    SciTech Connect

    Hagan, Michael F.; Chandler, David

    2006-02-09

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

  15. Mechanical oscillations of a viral capsid

    NASA Astrophysics Data System (ADS)

    Benson, Daryn; Sankey, Otto; Dykeman, Eric

    2010-03-01

    Viruses are sub-microscopic infectious agents that infect almost every living creature on Earth. They are unable to grow or reproduce outside of a host cell and are therefore parasitic in nature. A virus' internal genetic material is protected by an external protein coat (capsid). We developed a theoretical model which uses the interaction of light with a viral capsid to create large amplitude motions within the capsid. This work displays the results of the model on the tobacco mosaic virus (TMV) with attached RNA genome. The development of this model was motivated by the experimental work of Tsen et. al. [1] who used ultra-short laser pulses to inactivate viruses. [1] K-T. Tsen et al., J. of Physics -- Cond. Mat. 19, 472201 (2007).

  16. Toxicity of Gadolinium to Some Aquatic Microbes

    SciTech Connect

    Wilde, E.W.

    2001-01-24

    The toxicity of gadolinium to algae and bacteria was determined as part of an effort to develop a biological process to purify drums containing spent nuclear reactor heavy water moderator (D2O). This water was contaminated with high concentrations of gadolinium nitrate, a chemical used as a neutron poison during former nuclear reactor operations at the Savannah River Site (SRS) near Aiken, SC. Nuclear reactors were operated for approximately 30 years at the SRS to produce nuclear weapons materials for national defense. Throughout this period, a heavy water solution of gadolinium nitrate was utilized in a standby emergency shutdown system that could inject this chemical into the reactor moderator coolant water. The chemical was used for this purpose because the high neutron absorption cross sections of some gadolinium isotopes make gadolinium salts such as GdNO3 effective in controlling nuclear activity in aqueous systems (Gilbert et al. 1985; Rodenas et al. 1990). The use of this practice resulted in a large inventory of this degraded heavy water containing gadolinium nitrate. Microbiological and chemical studies were initiated to evaluate the potential use of bacteria and algae for water purification of the drums. Since metals are often toxic to microbes when present at concentrations substantially higher than natural environmental levels, it was hypothesized that Gd may be toxic to selected microorganisms (algae and bacteria) at the very high concentrations (average 80,000 mg/L, maximum 259,000 mg/L) present in most of the drums. Two principal components of the study included: (1) chemical and microbiological characterization of representative drums, and (2) an evaluation of the toxicity of gadolinium to selected species of algae. In addition to wastewater from nuclear production reactor operations, gadolinium waste is also generated from medical applications, especially MRI, and various electronic components including CD disks. Despite growing and widespread

  17. Tilable nature of virus capsids and the role of topological constraints in natural capsid design

    NASA Astrophysics Data System (ADS)

    Mannige, Ranjan V.; Brooks, Charles L., III

    2008-05-01

    Virus capsids are highly specific assemblies that are formed from a large number of often chemically identical capsid subunits. In the present paper we ask to what extent these structures can be viewed as mathematically tilable objects using a single two-dimensional tile. We find that spherical viruses from a large number of families—eight out of the twelve studied—qualitatively possess properties that allow their representation as two-dimensional monohedral tilings of a bound surface, where each tile represents a subunit. This we did by characterizing the extent to which individual spherical capsids display subunit-subunit (1) holes, (2) overlaps, and (3) gross structural variability. All capsids with T numbers greater than 1 from the Protein Data Bank, with homogeneous protein composition, were used in the study. These monohedral tilings, called canonical capsids due to their platonic (mathematical) form, offer a mathematical segue into the structural and dynamical understanding of not one, but a large number of virus capsids. From our data, it appears as though one may only break the long-standing rules of quasiequivalence by the introduction of subunit-subunit structural variability, holes, and gross overlaps into the shell. To explore the utility of canonical capsids in understanding structural aspects of such assemblies, we used graph theory and discrete geometry to enumerate the types of shapes that the tiles (and hence the subunits) must possess. We show that topology restricts the shape of the face to a limited number of five-sided prototiles, one of which is the “bisected trapezoid” that is a platonic representation of the most ubiquitous capsid subunit shape seen in nature (the trapezoidal jelly-roll motif). This motif is found in a majority of seemingly unrelated virus families that share little to no host, size, or amino acid sequence similarity. This suggests that topological constraints may exhibit dominant roles in the natural design of

  18. Modeling virus capsids and their protein binding -- the search for weak regions within the HIV capsid

    NASA Astrophysics Data System (ADS)

    Sankey, Otto F.; Benson, Daryn E.; Gilbert, C. Michael

    2011-03-01

    Viruses remain a threat to the health of humans worldwide with 33 million infected with HIV. Viruses are ubiquitous, infecting animals, plants, and bacteria. Each virus infects in its own unique manner making the problem seem intractable. However, some general physical steps apply to many viruses and the application of basic physical modeling can potentially have great impact. The aim of this theoretical study is to investigate the stability of the HIV viral capsid (protein shell). The structural shell can be compromised by physical probes such as pulsed laser light [1,2]. But, what are the weakest regions of the capsid so that we can begin to understand vulnerabilities of these deadly materials? The atomic structure of HIV capsids is not precisely known and we begin by describing our work to model the capsid structure. We have constructed three representative viral capsids of different CA protein number -- HIV-900, HIV-1260 and HIV-1740. The complexity of the assembly requires a course grained model to investigate protein interactions within the capsid which we will describe.

  19. Statistical mechanical models of virus capsid assembly

    NASA Astrophysics Data System (ADS)

    Hicks, Stephen Daniel

    Viruses have become an increasingly popular subject of physics investigation, particularly in the last decade. Advances in imaging of virus capsids---the protective protein shells---in a wide variety of stages of assembly have encouraged physical assembly models at a similarly wide variety of scales, while the apparent simplicity of the capsid system---typically, many identical units assembling spontaneously into an icosahedrally symmetric (rather than amorphous) shell---makes the problem particularly interesting. We take a look at the existing physical assembly models in light of the question of how a particular assembly target can be consistently achieved in the presence of so many possible incorrect results. This review leads us to pose our own model of fully irreversible virus assembly, which we study in depth using a large ensemble of simulated assembled capsids, generated under a variety of capsid shell elastic parameters. While this irreversible model (predictably) did not yield consistently symmetric results, we do glean some insight into the effect of elasticity on growth, as well as an understanding of common failure modes. In particular, we found that (i) capsid size depends strongly on the spontaneous curvature and weakly on the ratio of bending to stretching elastic stiffnesses, (ii) the probability of successful capsid completion decays exponentially with capsid size, and (iii) the degree of localization of Gaussian curvature depends heavily on the ratio of elastic stiffnesses. We then go on to consider more thoroughly the nature of the ensemble of symmetric and almost-symmetric capsids---ultimately computing a phase diagram of minimum-energy capsids as a function of the two above-mentioned elastic parameters---and also look at a number of modifications we can make to our irreversible model, finally putting forth a rather different type of model potentially appropriate for understanding immature HIV assembly, and concluding with a fit of this new

  20. Biodistribution of gadolinium-based contrast agents, including gadolinium deposition

    PubMed Central

    Aime, Silvio; Caravan, Peter

    2010-01-01

    The biodistribution of approved gadolinium (Gd) based contrast agents (GBCA) is reviewed. After intravenous injection GBCA distribute in the blood and the extracellular space and transiently through the excretory organs. Preclinical animal studies and the available clinical literature indicate that all these compounds are excreted intact. Elimination tends to be rapid and for the most part, complete. In renally insufficient patients the plasma elimination half-life increases substantially from hours to days depending on renal function. In patients with impaired renal function and nephrogenic systemic fibrosis (NSF), the agents gadodiamide, gadoversetamide, and gadopentetate dimeglumine have been shown to result in Gd deposition in the skin and internal organs. In these cases, it is likely that the Gd is no longer present as the GBCA, but this has still not been definitively shown. In preclinical models very small amounts of Gd are retained in the bone and liver, and the amount retained correlates with the kinetic and thermodynamic stability of the GBCA with respect to Gd release in vitro. The pattern of residual Gd deposition in NSF subjects may be different than that observed in preclinical rodent models. GBCA are designed to be used via intravenous administration. Altering the route of administration and/or the formulation of the GBCA can dramatically alter the biodistribution of the GBCA and can increase the likelihood of Gd deposition. PMID:19938038

  1. Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

    PubMed Central

    Kawase, M; Momoeda, M; Young, N S; Kajigaya, S

    1995-01-01

    In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus. PMID:7666560

  2. Dynamic Pathways for Viral Capsid Assembly

    PubMed Central

    Hagan, Michael F.; Chandler, David

    2006-01-01

    We develop a class of models with which we simulate the assembly of particles into T1 capsidlike objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile; for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes. PMID:16565055

  3. Mechanical properties of icosahedral virus capsids

    NASA Astrophysics Data System (ADS)

    Vliegenthart, G. A.; Gompper, G.

    2007-12-01

    Virus capsids are self-assembled protein shells in the size range of 10 to 100 nanometers. The shells of DNA-viruses have to sustain large internal pressures while encapsulating and protecting the viral DNA. We employ computer simulations to study the mechanical properties of crystalline shells with icosahedral symmetry that serve as a model for virus capsids. The shells are positioned on a substrate and deformed by a uni-axial force excerted by a small bead. We predict the elastic response for small deformations, and the buckling transitions at large deformations. Both are found to depend strongly on the number N of elementary building blocks (capsomers), and the Föppl-von Kármán number γ which characterizes the relative importance of shear and bending elasticity.

  4. Kinetics versus Thermodynamics in Virus Capsid Polymorphism.

    PubMed

    Moerman, Pepijn; van der Schoot, Paul; Kegel, Willem

    2016-07-01

    Virus coat proteins spontaneously self-assemble into empty shells in aqueous solution under the appropriate physicochemical conditions, driven by an interaction free energy per bond on the order of 2-5 times the thermal energy kBT. For this seemingly modest interaction strength, each protein building block nonetheless gains a very large binding free energy, between 10 and 20 kBT. Because of this, there is debate about whether the assembly process is reversible or irreversible. Here we discuss capsid polymorphism observed in in vitro experiments from the perspective of nucleation theory and of the thermodynamics of mass action. We specifically consider the potential contribution of a curvature free energy term to the effective interaction potential between the proteins. From these models, we propose experiments that may conclusively reveal whether virus capsid assembly into a mixture of polymorphs is a reversible or an irreversible process. PMID:27027925

  5. The structure of elongated viral capsids.

    PubMed

    Luque, Antoni; Reguera, David

    2010-06-16

    There are many viruses whose genetic material is protected by a closed elongated protein shell. Unlike spherical viruses, the structure and construction principles of these elongated capsids are not fully known. In this article, we have developed a general geometrical model to describe the structure of prolate or bacilliform capsids. We show that only a limited set of tubular architectures can be built closed by hemispherical icosahedral caps. In particular, the length and number of proteins adopt a very special set of discrete values dictated by the axial symmetry (fivefold, threefold, or twofold) and the triangulation number of the caps. The results are supported by experimental observations and simulations of simplified physical models. This work brings about a general classification of elongated viruses that will help to predict their structure, and to design viral cages with tailored geometrical properties for biomedical and nanotechnological applications. PMID:20550912

  6. Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

    NASA Astrophysics Data System (ADS)

    Michel, J. P.; Ivanovska, I. L.; Gibbons, M. M.; Klug, W. S.; Knobler, C. M.; Wuite, G. J. L.; Schmidt, C. F.

    2006-04-01

    The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids. atomic force microscopy | cowpea chlorotic mottle virus | finite element analysis | biomechanics

  7. Mechanostability of Proteins and Virus Capsids

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek

    2013-03-01

    Molecular dynamics of proteins within coarse grained models have become a useful tool in studies of large scale systems. The talk will discuss two applications of such modeling. The first is a theoretical survey of proteins' resistance to constant speed stretching as performed for a set of 17134 simple and 318 multidomain proteins. The survey has uncovered new potent force clamps. They involve formation of cysteine slipknots or dragging of a cystine plug through the cystine ring and lead to characteristic forces that are significantly larger than the common shear-based clamp such as observed in titin. The second application involves studies of nanoindentation processes in virus capsids and elucidates their molecular aspects by showing deviations in behavior compared to the continuum shell model. Across the 35 capsids studied, both the collapse force and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number < z > , defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with < z > - 6, where 6 is the minimum coordination for elastic stability in three dimensions. Supported by European Regional Development Fund, through Innovative Economy grant Nanobiom (POIG.01.01.02-00-008/08)

  8. Capsid modification strategies for detargeting adenoviral vectors.

    PubMed

    Parker, Alan L; Bradshaw, Angela C; Alba, Raul; Nicklin, Stuart A; Baker, Andrew H

    2014-01-01

    Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein. PMID:24132476

  9. Diversity of in-vivo assembled HIV-1 capsids

    NASA Astrophysics Data System (ADS)

    Lee, Se Il; Nguyen, Toan

    2008-03-01

    Understanding the capsid assembly process of Human Immunodeficiency Virus (HIV), the causative agent of Acute Immuno Deficiency Syndrom (AIDS), is very important because of recent intense interest in capsid-oriented viral therapy. The unique conical shapes of mature HIV-1 capsid have drawn significant interests in the biological community and started to attract attention from the physics community. Previous studies showed that in a free assembly process, the HIV-1 conical shape is not thermodynamically stable. However, if the volume of the capsid is constrained during assembly and the capsid protein shell has high spontaneous curvature, the conical shape is stable. In this work, we focus on in-vivo HIV-1 capsid assembly. For this case, the viral envelope membrane present during assembly imposes constraint on the length of the capsid. We use an elastic continuum shell theory to approximate the energies of various HIV-1 capsid shapes (spherical, cylindrical and conical). We show that for certain range of viral membrane diameter, the conical and cylindrical shapes are both thermodynamically stable. This result is supported by experimental observation that in-vivo assembled HIV-1 capsids are very heterogeneous in shapes and sizes. Numerical calculation is also performed to improve theoretical approximation.

  10. Prediction of stability changes upon mutation in an icosahedral capsid

    PubMed Central

    Hickman, Samuel J.

    2015-01-01

    ABSTRACT Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. Proteins 2015; 83:1733–1741. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc PMID:26178267

  11. Modeling HIV-1 viral capsid nucleation by dynamical systems.

    PubMed

    Sadre-Marandi, Farrah; Liu, Yuewu; Liu, Jiangguo; Tavener, Simon; Zou, Xiufen

    2015-12-01

    There are two stages generally recognized in the viral capsid assembly: nucleation and elongation. This paper focuses on the nucleation stage and develops mathematical models for HIV-1 viral capsid nucleation based on six-species dynamical systems. The Particle Swarm Optimization (PSO) algorithm is used for parameter fitting to estimate the association and dissociation rates from biological experiment data. Numerical simulations of capsid protein (CA) multimer concentrations demonstrate a good agreement with experimental data. Sensitivity and elasticity analysis of CA multimer concentrations with respect to the association and dissociation rates further reveals the importance of CA trimer-of- dimers in the nucleation stage of viral capsid self- assembly. PMID:26596714

  12. Large-Scale Functional Purification of Recombinant HIV-1 Capsid

    PubMed Central

    Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M.; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  13. Large-scale functional purification of recombinant HIV-1 capsid.

    PubMed

    Hung, Magdeleine; Niedziela-Majka, Anita; Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  14. A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability

    PubMed Central

    Mertens, Johann; Casado, Santiago; Mata, Carlos P.; Hernando-Pérez, Mercedes; de Pablo, Pedro J.; Carrascosa, José L.; Castón, José R.

    2015-01-01

    Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest RNP number (and best fitness) showed greatest capsid rigidity. When bound to dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid structural subunits that differ from the initial interactions during capsid assembly. Our results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsid-stabilizing and genetic information platforms. PMID:26336920

  15. Evaluation of absorbed dose in Gadolinium neutron capture therapy

    NASA Astrophysics Data System (ADS)

    Abdullaeva, Gayane; Djuraeva, Gulnara; Kim, Andrey; Koblik, Yuriy; Kulabdullaev, Gairatulla; Rakhmonov, Turdimukhammad; Saytjanov, Shavkat

    2015-02-01

    Gadolinium neutron capture therapy (GdNCT) is used for treatment of radioresistant malignant tumors. The absorbed dose in GdNCT can be divided into four primary dose components: thermal neutron, fast neutron, photon and natural gadolinium doses. The most significant is the dose created by natural gadolinium. The amount of gadolinium at the irradiated region is changeable and depends on the gadolinium delivery agent and on the structure of the location where the agent is injected. To de- fine the time dependence of the gadolinium concentration ρ(t) in the irradiated region the pharmacokinetics of gadolinium delivery agent (Magnevist) was studied at intratumoral injection in mice and intramuscular injection in rats. A polynomial approximation was applied to the experimental data and the influence of ρ(t) on the relative change of the absorbed dose of gadolinium was studied.

  16. Structural, optical and magnetic properties of gadolinium sesquioxide nanobars synthesized via thermal decomposition of gadolinium oxalate

    SciTech Connect

    Manigandan, R.; Giribabu, K.; Suresh, R.; Vijayalakshmi, L.; Stephen, A.; Narayanan, V.

    2013-10-15

    Graphical abstract: - Highlights: • The cubic Gd{sub 2}O{sub 3} nanobars are synthesized by decomposition of C{sub 6}H{sub 20}Gd{sub 2}O{sub 22}. • The nanoparticles are rectangular bar shape with high porous surface. • The combination of magnetic and optical properties within a single particle. • The Gd{sub 2}O{sub 3} nanobars have tailorable nanostructure, wide bandgap and are paramagnetic. - Abstract: Gadolinium oxide nanobars were obtained by thermal decomposition of gadolinium oxalate, which was synthesized by the chemical precipitation method along with glycerol. The functional group analysis and formation of gadolinium oxide from gadolinium oxalate were characterized by the Fourier transform infrared spectroscopy and thermo gravimetric analyzer. The crystal structure, average crystallite size, and lattice parameter were analyzed by X-ray diffraction technique. Moreover, Raman shifts, elemental composition and morphology of the gadolinium oxide was widely investigated by the laser Raman microscope, X-ray photoelectron spectroscopy, FE-SEM-EDAX and HR-TEM, respectively. Furthermore, the optical properties like band gap, absorbance measurement of the gadolinium oxide were extensively examined. In addition, the paramagnetic property of gadolinium oxide nanobars was explored by the vibrating sample magnetometer.

  17. Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

    SciTech Connect

    Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B.; Rossmann, Michael G.

    2010-07-22

    Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a 'glue' between neighboring hexameric capsomers, forming a 'cage' that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 {angstrom} resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

  18. Enhancement of the electron electric dipole moment in gadolinium garnets

    SciTech Connect

    Mukhamedjanov, T.N.; Dzuba, V.A.; Sushkov, O.P.

    2003-10-01

    Effects caused by the electron electric dipole moment (EDM) in gadolinium garnets are considered. Experimental studies of these effects could improve the current upper limit on the electron EDM by several orders of magnitude. We suggest a consistent theoretical model and perform calculations of observable effects in gadolinium gallium garnet and gadolinium iron garnet. Our calculation accounts for both direct and exchange diagrams.

  19. Neutron autoradiography of trace amounts of gadolinium

    SciTech Connect

    Shih, J.L.A.; Brugger, R.M. )

    1992-05-01

    This paper reports that application of gadolinium in neutron capture therapy is under evaluation. Crucial to development of this therapy is an imaging technique that would show the distribution and concentration of parts-per-million amounts of {sup 157}Gd in sliced samples. A technique that utilizes the principles of autoradiography and neutron radiography has been tried. The images taken with this method display the gadolinium distribution and its relative concentration in samples. Concentrations of {sup 157}Gd ranging from {approximately}20 to 500 ppm can be determined. The intrinsic spatial resolution of the system used in the Missouri University Research Reactor is 70 {mu}m.

  20. Mx oligomer: a novel capsid pattern sensor?

    PubMed

    Kong, Jia; Ma, Min; He, Shuangyi; Qin, Xiaohong

    2016-08-01

    Myxovirus resistance proteins represent a family of interferon-induced restriction factors of the innate and adaptive immune system. Human MxB acts as a novel restriction factor with antiviral activity against a range of HIV-1 and other retroviruses mainly by inhibiting the uncoating process after reverse transcription but prior to integration. Based on published data and conservation analysis, we propose a novel hypothesis, in which MxB dimers form higher order oligomers that restrict retroviral replication by binding to the viral capsid. Insights into the mechanistic basis of structural and functional characteristics of MxB will greatly advance our understanding of MxB. PMID:27492442

  1. Packaging of Polyelectrolytes in Viral Capsids: The Interplay Between Polymer Length and Capsid Size

    NASA Astrophysics Data System (ADS)

    Knobler, Charles

    2008-03-01

    Each particle of the Cowpea Chlorotic Mottle Virus (CCMV) has a very small ``parts list,'' consisting of two components: a molecule of single-stranded RNA and a 190-residue protein that makes up the 28-nm diameter icosahedral capsid. When purified viral RNA and capsid protein are mixed in solution at an appropriate pH and ionic strength, infectious wild-type viruses form spontaneously. Virus-like particles (VLPs) are formed when the protein self assembles around other anionic polymers such as poly(styrene sulfonate) (PSS). Under different pH and ionic strength conditions the capsid protein can assemble by itself into empty capsids, multishell structures, tubes and sheets. To explore the effect on virion size of the competition between the preferred curvature of the protein and the size of the packaged cargo we have examined the formation of VLPs around PSS polymers with molecular weights ranging from 400 kDa to 3.4 MDa. Two distinct sizes are observed -- 22 nm for the lower molecular weights, jumping to 27 nm at 2 MDa. While under given conditions the size of PSS in solution is directly determined by its molecular weight, the self-complementarity of RNA makes its solution structure dependent on the nucleotide sequence as well. We have therefore employed Small-Angle X-ray Scattering and Fluorescence Correlation Spectroscopy to examine the sizes of viral and non-viral RNAs of identical lengths. A model for the assembly that includes both the self-interactions of the polyelectrolyte and the capsid proteins and the interactions between them provides insight into the experimental results.

  2. Multivalent Viral Capsids with Internal Cargo for Fibrin Imaging

    PubMed Central

    Obermeyer, Allie C.; Capehart, Stacy L.; Jarman, John B.; Francis, Matthew B.

    2014-01-01

    Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications. PMID:24960118

  3. A toxicological study of gadolinium nitrate

    SciTech Connect

    London, J.E.

    1988-05-01

    The sensitization study in the guinea pig did not show gadolinium nitrate to have potential sensitizing properties. Skin application studies in the rabbit demonstrated that it was cutaneously a severe irritant. This material was considered an irritant in the rabbit eye application studies. 3 refs., 1 tab.

  4. Gadolinium: Central Metal of the Lanthanoids

    ERIC Educational Resources Information Center

    Laing, Michael

    2009-01-01

    The physical and chemical properties of gadolinium are compared with those of the other lanthanoids. Some properties are intermediate between those of lanthanum and lutetium; some between those of barium and hafnium; and others (unexpectedly) between those of ytterbium and lutetium. Both the remarkably high molar heat capacity of the metal and the…

  5. Bluetongue virus capsid assembly and maturation.

    PubMed

    Mohl, Bjorn-Patrick; Roy, Polly

    2014-08-01

    Maturation is an intrinsic phase of the viral life cycle and is often intertwined with egress. In this review we focus on orbivirus maturation by using Bluetongue virus (BTV) as a representative. BTV, a member of the genus Orbivirus within the family Reoviridae, has over the last three decades been subjected to intense molecular study and is thus one of the best understood viruses. BTV is a non-enveloped virus comprised of two concentric protein shells that encapsidate 10 double-stranded RNA genome segments. Upon cell entry, the outer capsid is shed, releasing the core which does not disassemble into the cytoplasm. The polymerase complex within the core then synthesizes transcripts from each genome segment and extrudes these into the cytoplasm where they act as templates for protein synthesis. Newly synthesized ssRNA then associates with the replicase complex prior to encapsidation by inner and outer protein layers of core within virus-triggered inclusion bodies. Maturation of core occurs outside these inclusion bodies (IBs) via the addition of the outer capsid proteins, which appears to be coupled to a non-lytic, exocytic pathway during early infection. Similar to the enveloped viruses, BTV hijacks the exocytosis and endosomal sorting complex required for trafficking (ESCRT) pathway via a non-structural glycoprotein. This exquisitely detailed understanding is assembled from a broad array of assays, spanning numerous and diverse in vitro and in vivo studies. Presented here are the detailed insights of BTV maturation and egress. PMID:25196482

  6. Structure of the Triatoma virus capsid.

    PubMed

    Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S; Costabel, Marcelo D; Marti, Gerardo A; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M A; Rey, Felix A

    2013-06-01

    The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed. PMID:23695247

  7. Structure of the Triatoma virus capsid

    PubMed Central

    Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S.; Costabel, Marcelo D.; Marti, Gerardo A.; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M. A.; Rey, Felix A.

    2013-01-01

    The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed. PMID:23695247

  8. Monte Carlo simulations of polyelectrolytes inside viral capsids

    NASA Astrophysics Data System (ADS)

    Angelescu, Daniel George; Bruinsma, Robijn; Linse, Per

    2006-04-01

    Structural features of polyelectrolytes as single-stranded RNA or double-stranded DNA confined inside viral capsids and the thermodynamics of the encapsidation of the polyelectrolyte into the viral capsid have been examined for various polyelectrolyte lengths by using a coarse-grained model solved by Monte Carlo simulations. The capsid was modeled as a spherical shell with embedded charges and the genome as a linear jointed chain of oppositely charged beads, and their sizes corresponded to those of a scaled-down T=3 virus. Counterions were explicitly included, but no salt was added. The encapisdated chain was found to be predominantly located at the inner capsid surface, in a disordered manner for flexible chains and in a spool-like structure for stiff chains. The distribution of the small ions was strongly dependent on the polyelectrolyte-capsid charge ratio. The encapsidation enthalpy was negative and its magnitude decreased with increasing polyelectrolyte length, whereas the encapsidation entropy displayed a maximum when the capsid and polyelectrolyte had equal absolute charge. The encapsidation process remained thermodynamically favorable for genome charges ca. 3.5 times the capsid charge. The chain stiffness had only a relatively weak effect on the thermodynamics of the encapsidation.

  9. Structure of the Triatoma virus capsid

    SciTech Connect

    Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S.; Costabel, Marcelo D.; Marti, Gerardo A.; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M. A.; Rey, Felix A.

    2013-06-01

    The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

  10. Gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres for gadolinium neutron-capture therapy.

    PubMed

    Saha, Tapan Kumar; Ichikawa, Hideki; Fukumori, Yoshinobu

    2006-12-11

    In order to provide a suitable device that would contain water-soluble drugs, highly water-soluble gadolinium diethylenetriaminopentaacetic acid-loaded chitosan microspheres (CMS-Gd-DTPA) were prepared by the emulsion method using glutaraldehyde as a cross-linker and Span 80 as a surfactant for gadolinium neutron-capture therapy of cancer. The gadolinium content and the mass median diameter of CMS-Gd-DTPA were estimated. The size and morphology of the CMS-Gd-DTPA were strongly influenced by the initial applied weight ratio of Gd-DTPA:chitosan. FTIR spectra showed that the electrostatic interaction between chitosan and Gd-DTPA accelerated the formation of gadolinium-enriched chitosan microspheres. Sufficient amounts of glutaraldehyde and Span 80 were necessary for producing discrete CMS-Gd-DTPA. The CMS-Gd-DTPA having a mass median diameter 11.7microm and 11.6% of gadolinium could be used in Gd-NCT following intratumoral injection. PMID:17045253

  11. Method of separating and purifying gadolinium-153

    DOEpatents

    Bray, Lane A [Richland, WA; Corneillie, Todd M [Davis, CA

    2001-01-01

    The present invention is an improvement to the method of separating and purifying gadolinium from a mixture of gadolinium and europium having the steps of (a) dissolving the mixture in an acid; (b) reducing europium+3 to europium+2; and (c) precipitating the europium+2 with a sulfate ion in a superstoichiometric amount; wherein the improvement is achieved by using one or more of the following: (i) the acid is an anoic acid; (ii) the reducing is with zinc metal in the absence of a second metal or with an amount of the second metal that is ineffective in the reducing; (iii) adding a group IIA element after step (c) for precipitating the excess sulfate prior to repeating step (c); (iv) the sulfate is a sulfate salt with a monovalent cation; (v) adding cold europium+3 prior to repeating step (c).

  12. Progress Towards Laser Cooling and Trapping Gadolinium

    NASA Astrophysics Data System (ADS)

    Adhikari, Upendra; Simien, Clayton

    2016-05-01

    Lanthanide elements are of interest because of their potential for investigating next generation optical clock transitions, novel non-S ground state ultracold collisions, and the physics of quantum degenerate dipolar gases. We present our progress towards laser cooling and trapping atomic Gadolinium (Gd). A magneto-optical trap is the first step towards precision measurements, ultracold collision studies, and for probing dipolar physics of Gd. The design, construction, and performance of the apparatus will be presented.

  13. Emerging concepts for myocardial late gadolinium enhancement MRI.

    PubMed

    Doltra, Adelina; Amundsen, Brage Hoyem; Gebker, Rolf; Fleck, Eckart; Kelle, Sebastian

    2013-08-01

    Late gadolinium enhancement is a useful tool for scar detection, based on differences in the volume of distribution of gadolinium, an extracellular agent. The presence of fibrosis in the myocardium amenable to be detected with late gadolinium enhancement MRI is found not only in ischemic cardiomyopathy, in which it offers information regarding viability and prognosis, but also in a wide variety of non-ischemic cardiomyopathies. In the following review we will discuss the methodological aspects of gadolinium-based imaging, as well as its applications and anticipated future developments. PMID:23909638

  14. Viral genome structures are optimal for capsid assembly

    PubMed Central

    Perlmutter, Jason D; Qiao, Cong; Hagan, Michael F

    2013-01-01

    Understanding how virus capsids assemble around their nucleic acid (NA) genomes could promote efforts to block viral propagation or to reengineer capsids for gene therapy applications. We develop a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics. In contrast to recent theoretical models, we find that capsids spontaneously ‘overcharge’; that is, the negative charge of the NA exceeds the positive charge on capsid. When applied to specific viruses, the optimal NA lengths closely correspond to the natural genome lengths. Calculations based on linear polyelectrolytes rather than base-paired NAs underpredict the optimal length, demonstrating the importance of NA structure to capsid assembly. These results suggest that electrostatics, excluded volume, and NA tertiary structure are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained, at least in part, on a thermodynamic basis. DOI: http://dx.doi.org/10.7554/eLife.00632.001 PMID:23795290

  15. All-Atom Molecular Dynamics of Virus Capsids as Drug Targets.

    PubMed

    Perilla, Juan R; Hadden, Jodi A; Goh, Boon Chong; Mayne, Christopher G; Schulten, Klaus

    2016-05-19

    Virus capsids are protein shells that package the viral genome. Although their morphology and biological functions can vary markedly, capsids often play critical roles in regulating viral infection pathways. A detailed knowledge of virus capsids, including their dynamic structure, interactions with cellular factors, and the specific roles that they play in the replication cycle, is imperative for the development of antiviral therapeutics. The following Perspective introduces an emerging area of computational biology that focuses on the dynamics of virus capsids and capsid-protein assemblies, with particular emphasis on the effects of small-molecule drug binding on capsid structure, stability, and allosteric pathways. When performed at chemical detail, molecular dynamics simulations can reveal subtle changes in virus capsids induced by drug molecules a fraction of their size. Here, the current challenges of performing all-atom capsid-drug simulations are discussed, along with an outlook on the applicability of virus capsid simulations to reveal novel drug targets. PMID:27128262

  16. Resonance parameter measurements and analysis of gadolinium

    SciTech Connect

    Leinweber, G.; Barry, D. P.; Trbovich, M. J.; Burke, J. A.; Drindak, N. J.; Knox, H. D.; Ballad, R. V.; Block, R. C.; Danon, Y.; Severnyak, L. I.

    2006-07-01

    The purpose of the present work is to measure the neutron cross sections of gadolinium accurately. Gd has the highest thermal absorption cross section of any natural element. Therefore it is an important element for thermal reactor applications Neutron capture and transmission measurements were performed by the time-of-flight technique at the Rensselaer Polytechnic Inst. (RPI) LINAC facility using metallic and liquid Gd samples. The liquid samples were isotopically-enriched in either {sup 155}Gd or {sup 157}Gd. The capture measurements were made at the 25-m flight station with a sodium iodide detector, and the transmission measurements were performed at 15- and 25-m flight stations with {sup 6}Li glass scintillation detectors. The multilevel R-matrix Bayesian code SAMMY was used to extract resonance parameters. The results of the thermal region analysis are significant. Resonance parameters for the low energy doublet, at 0.025 and 0.032 eV, are presented. The thermal (2200 m/s) capture cross section of {sup 157}Gd has been measured to be 11% smaller than that calculated from ENDF/B-VI updated through release 8. Thermal capture cross sections and capture resonance integrals for each isotope as well as elemental gadolinium are presented. In the epithermal region, natural metal samples were measured in capture and transmission. Neutron interaction data up to 300 eV have been analyzed. Substantial improvement to the understanding of gadolinium cross sections is presented, particularly above 180 eV where the ENDF resolved region for {sup 155}Gd ends. (authors)

  17. Extraction-chromatographic affinage in gadolinium-153 preparation production technology

    SciTech Connect

    Melnik, M.I.; Karelin, E.A.; Kuznetsov, R.A.

    1993-12-31

    The gadolinium 153 preparation is used for production of medical gamma-sources which are applicable in bone densimeters for early diagnostics of osteoporosis. This preparation must meet strict requirements with respect to the content of europium radionuclides and specific activity. In The Research Institute of Atomic Reactors (RIAR) the gadolinium 153 is produced by neutron irradiation of Europium 151. This process is described.

  18. Light deflection in gadolinium molybdate ferroelastic crystals

    NASA Astrophysics Data System (ADS)

    Staniorowski, Piotr; Bornarel, Jean

    2000-02-01

    The deflection of a He-Ne light beam by polydomain gadolinium molybdate (GMO) crystals has been studied with respect to incidence angle icons/Journals/Common/alpha" ALT="alpha" ALIGN="TOP"/> i on the sample at room temperature. The A and B deflected beams do not cross each other during the icons/Journals/Common/alpha" ALT="alpha" ALIGN="TOP"/> i variation, in contrast to results and calculations previously published. The model using the Fresnel equation confirms this result. The model presented is more accurate for numerical calculation than that using the Huygens construction.

  19. Towards modeling gadolinium-lead-borate glasses

    SciTech Connect

    Rada, S.; Ristoiu, T.; Rada, M.; Coroiu, I.; Maties, V.; Culea, E.

    2010-01-15

    Infrared spectra of gadolinium-lead-borate glasses of the xGd{sub 2}O{sub 3}.(100 - x)[3B{sub 2}O{sub 3}.PbO] system, where x = 0, 5, 10, 15, 25, 35 and 50 mol.%, have been recorded to explore the role of content of gadolinium ions behaving as glass modifier. The FTIR spectroscopy data for the xGd{sub 2}O{sub 3}.(1 - x)[3B{sub 2}O{sub 3}.PbO] glasses show the structural role of lead ions as a network-formers and of the gadolinium ions network modifiers. Adding of the rare earth ion up to 35 mol.% into the glass matrix, the IR bands characteristic to the studied glasses become sharper and more pronounced. Structural changes, as recognized by analyzing band shapes of IR spectra, revealed that Gd{sub 2}O{sub 3} causes a change from the continuous borate network to the continuous lead-borate network interconnected through Pb-O-B and B-O-B bridges and the transformation of some tetrahedral [BO{sub 4}] units into trigonal [BO{sub 3}] units. Then, gadolinium ions have affinity towards [BO{sub 3}] structural units which contain non-bridging oxygens necessary for the charge compensation because the more electronegative [BO{sub 3}] structural units were implied in the formation of B-O-Gd bonds and the transformation of glass network into a glass ceramic. We propose a possible structural model of building blocks for the formation of continuous random 3B{sub 2}O{sub 3}.PbO network glass used by density functional theory (DFT) calculations. DFT calculations show that lead atoms occupy three different sites in the proposed model. The first is coordinated with six oxygen atoms forming distorted octahedral geometries. The second lead atom has an octahedral oxygen environment and the five longer Pb-O bonds are considered as participating in the metal coordination scheme. The third lead atom has ionic character. In agreement with the results offered by the experimental FTIR data, the theoretical IR data confirm that our proposed structure is highly possible.

  20. Building a viral capsid in the presence of genomic RNA

    NASA Astrophysics Data System (ADS)

    Dykeman, Eric C.; Stockley, Peter G.; Twarock, Reidun

    2013-02-01

    Virus capsid assembly has traditionally been considered as a process that can be described primarily via self-assembly of the capsid proteins, neglecting interactions with other viral or cellular components. Our recent work on several ssRNA viruses, a major class of viral pathogens containing important human, animal, and plant viruses, has shown that this protein-centric view is too simplistic. Capsid assembly for these viruses relies strongly on a number of cooperative roles played by the genomic RNA. This realization requires a new theoretical framework for the modeling and prediction of the assembly behavior of these viruses. In a seminal paper Zlotnick [J. Mol. Biol.0022-283610.1006/jmbi.1994.1473 241, 59 (1994)] laid the foundations for the modeling of capsid assembly as a protein-only self-assembly process, illustrating his approach using the example of a dodecahedral study system. We describe here a generalized framework for modeling assembly that incorporates the regulatory functions provided by cognate protein-nucleic-acid interactions between capsid proteins and segments of the genomic RNA, called packaging signals, into the model. Using the same dodecahedron system we demonstrate, using a Gillespie-type algorithm to deal with the enhanced complexity of the problem instead of a master equation approach, that assembly kinetics and yield strongly depend on the distribution and nature of the packaging signals, highlighting the importance of the crucial roles of the RNA in this process.

  1. Polymorphism in the assembly of polyomavirus capsid protein VP1.

    PubMed Central

    Salunke, D M; Caspar, D L; Garcea, R L

    1989-01-01

    Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:2557933

  2. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    NASA Astrophysics Data System (ADS)

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-05-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

  3. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly.

    PubMed

    Grime, John M A; Dama, James F; Ganser-Pornillos, Barbie K; Woodward, Cora L; Jensen, Grant J; Yeager, Mark; Voth, Gregory A

    2016-01-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies. PMID:27174390

  4. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    PubMed Central

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-01-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies. PMID:27174390

  5. Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

    PubMed Central

    2012-01-01

    Background Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection. Results To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α. Conclusions The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability

  6. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    PubMed

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. PMID:24878641

  7. Icosahedral capsid formation by capsomers and short polyions

    NASA Astrophysics Data System (ADS)

    Zhang, Ran; Linse, Per

    2013-04-01

    Kinetical and structural aspects of the capsomer-polyion co-assembly into icosahedral viruses have been simulated by molecular dynamics using a coarse-grained model comprising cationic capsomers and short anionic polyions. Conditions were found at which the presence of polyions of a minimum length was necessary for capsomer formation. The largest yield of correctly formed capsids was obtained at which the driving force for capsid formation was relatively weak. Relatively stronger driving forces, i.e., stronger capsomer-capsomer short-range attraction and/or stronger electrostatic interaction, lead to larger fraction of kinetically trapped structures and aberrant capsids. The intermediate formation was investigated and different evolving scenarios were found by just varying the polyion length.

  8. Modelling the self-assembly of virus capsids

    NASA Astrophysics Data System (ADS)

    Johnston, Iain G.; Louis, Ard A.; Doye, Jonathan P. K.

    2010-03-01

    We use computer simulations to study a model, first proposed by Wales (2005 Phil. Trans. R. Soc. A 363 357), for the reversible and monodisperse self-assembly of simple icosahedral virus capsid structures. The success and efficiency of assembly as a function of thermodynamic and geometric factors can be qualitatively related to the potential energy landscape structure of the assembling system. Even though the model is strongly coarse-grained, it exhibits a number of features also observed in experiments, such as sigmoidal assembly dynamics, hysteresis in capsid formation and numerous kinetic traps. We also investigate the effect of macromolecular crowding on the assembly dynamics. Crowding agents generally reduce capsid yields at optimal conditions for non-crowded assembly, but may increase yields for parameter regimes away from the optimum. Finally, we generalize the model to a larger triangulation number T = 3, and observe assembly dynamics more complex than that seen for the original T = 1 model.

  9. A Simple Model for Immature Retrovirus Capsid Assembly

    NASA Astrophysics Data System (ADS)

    Paquay, Stefan; van der Schoot, Paul; Dragnea, Bogdan

    In this talk I will present simulations of a simple model for capsomeres in immature virus capsids, consisting of only point particles with a tunable range of attraction constrained to a spherical surface. We find that, at sufficiently low density, a short interaction range is sufficient for the suppression of five-fold defects in the packing and causes instead larger tears and scars in the capsid. These findings agree both qualitatively and quantitatively with experiments on immature retrovirus capsids, implying that the structure of the retroviral protein lattice can, for a large part, be explained simply by the effective interaction between the capsomeres. We thank the HFSP for funding under Grant RGP0017/2012.

  10. Calibrating elastic parameters from molecular dynamics simulations of capsid proteins

    NASA Astrophysics Data System (ADS)

    Hicks, Stephen; Henley, Christopher

    2008-03-01

    Virus capsids are modeled with elastic network models in which a handful of parameters determine transitions in assembly [1] and morphology [2]. We introduce an approach to compute these parameters from the microscopic structure of the proteins involved. We consider each protein as one or a few rigid bodies with very general interactions, which we parameterize by fitting the simulated equilibrium fluctuations (relative translations and rotations) of a pair of proteins (or fragments) to a 6-dimensional Gaussian. We can then compose these generalized springs into the global capsid structure to determine the continuum elastic parameters. We demonstrate our approach on HIV capsid protein and compare our results with the observed lattice structure (from cryo-EM [3] and AFM indentation studies). [1] R. Zandi et al, PNAS 101 (2004) 15556. [2] J. Lidmar, L. Mirny, and D. R. Nelson, PRE 68 (2003) 051910. [3] B. K. Ganser-Pornillos et al, Cell 131 (2007) 70.

  11. C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly

    PubMed Central

    Castón, José R.; Martínez-Torrecuadrada, Jorge L.; Maraver, Antonio; Lombardo, Eleuterio; Rodríguez, José F.; Casal, J. Ignacio; Carrascosa, José L.

    2001-01-01

    Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis. PMID:11602723

  12. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    PubMed Central

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  13. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    SciTech Connect

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  14. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    NASA Astrophysics Data System (ADS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  15. Alkali metal and alkali earth metal gadolinium halide scintillators

    DOEpatents

    Bourret-Courchesne, Edith; Derenzo, Stephen E.; Parms, Shameka; Porter-Chapman, Yetta D.; Wiggins, Latoria K.

    2016-08-02

    The present invention provides for a composition comprising an inorganic scintillator comprising a gadolinium halide, optionally cerium-doped, having the formula A.sub.nGdX.sub.m:Ce; wherein A is nothing, an alkali metal, such as Li or Na, or an alkali earth metal, such as Ba; X is F, Br, Cl, or I; n is an integer from 1 to 2; m is an integer from 4 to 7; and the molar percent of cerium is 0% to 100%. The gadolinium halides or alkali earth metal gadolinium halides are scintillators and produce a bright luminescence upon irradiation by a suitable radiation.

  16. Gadolinium enrichment technology at Lawrence Livermore National Laboratory

    NASA Astrophysics Data System (ADS)

    Haynam, Christopher A.; Comaskey, Brian J.; Conway, John; Eggert, Jon; Glaser, Joseph; Ng, Edmund W.; Paisner, Jeffrey A.; Solarz, Richard W.; Worden, Earl F.

    1993-05-01

    A method based on polarization selectivity and three step laser photoionization is presented for separation of the odd isotopes of gadolinium. Measurements of the spectroscopic parameters needed to quantify the excitation pathway are discussed. Model results are presented for the efficiency of photoionization. The vapor properties of electron beam vaporized gadolinium are presented which show dramatic cooling during the expansion of the hot dense vapor into a vacuum. This results in a significant increase in the efficiency of conversion of natural feed into enriched product in the AVLIS process. Production of enriched gadolinium for use in commercial power reactors appears to be economically viable using technology in use at LLNL.

  17. Removal of gadolinium nitrate from heavy water

    SciTech Connect

    Wilde, E.W.

    2000-03-22

    Work was conducted to develop a cost-effective process to purify 181 55-gallon drums containing spent heavy water moderator (D2O) contaminated with high concentrations of gadolinium nitrate, a chemical used as a neutron poison during former nuclear reactor operations at the Savannah River Site (SRS). These drums also contain low level radioactive contamination, including tritium, which complicates treatment options. Presently, the drums of degraded moderator are being stored on site. It was suggested that a process utilizing biological mechanisms could potentially lower the total cost of heavy water purification by allowing the use of smaller equipment with less product loss and a reduction in the quantity of secondary waste materials produced by the current baseline process (ion exchange).

  18. STRUCTURAL DYNAMICS, AN INTRINSIC PROPERTY OF VIRAL CAPSIDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this review, we emphasize that high-resolution models of the structures of small plant and animal viruses obtained by X-ray crystallography are static and insufficient to describe the behavior of these virions. Viral capsids are highly flexible and may undergo conformational changes under physio...

  19. Molecular Evolution of the Capsid Gene in Norovirus Genogroup I.

    PubMed

    Kobayashi, Miho; Yoshizumi, Shima; Kogawa, Sayaka; Takahashi, Tomoko; Ueki, Yo; Shinohara, Michiyo; Mizukoshi, Fuminori; Tsukagoshi, Hiroyuki; Sasaki, Yoshiko; Suzuki, Rieko; Shimizu, Hideaki; Iwakiri, Akira; Okabe, Nobuhiko; Shirabe, Komei; Shinomiya, Hiroto; Kozawa, Kunihisa; Kusunoki, Hideki; Ryo, Akihide; Kuroda, Makoto; Katayama, Kazuhiko; Kimura, Hirokazu

    2015-01-01

    We studied the molecular evolution of the capsid gene in all genotypes (genotypes 1-9) of human norovirus (NoV) genogroup I. The evolutionary time scale and rate were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also performed selective pressure analysis and B-cell linear epitope prediction in the deduced NoV GI capsid protein. Furthermore, we analysed the effective population size of the virus using Bayesian skyline plot (BSP) analysis. A phylogenetic tree by MCMC showed that NoV GI diverged from the common ancestor of NoV GII, GIII, and GIV approximately 2,800 years ago with rapid evolution (about 10(-3) substitutions/site/year). Some positive selection sites and over 400 negative selection sites were estimated in the deduced capsid protein. Many epitopes were estimated in the deduced virus capsid proteins. An epitope of GI.1 may be associated with histo-blood group antigen binding sites (Ser377, Pro378, and Ser380). Moreover, BSP suggested that the adaptation of NoV GI strains to humans was affected by natural selection. The results suggested that NoV GI strains evolved rapidly and date back to many years ago. Additionally, the virus may have undergone locally affected natural selection in the host resulting in its adaptation to humans. PMID:26338545

  20. Nonlinear finite-element analysis of nanoindentation of viral capsids

    NASA Astrophysics Data System (ADS)

    Gibbons, Melissa M.; Klug, William S.

    2007-03-01

    Recent atomic force microscope (AFM) nanoindentation experiments measuring mechanical response of the protein shells of viruses have provided a quantitative description of their strength and elasticity. To better understand and interpret these measurements, and to elucidate the underlying mechanisms, this paper adopts a course-grained modeling approach within the framework of three-dimensional nonlinear continuum elasticity. Homogeneous, isotropic, elastic, thick-shell models are proposed for two capsids: the spherical cowpea chlorotic mottle virus (CCMV), and the ellipsocylindrical bacteriophage ϕ29 . As analyzed by the finite-element method, these models enable parametric characterization of the effects of AFM tip geometry, capsid dimensions, and capsid constitutive descriptions. The generally nonlinear force response of capsids to indentation is shown to be insensitive to constitutive particulars, and greatly influenced by geometric and kinematic details. Nonlinear stiffening and softening of the force response is dependent on the AFM tip dimensions and shell thickness. Fits of the models capture the roughly linear behavior observed in experimental measurements and result in estimates of Young’s moduli of ≈280-360MPa for CCMV and ≈4.5GPa for ϕ29 .

  1. [Structure, Immunogenicity and Clinical Value of Chlamydiaphage Capsid Protein 3].

    PubMed

    Yao, Weifeng; Li, Yiju; Yuan, Jing; Wang, Lei; Li, Qunyan; Song, Mengmeng; Lu, Guiling; Zhang, Litao

    2015-07-01

    We wished to assess the role of chlamydia micro virus capsid protein Vp3 in recombinant molecules, chart its molecular evolution, screen the wild-type strain, and reveal its value in clinical research. Using a protein BLAST multiple-alignment program, we compared various strains of Chlamydia micro virus capsid protein Vp3 sequences. Using a "distance tree" of those results, we created a phylogenetic tree. We applied the Karplus-Schulz method of flexible-region analyses for highly conserved alignments of amino-acid sequences. Gamier-Robson and Chou-Fasman methods were employed to analyze two-level structures of sequences. The Emini method was used for analyses of the accessibility of surface epitopes. Studies of hydrophilic proteins were undertaken using Kyte-Doolittle and Hopp-Woods methods. Analyses of antigen epitopes helped to reveal the antigen index using the Jameson-Wolf method. All sequences in the six strains of chlamydia micro virus capsid protein Vp3 were highly conserved, with the main differences being between Vp3 protein in Chp1 and the other five strains of the micro virus. The viral strain of Vp3 protein was based mainly on micro-alpha helix structures, and multiple epitopes were noted in highly conserved regions. Vp3 protein was highly conserved structurally, and was an important protein of the chlamydiaphage capsid. Vp3 protein has a complicated molecular structure, highly conserved regions with strong immunogenicity, and has considerable research value. PMID:26524915

  2. L2, the minor capsid protein of papillomavirus

    SciTech Connect

    Wang, Joshua W.; Roden, Richard B.S.

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  3. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

    PubMed Central

    Roos, Wouter H.; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J. L.

    2009-01-01

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

  4. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids.

    PubMed

    Roos, Wouter H; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J L

    2009-06-16

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

  5. Periodic Table of Virus Capsids: Implications for Natural Selection and Design

    PubMed Central

    Mannige, Ranjan V.; Brooks, Charles L.

    2010-01-01

    Background For survival, most natural viruses depend upon the existence of spherical capsids: protective shells of various sizes composed of protein subunits. So far, general evolutionary pressures shaping capsid design have remained elusive, even though an understanding of such properties may help in rationally impeding the virus life cycle and designing efficient nano-assemblies. Principal Findings This report uncovers an unprecedented and species-independent evolutionary pressure on virus capsids, based on the the notion that the simplest capsid designs (or those capsids with the lowest “hexamer complexity”, ) are the fittest, which was shown to be true for all available virus capsids. The theories result in a physically meaningful periodic table of virus capsids that uncovers strong and overarching evolutionary pressures, while also offering geometric explanations to other capsid properties (rigidity, pleomorphy, auxiliary requirements, etc.) that were previously considered to be unrelatable properties of the individual virus. Significance Apart from describing a universal rule for virus capsid evolution, our work (especially the periodic table) provides a language with which highly diverse virus capsids, unified only by geometry, may be described and related to each other. Finally, the available virus structure databases and other published data reiterate the predicted geometry-derived rules, reinforcing the role of geometry in the natural selection and design of virus capsids. PMID:20209096

  6. Synthesis of gadolinium oxide magnetoliposomes for magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Roberts, Danielle; Zhu, Weibe L.; Frommen, Christoph M.; Rosenzweig, Zeev

    2000-05-01

    A method for the synthesis of gadolinium oxide magnetoliposomes, i.e., nanosized gadolinium oxide magnetic particles coated by a phospholipid membrane, is presented. Magnetoliposomes were prepared by reacting lauric acid coated gadolinium oxide nanoparticles with dimyristoylphosphatidylcholine liposomes prepared using a direct injection method. The gadolinium oxide magnetoliposomes were characterized using transmission electron microscopy imaging, x-ray diffraction, and fluorescence. The magnetic properties of the magnetoliposomes were investigated with a superconducting quantum interference device magnetometer and nuclear magnetic resonance (NMR) spectrometry. Our results indicate that the magnetoliposomes contain approximately spherical nanoparticles averaging 20 nm in diameter. The occurrence of a phospholipid bilayer surrounding the magnetic particles is confirmed both by transmission electron micrographs of samples negatively stained with uranyl acetate and by digital fluorescence imaging microscopy measurements of magnetoliposomes labeled with fluorescein. The particles are paramagnetic at room temperature. NMR measurements show that the ratio between the relaxivities of the particles depends largely on their preparation.

  7. All-Atom Molecular Dynamics of Virus Capsids as Drug Targets

    PubMed Central

    2016-01-01

    Virus capsids are protein shells that package the viral genome. Although their morphology and biological functions can vary markedly, capsids often play critical roles in regulating viral infection pathways. A detailed knowledge of virus capsids, including their dynamic structure, interactions with cellular factors, and the specific roles that they play in the replication cycle, is imperative for the development of antiviral therapeutics. The following Perspective introduces an emerging area of computational biology that focuses on the dynamics of virus capsids and capsid–protein assemblies, with particular emphasis on the effects of small-molecule drug binding on capsid structure, stability, and allosteric pathways. When performed at chemical detail, molecular dynamics simulations can reveal subtle changes in virus capsids induced by drug molecules a fraction of their size. Here, the current challenges of performing all-atom capsid–drug simulations are discussed, along with an outlook on the applicability of virus capsid simulations to reveal novel drug targets. PMID:27128262

  8. Electrostatics of capsid-induced viral RNA organization

    PubMed Central

    Forrey, Christopher; Muthukumar, M.

    2009-01-01

    We have addressed the role of electrostatics in the formation of genome structure in the Pariacoto virus, where substantial experimental data are available. We have used Langevin dynamics simulation of a coarse-grained model, based on the published crystal structure of the rigid portion of the Pariacoto capsid and including flexible N-terminal protein arms, attached to the rigid capsid at the appropriate locations. The inclusion of charged residues in our model was dictated solely by the location of charges inherent in the Pariacoto sequence itself. Although the viral genome and other exogenous RNA sequences used in experimental studies can assume secondary structures, we have intentionally used uniformly charged flexible polyelectrolyte lacking predetermined secondary structures as the substitute for the viral genome, in order to see whether the same final assembled genome structure emerges without invoking secondary RNA structures. The intent of our study was to investigate the internal environment presented by the capsid proteins of Pariacoto virus, specifically whether the topological features and electrostatic potential at the inner capsid surface can induce complexation of generic negatively charged polyelectrolyte into structures similar to those observed experimentally with packaged RNA. We find that the charge decoration on the interior of the capsid templates the assembly of the flexible polyelectrolyte, allowing hybridizationlike folding of similarly charged strands, and eventually organizing dodecahedral assembly of the polymer. Our results from a generic flexible polyelectrolyte for the assembled structure and bimodal monomer distribution are remarkably matched to that of the viral RNA found experimentally. Results of our work can be interpreted primarily as a consequence of electrostatics, as consideration of base-pairing has been omitted. We propose that our work supports the growing body of evidence that electrostatic interactions play a crucial

  9. Antigenic properties of avian hepatitis E virus capsid protein.

    PubMed

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail. PMID:26340899

  10. CapsidMaps: Protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps

    PubMed Central

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L.; Reddy, Vijay

    2016-01-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu. PMID:25697908

  11. Gadolinium-153 as a brachytherapy isotope

    NASA Astrophysics Data System (ADS)

    Enger, Shirin A.; Fisher, Darrell R.; Flynn, Ryan T.

    2013-02-01

    The purpose of this work was to present the fundamental dosimetric characteristics of a hypothetical 153Gd brachytherapy source using the AAPM TG-43U1 dose-calculation formalism. Gadolinium-153 is an intermediate-energy isotope that emits 40-100 keV photons with a half-life of 242 days. The rationale for considering 153Gd as a brachytherapy source is for its potential of patient specific shielding and to enable reduced personnel shielding requirements relative to 192Ir, and as an isotope for interstitial rotating shield brachytherapy (I-RSBT). A hypothetical 153Gd brachytherapy source with an active core of 0.84 mm diameter, 10 mm length and specific activity of 5.55 TBq of 153Gd per gram of Gd was simulated with Geant4. The encapsulation material was stainless steel with a thickness of 0.08 mm. The radial dose function, anisotropy function and photon spectrum in water were calculated for the 153Gd source. The simulated 153Gd source had an activity of 242 GBq and a dose rate in water 1 cm off axis of 13.12 Gy h-1, indicating that it would be suitable as a low-dose-rate or pulsed-dose-rate brachytherapy source. The beta particles emitted have low enough energies to be absorbed in the source encapsulation. Gadolinium-153 has an increasing radial dose function due to multiple scatter of low-energy photons. Scattered photon dose takes over with distance from the source and contributes to the majority of the absorbed dose. The anisotropy function of the 153Gd source decreases at low polar angles, as a result of the long active core. The source is less anisotropic at polar angles away from the longitudinal axes. The anisotropy function increases with increasing distance. The 153Gd source considered would be suitable as an intermediate-energy low-dose-rate or pulsed-dose-rate brachytherapy source. The source could provide a means for I-RSBT delivery and enable brachytherapy treatments with patient specific shielding and reduced personnel shielding requirements relative to

  12. Gadolinium-153 as a brachytherapy isotope.

    PubMed

    Enger, Shirin A; Fisher, Darrell R; Flynn, Ryan T

    2013-02-21

    The purpose of this work was to present the fundamental dosimetric characteristics of a hypothetical (153)Gd brachytherapy source using the AAPM TG-43U1 dose-calculation formalism. Gadolinium-153 is an intermediate-energy isotope that emits 40-100 keV photons with a half-life of 242 days. The rationale for considering (153)Gd as a brachytherapy source is for its potential of patient specific shielding and to enable reduced personnel shielding requirements relative to (192)Ir, and as an isotope for interstitial rotating shield brachytherapy (I-RSBT). A hypothetical (153)Gd brachytherapy source with an active core of 0.84 mm diameter, 10 mm length and specific activity of 5.55 TBq of (153)Gd per gram of Gd was simulated with Geant4. The encapsulation material was stainless steel with a thickness of 0.08 mm. The radial dose function, anisotropy function and photon spectrum in water were calculated for the (153)Gd source. The simulated (153)Gd source had an activity of 242 GBq and a dose rate in water 1 cm off axis of 13.12 Gy h(-1), indicating that it would be suitable as a low-dose-rate or pulsed-dose-rate brachytherapy source. The beta particles emitted have low enough energies to be absorbed in the source encapsulation. Gadolinium-153 has an increasing radial dose function due to multiple scatter of low-energy photons. Scattered photon dose takes over with distance from the source and contributes to the majority of the absorbed dose. The anisotropy function of the (153)Gd source decreases at low polar angles, as a result of the long active core. The source is less anisotropic at polar angles away from the longitudinal axes. The anisotropy function increases with increasing distance. The (153)Gd source considered would be suitable as an intermediate-energy low-dose-rate or pulsed-dose-rate brachytherapy source. The source could provide a means for I-RSBT delivery and enable brachytherapy treatments with patient specific shielding and reduced personnel

  13. Nucleoporin 153 Arrests the Nuclear Import of Hepatitis B Virus Capsids in the Nuclear Basket

    PubMed Central

    Foss, Michael; Zhou, Lixin; Rabe, Birgit; Hoellenriegel, Julia; Stoeber, Miriam; Panté, Nelly; Kann, Michael

    2010-01-01

    Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin β (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin β. The binding sites of importin β and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin β. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos. PMID:20126445

  14. Studies of narrow autoionizing resonances in gadolinium

    SciTech Connect

    Bushaw, Bruce A.; Nortershauser, W.; Blaum, K.; Wendt, Klaus

    2003-06-30

    The autoionization (AI) spectrum of gadolinium between the first and second limits has been investigated by triple-resonance excitation with high-resolution cw lasers. A large number of narrow AI resonances have been observed and assigned total angular momentum J values. The resonances are further divided into members of AI Rydberg series converging to the second limit or other ''interloping'' levels. Fine structure in the Rydberg series has been identified and interpreted in terms of Jc j coupling. A number of detailed studies have been performed on the interloping resonances: These include lifetime determination by lineshape analysis, isotope shifts, hyperfine structure, and photoionization saturation parameters. The electronic structure of the interloping levels is discussed in terms of these studies. Linewidths generally decrease with increasing total angular momentum and the J = 7 resonances are extremely narrow with Lorentzian widths ranging from < 1 MHz up to 157 MHz. The strongest resonances are found to have cross-sections of {approx}10-12 cm{sup 2} and photoionization can be saturated with powers available from cw diode lasers.

  15. Distribution profile of gadolinium in gadolinium chelate-treated renally-impaired rats: role of pharmaceutical formulation.

    PubMed

    Fretellier, Nathalie; Salhi, Mariem; Schroeder, Josef; Siegmund, Heiko; Chevalier, Thibaut; Bruneval, Patrick; Jestin-Mayer, Gaëlle; Delaloge, Francette; Factor, Cécile; Mayer, Jean-François; Fabicki, Jean-Michel; Robic, Caroline; Bonnemain, Bruno; Idée, Jean-Marc; Corot, Claire

    2015-05-25

    While not acutely toxic, chronic hepatic effect of certain gadolinium chelates (GC), used as contrast agent for magnetic resonance imaging, might represent a risk in renally-impaired patients due to free gadolinium accumulation in the liver. To answer this question, this study investigated the consequences of the presence of small amounts of either a soluble gadolinium salt ("free" Gd) or low-stability chelating impurity in the pharmaceutical solution of gadoteric acid, a macrocyclic GC with high thermodynamic and kinetic stabilities, were investigated in renally-impaired rats. Renal failure was induced by adding 0.75% adenine in the diet for three weeks. The pharmaceutical and commercial solution of gadoteric acid was administered (5 daily intravenous injections of 2.5 mmol Gd/kg) either alone or after being spiked with either "free" gadolinium (i.e., 0.04% w/v) or low-stability impurity (i.e., 0.06 w/v). Another GC, gadodiamide (low thermodynamic and kinetic stabilities) was given as its commercial solution at a similar dose. Non-chelated gadolinium was tested at two doses (0.005 and 0.01 mmol Gd/kg) as acetate salt. Gadodiamide induced systemic toxicity (mortality, severe epidermal and dermal lesions) and substantial tissue Gd retention. The addition of very low amounts of "free", non-chelated gadolinium or low thermodynamic stability impurity to the pharmaceutical solution of the thermodynamically stable GC gadoteric acid resulted in substantial capture of metal by the liver, similar to what was observed in "free" gadolinium salt-treated rats. Relaxometry studies strongly suggested the presence of free and soluble gadolinium in the liver. Electron microscopy examinations revealed the presence of free and insoluble gadolinium deposits in hepatocytes and Kupffer cells of rats treated with gadoteric acid solution spiked with low-stability impurity, free gadolinium and gadodiamide, but not in rats treated with the pharmaceutical solution of gadoteric acid. The

  16. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    SciTech Connect

    Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

    2011-03-01

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting {beta}-hairpin observed in other polyomaviruses. We postulate that the terminal {beta}-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  17. Determination of Viral Capsid Elastic Properties from Equilibrium Thermal Fluctuations

    NASA Astrophysics Data System (ADS)

    May, Eric R.; Brooks, Charles L., III

    2011-05-01

    We apply two-dimensional elasticity theory to viral capsids to develop a framework for calculating elastic properties of viruses from equilibrium thermal fluctuations of the capsid surface in molecular dynamics and elastic network model trajectories. We show that the magnitudes of the long wavelength modes of motion available in a simulation with all atomic degrees of freedom are recapitulated by an elastic network model. For the mode spectra to match, the elastic network model must be scaled appropriately by a factor which can be determined from an icosahedrally constrained all-atom simulation. With this method we calculate the two-dimensional Young’s modulus Y, bending modulus κ, and Föppl-von Kármán number γ, for the T=1 mutant of the Sesbania mosaic virus. The values determined are in the range of previous theoretical estimates.

  18. Interrogating viral capsid assembly with ion mobility-mass spectrometry

    NASA Astrophysics Data System (ADS)

    Uetrecht, Charlotte; Barbu, Ioana M.; Shoemaker, Glen K.; van Duijn, Esther; Heck, Albert J. R.

    2011-02-01

    Most proteins fulfil their function as part of large protein complexes. Surprisingly, little is known about the pathways and regulation of protein assembly. Several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virion and thus resemble ideal model systems for studying protein complex formation. Even for these systems, the mechanism for self-assembly is still poorly understood, although it is generally thought that smaller oligomeric structures form key intermediates. This assembly nucleus and larger viral assembly intermediates are typically low abundant and difficult to monitor. Here, we characterised small oligomers of Hepatitis B virus (HBV) and norovirus under equilibrium conditions using native ion mobility mass spectrometry. This data in conjunction with computational modelling enabled us to elucidate structural features of these oligomers. Instead of more globular shapes, the intermediates exhibit sheet-like structures suggesting that they are assembly competent. We propose pathways for the formation of both capsids.

  19. Useful scars: Physics of the capsids of archaeal viruses.

    PubMed

    Perotti, L E; Dharmavaram, S; Klug, W S; Marian, J; Rudnick, J; Bruinsma, R F

    2016-07-01

    We propose a physical model for the capsids of tailed archaeal viruses as viscoelastic membranes under tension. The fluidity is generated by thermal motion of scarlike structures that are an intrinsic feature of the ground state of large particle arrays covering surfaces with nonzero Gauss curvature. The tension is generated by a combination of the osmotic pressure of the enclosed genome and an extension force generated by filamentous structure formation that drives the formation of the tails. In continuum theory, the capsid has the shape of a surface of constant mean curvature: an unduloid. Particle arrays covering unduloids are shown to exhibit pronounced subdiffusive and diffusive single-particle transport at temperatures that are well below the melting temperature of defect-free particle arrays on a surface with zero Gauss curvature. PMID:27575161

  20. Useful scars: Physics of the capsids of archaeal viruses

    NASA Astrophysics Data System (ADS)

    Perotti, L. E.; Dharmavaram, S.; Klug, W. S.; Marian, J.; Rudnick, J.; Bruinsma, R. F.

    2016-07-01

    We propose a physical model for the capsids of tailed archaeal viruses as viscoelastic membranes under tension. The fluidity is generated by thermal motion of scarlike structures that are an intrinsic feature of the ground state of large particle arrays covering surfaces with nonzero Gauss curvature. The tension is generated by a combination of the osmotic pressure of the enclosed genome and an extension force generated by filamentous structure formation that drives the formation of the tails. In continuum theory, the capsid has the shape of a surface of constant mean curvature: an unduloid. Particle arrays covering unduloids are shown to exhibit pronounced subdiffusive and diffusive single-particle transport at temperatures that are well below the melting temperature of defect-free particle arrays on a surface with zero Gauss curvature.

  1. Production of porcine parvovirus empty capsids with high immunogenic activity.

    PubMed

    Martínez, C; Dalsgaard, K; López de Turiso, J A; Cortés, E; Vela, C; Casal, J I

    1992-01-01

    The VP2 gene of porcine parvovirus was cloned in the baculovirus system and expressed in insect cells. The resulting product was present in high yield. It self-assembled into particles which were structurally and antigenically indistinguishable from regular PPV capsids. A high degree of purity of the recombinant capsids was obtained by ammonium sulphate precipitation of cell lysates. These virus-like particles were used as antigen in the immunization of two pigs. The pigs elicited an immune response which, when assayed by standard serological techniques, was identical to that of a commercial vaccine. The amount of recombinant antigen needed in a vaccine dose was only 3 micrograms in a primary dose and 1.5 micrograms in the booster. PMID:1523879

  2. Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies

    PubMed Central

    Wang, Wen-Hung; Kuo, Chung-Wen; Chang, Li-Kwan; Hung, Chen-Chia; Chang, Tzu-Hsuan

    2015-01-01

    ABSTRACT The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA

  3. Chemical Reactivity of Brome Mosaic Virus Capsid Protein

    PubMed Central

    Running, W. E.; Ni, P.; Kao, C. C.; Reilly, J. P.

    2012-01-01

    Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine resides with S-methylthioacetimidate (SMTA) across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of Brome Mosaic Virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from six to twelve, correlating well with the known pH dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein’s lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range. PMID:22750573

  4. Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

    SciTech Connect

    Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.; Wills, John W.; Courtney, Richard J. . E-mail: rcourtney@psu.edu

    2007-05-10

    The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.

  5. PARTITIONING OF GADOLINIUM IN THE CHEMICAL PROCESSING CELL

    SciTech Connect

    Reboul, S.; Best, D.; Stone, M.; Click, D.

    2011-04-27

    A combination of short-term beaker tests and longer-duration Sludge Receipt and Adjustment Tank (SRAT) simulations were performed to investigate the relative partitioning behaviors of gadolinium and iron under conditions applicable to the Chemical Processing Cell (CPC). The testing was performed utilizing non-radioactive simple Fe-Gd slurries, non-radioactive Sludge Batch 6 simulant slurries, and a radioactive real-waste slurry representative of Sludge Batch 7 material. The testing focused on the following range of conditions: (a) Fe:Gd ratios of 25-100; (b) pH values of 2-6; (c) acidification via addition of nitric, formic, and glycolic acids; (d) temperatures of {approx}93 C and {approx}22 C; and (e) oxalate concentrations of <100 mg/kg and {approx}10,000 mg/kg. The purpose of the testing was to provide data for assessing the potential use of gadolinium as a supplemental neutron poison when dispositioning excess plutonium. Understanding of the partitioning behavior of gadolinium in the CPC was the first step in assessing gadolinium's potential applicability. Significant fractions of gadolinium partitioned to the liquid-phase at pH values of 4.0 and below, regardless of the Fe:Gd ratio. In SRAT simulations targeting nitric and formic acid additions of 150% acid stoichiometry, the pH dropped to a minimum of 3.5-4.0, and the maximum fractions of gadolinium and iron partitioning to solution were both {approx}20%. In contrast, in a SRAT simulation utilizing a nitric and formic acid addition under atypical conditions (due to an anomalously low insoluble solids content), the pH dropped to a minimum of 3.7, and the maximum fractions of gadolinium and iron partitioning to solution were {approx}60% and {approx}70%, respectively. When glycolic acid was used in combination with nitric and formic acids at 100% acid stoichiometry, the pH dropped to a minimum of 3.6-4.0, and the maximum fractions of gadolinium and iron partitioning to solution were 60-80% and 3-5%, respectively

  6. Nanoindentation of virus capsids in a molecular model

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Robbins, Mark O.

    2010-01-01

    A molecular-level model is used to study the mechanical response of empty cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV) capsids. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the Cα atoms. Nanoindentation by a large tip is modeled as compression between parallel plates. Plots of the compressive force versus plate separation for CCMV are qualitatively consistent with continuum models and experiments, showing an elastic region followed by an irreversible drop in force. The mechanical response of CPMV has not been studied, but the molecular model predicts an order of magnitude higher stiffness and a much shorter elastic region than for CCMV. These large changes result from small structural changes that increase the number of bonds by only 30% and would be difficult to capture in continuum models. Direct comparison of local deformations in continuum and molecular models of CCMV shows that the molecular model undergoes a gradual symmetry breaking rotation and accommodates more strain near the walls than the continuum model. The irreversible drop in force at small separations is associated with rupturing nearly all of the bonds between capsid proteins in the molecular model, while a buckling transition is observed in continuum models.

  7. Nanoindentation of virus capsids in a molecular model.

    PubMed

    Cieplak, Marek; Robbins, Mark O

    2010-01-01

    A molecular-level model is used to study the mechanical response of empty cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV) capsids. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the C(alpha) atoms. Nanoindentation by a large tip is modeled as compression between parallel plates. Plots of the compressive force versus plate separation for CCMV are qualitatively consistent with continuum models and experiments, showing an elastic region followed by an irreversible drop in force. The mechanical response of CPMV has not been studied, but the molecular model predicts an order of magnitude higher stiffness and a much shorter elastic region than for CCMV. These large changes result from small structural changes that increase the number of bonds by only 30% and would be difficult to capture in continuum models. Direct comparison of local deformations in continuum and molecular models of CCMV shows that the molecular model undergoes a gradual symmetry breaking rotation and accommodates more strain near the walls than the continuum model. The irreversible drop in force at small separations is associated with rupturing nearly all of the bonds between capsid proteins in the molecular model, while a buckling transition is observed in continuum models. PMID:20078182

  8. Assembly of Recombinant Israeli Acute Paralysis Virus Capsids

    PubMed Central

    Ren, Junyuan; Cone, Abigail; Willmot, Rebecca; Jones, Ian M.

    2014-01-01

    The dicistrovirus Israeli Acute Paralysis Virus (IAPV) has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss. PMID:25153716

  9. Hierarchical multi-step organization during viral capsid assembly.

    PubMed

    Lampel, Ayala; Varenik, Maxim; Regev, Oren; Gazit, Ehud

    2015-12-01

    Formation of the HIV-1 core by the association of capsid proteins is a critical, not fully understood, step in the viral life cycle. Understanding the early stages of the mechanism may improve treatment opportunities. Here, spectroscopic analysis (opacity) is used to follow the kinetics of capsid protein assembly, which shows three stages: a lag phase, followed by a linear increase stage and terminated by a plateau. Adding pre-incubated capsid proteins at the start of the lag phase shortens it and increases the rate of assembly at the linear stage, demonstrating autoacceleration and cooperative assembly. Cryogenic transmission electron microscopy is used to probe structural evolution at these three stages. At the beginning of the lag phase, short tubular assemblies are found alongside micron long tubes. Their elongation continues all throughout the lag phase, at the end of which tubes start to assemble into bundles. Based on these results, we suggest a multi-step self-assembly process including fast nucleation and elongation followed by tubes packing into arrays. PMID:26497114

  10. Gadolinium-Based Contrast Agent Accumulation and Toxicity: An Update.

    PubMed

    Ramalho, J; Semelka, R C; Ramalho, M; Nunes, R H; AlObaidy, M; Castillo, M

    2016-07-01

    In current practice, gadolinium-based contrast agents have been considered safe when used at clinically recommended doses in patients without severe renal insufficiency. The causal relationship between gadolinium-based contrast agents and nephrogenic systemic fibrosis in patients with renal insufficiency resulted in new policies regarding the administration of these agents. After an effective screening of patients with renal disease by performing either unenhanced or reduced-dose-enhanced studies in these patients and by using the most stable contrast agents, nephrogenic systemic fibrosis has been largely eliminated since 2009. Evidence of in vivo gadolinium deposition in bone tissue in patients with normal renal function is well-established, but recent literature showing that gadolinium might also deposit in the brain in patients with intact blood-brain barriers caught many individuals in the imaging community by surprise. The purpose of this review was to summarize the literature on gadolinium-based contrast agents, tying together information on agent stability and animal and human studies, and to emphasize that low-stability agents are the ones most often associated with brain deposition. PMID:26659341

  11. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    NASA Technical Reports Server (NTRS)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  12. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly.

    PubMed Central

    Rodgers, R E; Chang, D; Cai, X; Consigli, R A

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents. Images PMID:8151798

  13. The fate of HIV-1 capsid: a biochemical assay for HIV-1 uncoating.

    PubMed

    Yang, Yang; Luban, Jeremy; Diaz-Griffero, Felipe

    2014-01-01

    The uncoating process of HIV-1 is a poorly understood process, so the development of a reliable assay to study uncoating is critical for moving the field forward. Here we describe an uncoating assay that currently represents the state-of-the-art biochemical procedure for monitoring uncoating and core stability during infection. This assay is based on the biochemical separation of soluble capsid protein from particulate capsid cores and provides information about the fate of the capsid during infection. PMID:24158811

  14. Gadolinium metallo nanocongregates as potential magnetosensors for detecting early stage cancers

    SciTech Connect

    Dutta, Ranu; Pandey, Avinash C.

    2015-04-27

    Gadolinium chelates and gadolinium based inorganic nanoparticles have been extensively studied, because of the high magnetic moment of gadolinium. Here, metallic gadolinium nanocongregates have been developed. Upon injecting these nanoparticles in the mice, they initially circulate in the blood stream and are localized at the cancer site, which could be visualized upon application of magnetic field hence acting as small magnetic nanosensors searching for even small cancers, detecting cancers at a very early stage.

  15. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids.

    PubMed

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. PMID:27253068

  16. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    PubMed Central

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  17. Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus Results in the Formation of Multiple Capsid Species: Isolation and Molecular Characterization of A, B, and C Capsids from a Gammaherpesvirus

    PubMed Central

    Nealon, K.; Newcomb, W. W.; Pray, T. R.; Craik, C. S.; Brown, J. C.; Kedes, D. H.

    2001-01-01

    Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome. PMID:11222712

  18. The tripartite capsid gene of Salmonella phage Gifsy-2 yields a capsid assembly pathway engaging features from HK97 and {lambda}

    SciTech Connect

    Effantin, Gregory; Figueroa-Bossi, Nara; Schoehn, Guy; Bossi, Lionello; Conway, James F.

    2010-07-05

    Phage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a {approx} 200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold-capsid fusion) and {lambda} (protease-scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12 A. The capsids have lambdoid T = 7 geometry and are well modeled with the atomic structures of HK97 mcp and phage {lambda} gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and {lambda}.

  19. Growth of bulk gadolinium pyrosilicate single crystals for scintillators

    NASA Astrophysics Data System (ADS)

    Gerasymov, I.; Sidletskiy, O.; Neicheva, S.; Grinyov, B.; Baumer, V.; Galenin, E.; Katrunov, K.; Tkachenko, S.; Voloshina, O.; Zhukov, A.

    2011-03-01

    Ce, Pr, and La-doped gadolinium pyrosilicate Gd2Si2O7 (GPS) single crystals were grown by the Czochralski and Top Seeded Solution Growth (TSSG) techniques for the first time. Formation conditions of different pyrosilicate phases were determined. X-ray luminescence integral intensity of Ce-doped GPS is about one order of magnitude higher in comparison with gadolinium oxyorthosilicate Gd2SiO5:Ce (GSO:Ce). All samples demonstrate temperature stability of luminescence yield up to 400 K.

  20. Production, Purification, and Capsid Stability of Rhinovirus C Types

    PubMed Central

    Griggs, Theodor F.; Bochkov, Yury A.; Nakagome, Kazuyuki; Palmenberg, Ann C.; Gern, James E.

    2015-01-01

    The Rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher-yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification. PMID:25724434

  1. Discovery of samarium, europium, gadolinium, and terbium isotopes

    SciTech Connect

    May, E.; Thoennessen, M.

    2013-01-15

    Currently, thirty-four samarium, thirty-four europium, thirty-one gadolinium, and thirty-one terbium isotopes have been observed and the discovery of these isotopes is described here. For each isotope a brief synopsis of the first refereed publication, including the production and identification method, is presented.

  2. Development of gadolinium based nanoparticles having an affinity towards melanin

    NASA Astrophysics Data System (ADS)

    Morlieras, Jessica; Chezal, Jean-Michel; Miot-Noirault, Elisabeth; Roux, Amandine; Heinrich-Balard, Laurence; Cohen, Richard; Tarrit, Sébastien; Truillet, Charles; Mignot, Anna; Hachani, Roxanne; Kryza, David; Antoine, Rodolphe; Dugourd, Philippe; Perriat, Pascal; Janier, Marc; Sancey, Lucie; Lux, François; Tillement, Olivier

    2013-01-01

    Small Rigid Platforms (SRPs) are sub-5 nanometre gadolinium based nanoparticles that have been developed for multimodal imaging and theranostic applications. They are composed of a polysiloxane network surrounded by gadolinium chelates. A covalent coupling with quinoxaline derivatives has been performed. Such derivatives have proven their affinity for melanin frequently expressed in primary melanoma cases. Three different quinoxaline derivatives have been synthesised and coupled to the nanoparticles. The affinity of the grafted nanoparticles for melanin has then been shown in vitro by surface plasmon resonance on a homemade melanin grafted gold chip.Small Rigid Platforms (SRPs) are sub-5 nanometre gadolinium based nanoparticles that have been developed for multimodal imaging and theranostic applications. They are composed of a polysiloxane network surrounded by gadolinium chelates. A covalent coupling with quinoxaline derivatives has been performed. Such derivatives have proven their affinity for melanin frequently expressed in primary melanoma cases. Three different quinoxaline derivatives have been synthesised and coupled to the nanoparticles. The affinity of the grafted nanoparticles for melanin has then been shown in vitro by surface plasmon resonance on a homemade melanin grafted gold chip. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33457g

  3. Purification of cerium, neodymium and gadolinium for low background experiments

    NASA Astrophysics Data System (ADS)

    Boiko, R. S.; Barabash, A. S.; Belli, P.; Bernabei, R.; Cappella, F.; Cerulli, R.; Danevich, F. A.; Incicchitti, A.; Laubenstein, M.; Mokina, V. M.; Nisi, S.; Poda, D. V.; Polischuk, O. G.; Tretyak, V. I.

    2014-01-01

    Cerium, neodymium and gadolinium contain double beta active isotopes. The most interesting are 150Nd and 160Gd (promising for 0ν2β search), 136Ce (2β+ candidate with one of the highest Q2β). The main problem of compounds containing lanthanide elements is their high radioactive contamination by uranium, radium, actinium and thorium. The new generation 2β experiments require development of methods for a deep purification of lanthanides from the radioactive elements. A combination of physical and chemical methods was applied to purify cerium, neodymium and gadolinium. Liquid-liquid extraction technique was used to remove traces of Th and U from neodymium, gadolinium and for purification of cerium from Th, U, Ra and K. Co-precipitation and recrystallization methods were utilized for further reduction of the impurities. The radioactive contamination of the samples before and after the purification was tested by using ultra-low-background HPGe gamma spectrometry. As a result of the purification procedure the radioactive contamination of gadolinium oxide (a similar purification efficiency was reached also with cerium and neodymium oxides) was decreased from 0.12 Bq/kg to 0.007 Bq/kg in 228Th, from 0.04 Bq/kg to <0.006 Bq/kg in 226Ra, and from 0.9 Bq/kg to 0.04 Bq/kg in 40K. The purification methods are much less efficient for chemically very similar radioactive elements like actinium, lanthanum and lutetium.

  4. Dynamic and Kinetic Assembly Studies of an Icosahedral Virus Capsid

    NASA Astrophysics Data System (ADS)

    Lee, Kelly

    2011-03-01

    Hepatitis B virus has an icosahedrally symmetrical core particle (capsid), composed of either 90 or 120 copies of a dimeric protein building block. We are using time-resolved, solution small-angle X-ray scattering and single-molecule fluorescence microscopy to probe the core particle assembly reaction at the ensemble and individual assembly levels. Our experiments to date reveal the assembly process to be highly cooperative with minimal population of stable intermediate species. Solution conditions, particularly salt concentration, appears to influence the partitioning of assembly products into the two sizes of shells. Funding from NIH R00-GM080352 and University of Washington.

  5. Structural and magnetic phase transitions in gadolinium under high pressures and low temperatures

    SciTech Connect

    Samudrala, Gopi K.; Tsoi, Georgiy M.; Weir, Samuel T.; Vohra, Yogesh K.

    2014-11-07

    High pressure structural transition studies have been carried out on rare earth metal gadolinium in a diamond anvil cell at room temperature to 169 GPa. Gadolinium has been compressed to 38% of its initial volume at this pressure. With increasing pressure, a crystal structure sequence of hcp → Smtype→ dhcp → fcc → dfcc → monoclinic has been observed in our studies on gadolinium. The measured equation of state of gadolinium is presented to 169 GPa at ambient temperature. Magnetic ordering temperature of gadolinium has been studied using designer diamond anvils to a pressure of 25 GP and a temperature of 10 K. The magnetic ordering temperature has been determined from the four-point electrical resistivity measurements carried out on gadolinium. Furthermore, our experiments show that the magnetic transition temperature decreases with increasing pressure to 19 GPa and then increases when gadolinium is subjected to higher pressures.

  6. Structural and magnetic phase transitions in gadolinium under high pressures and low temperatures

    DOE PAGESBeta

    Samudrala, Gopi K.; Tsoi, Georgiy M.; Weir, Samuel T.; Vohra, Yogesh K.

    2014-11-07

    High pressure structural transition studies have been carried out on rare earth metal gadolinium in a diamond anvil cell at room temperature to 169 GPa. Gadolinium has been compressed to 38% of its initial volume at this pressure. With increasing pressure, a crystal structure sequence of hcp → Smtype→ dhcp → fcc → dfcc → monoclinic has been observed in our studies on gadolinium. The measured equation of state of gadolinium is presented to 169 GPa at ambient temperature. Magnetic ordering temperature of gadolinium has been studied using designer diamond anvils to a pressure of 25 GP and a temperaturemore » of 10 K. The magnetic ordering temperature has been determined from the four-point electrical resistivity measurements carried out on gadolinium. Furthermore, our experiments show that the magnetic transition temperature decreases with increasing pressure to 19 GPa and then increases when gadolinium is subjected to higher pressures.« less

  7. All-atom Multiscale Simulation of Cowpea Chlorotic Mottle Virus Capsid Swelling

    PubMed Central

    Miao, Yinglong; Johnson, John E.; Ortoleva, Peter J.

    2010-01-01

    An all-atom multiscale computational modeling approach, Molecular Dynamics/Order Parameter eXtrapolation (MD/OPX), has recently been developed for simulating large bionanosystems. It accelerates MD simulations and addresses rapid atomistic fluctuations and slowly-varying nanoscale dynamics of bionanosystems simultaneously. With modules added to account for water molecules and ions, MD/OPX is applied to simulate the swelling of cowpea chlorotic mottle virus (CCMV) capsid solvated in a host medium in this study. Simulation results show that the N-terminal arms of capsid proteins undergo large deviations from the initial configurations with their length extended quickly during the early stage of capsid swelling. The capsid swelling is a symmetry-breaking process involving local initiation and front propagation. The capsid swelling rate is ~0.25 nm/ns (npn) during early stage of the simulation and propagation of the structural transition across the capsid is roughly 0.6npn. The system conditions that affect swelling of the capsid are analyzed. Prospects for creating a phase diagram for CCMV capsid swelling and using predictions to guide experiments are discussed. PMID:20695471

  8. Viral capsids: Kinetics of assembly under transient conditions and kinetics of disassembly

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2014-10-01

    The available kinetic models of assembly of viral protein capsids are focused primarily on the situations in vitro where the amount of protein is fixed. In vivo, however, the viral protein synthesis and capsid assembly occur under transient conditions in parallel with viral genome replication. Herein, a kinetic model describing the latter case of capsid assembly is proposed with emphasis on the period corresponding to the initial stage of viral genome replication. The analysis is aimed at small icosahedral capsids. With biologically reasonable values of model parameters, the model predicts rapid exponential growth of the populations of monomers and fully assembled capsids during the transient period of genome replication. Under the subsequent steady-state conditions with respect to replication, the monomer population is predicted to be nearly constant while the number of fully assembled capsids increases linearly. The kinetics of capsid disassembly, described briefly as well under conditions of negligible monomer concentration, exhibit a short induction period when the number of proteins in a capsid is only slightly smaller than in the beginning, followed by more rapid protein detachment. According to calculations, the latter kinetics may strongly depend on protein degradation.

  9. Analysis of the protein-protein interactions in the parvovirus H-1 capsid.

    PubMed Central

    Paradiso, P R

    1983-01-01

    The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell. Images PMID:6827655

  10. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    SciTech Connect

    Lebrun, Marielle; Thelen, Nicolas; Thiry, Marc; Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia; Di Valentin, Emmanuel; Bontems, Sébastien; Sadzot-Delvaux, Catherine

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  11. Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26

    PubMed Central

    Döhner, Katinka; Radtke, Kerstin; Schmidt, Simone; Sodeik, Beate

    2006-01-01

    Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin. PMID:16873277

  12. Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.

    PubMed

    Lad, Latesh; Clancy, Sheila; Koditek, David; Wong, Melanie H; Jin, Debi; Niedziela-Majka, Anita; Papalia, Giuseppe A; Hung, Magdeleine; Yant, Stephen; Somoza, John R; Hu, Eric; Chou, Chienhung; Tse, Winston; Halcomb, Randall; Sakowicz, Roman; Pagratis, Nikos

    2015-04-01

    HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function. PMID:25774576

  13. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery.

    PubMed

    Huet, Alexis; Makhov, Alexander M; Huffman, Jamie B; Vos, Matthijn; Homa, Fred L; Conway, James F

    2016-06-01

    The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all of which are essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large functional complexes. Here we report two high-quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7-Å resolution. From these, we developed a complete model of subunit and domain organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface, thereby initiating nuclear egress. Differences in the folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions. PMID:27111889

  14. Mutational analysis of the capsid protein of Leishmania RNA virus LRV1-4.

    PubMed Central

    Cadd, T L; MacBeth, K; Furlong, D; Patterson, J L

    1994-01-01

    The virion of Leishmania RNA virus is predicted to be composed of a 742-amino-acid major capsid protein and a small percentage of capsid-polymerase fusion molecules. Recently, the capsid protein alone was expressed and shown to spontaneously assemble into viruslike particles. Since the major structural protein of the virion shell self-assembles into viruslike particles when expressed in the baculovirus expression system, assembly of the virion can be studied by mutational analysis and expression of a single open reading frame. In this study, several deletions and one addition of the capsid protein of Leishmania RNA virus LRV1-4 were generated. These mutants show different degrees of assembly. Assembly domains are being identified such that the capsid protein may be used as a macromolecular packaging and delivery system for Leishmania species. Images PMID:7966563

  15. Binary alloy of virus capsids and gold nanoparticles as a Mie-resonance-based optical metamaterial

    NASA Astrophysics Data System (ADS)

    Yannopapas, Vassilios

    2015-02-01

    We present a metamaterial design based on a binary alloy of gold nanoparticles and virus capsids (protein nanoparticles) which possesses metamaterial functionalities in the optical regime. Such binary alloys have already been realized in the laboratory by means of DNA-programmed crystallization of metallic nanoparticles and virus capsids with suitable DNA linkers. The resulting binary alloy has a NaTl-lattice symmetry and operates as Mie resonance-based metamaterial thanks to the extremely high values of the electric permittivity of the virus capsids. By employing an effective-medium theory and rigorous electrodynamic calculations we identify regions of photo-induced magnetic activity stemming from the Mie resonances of the virus capsids. The magnetic activity of the virus particles accompanied by the ordinary electric activity of the gold nanoparticles results in spectral regions of negative refractive index which can be tuned to a desired spectral window by varying the concentration of the RNA within the virus capsids.

  16. The Role of Solution Conditions in the Bacteriophage PP7 Capsid Charge Regulation

    PubMed Central

    Nap, Rikkert J.; Božič, Anže Lošdorfer; Szleifer, Igal; Podgornik, Rudolf

    2014-01-01

    We investigate and quantify the effects of pH and salt concentration on the charge regulation of the bacteriophage PP7 capsid. These effects are found to be extremely important and substantial, introducing qualitative changes in the charge state of the capsid such as a transition from net-positive to net-negative charge depending on the solution pH. The overall charge of the virus capsid arises as a consequence of a complicated balance with the chemical dissociation equilibrium of the amino acids and the electrostatic interaction between them, and the translational entropy of the mobile solution ions, i.e., counterion release. We show that to properly describe and predict the charging equilibrium of viral capsids in general, one needs to include molecular details as exemplified by the acid-base equilibrium of the detailed distribution of amino acids in the proteinaceous capsid shell. PMID:25418178

  17. Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids.

    PubMed

    Ahadi, Aylin; Johansson, Dan; Evilevitch, Alex

    2013-03-01

    Viruses can be described as biological objects composed mainly of two parts: a stiff protein shell called a capsid, and a core inside the capsid containing the nucleic acid and liquid. In many double-stranded DNA bacterial viruses (aka phage), the volume ratio between the liquid and the encapsidated DNA is approximately 1:1. Due to the dominant DNA hydration force, water strongly mediates the interaction between the packaged DNA strands. Therefore, water that hydrates the DNA plays an important role in nanoindentation experiments of DNA-filled viral capsids. Nanoindentation measurements allow us to gain further insight into the nature of the hydration and electrostatic interactions between the DNA strands. With this motivation, a continuum-based numerical model for simulating the nanoindentation response of DNA-filled viral capsids is proposed here. The viral capsid is modeled as large- strain isotropic hyper-elastic material, whereas porous elasticity is adopted to capture the mechanical response of the filled viral capsid. The voids inside the viral capsid are assumed to be filled with liquid, which is modeled as a homogenous incompressible fluid. The motion of a fluid flowing through the porous medium upon capsid indentation is modeled using Darcy's law, describing the flow of fluid through a porous medium. The nanoindentation response is simulated using three-dimensional finite element analysis and the simulations are performed using the finite element code Abaqus. Force-indentation curves for empty, partially and completely DNA-filled capsids are directly compared to the experimental data for bacteriophage λ. Material parameters such as Young's modulus, shear modulus, and bulk modulus are determined by comparing computed force-indentation curves to the data from the atomic force microscopy (AFM) experiments. Predictions are made for pressure distribution inside the capsid, as well as the fluid volume ratio variation during the indentation test. PMID:23860868

  18. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    SciTech Connect

    Kim, Yoon Sik Seo, Hyun Wook Jung, Guhung

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  19. Linear Gadolinium-Based Contrast Agents Are Associated With Brain Gadolinium Retention in Healthy Rats

    PubMed Central

    Robert, Philippe; Violas, Xavier; Grand, Sylvie; Lehericy, Stéphane; Idée, Jean-Marc; Ballet, Sébastien; Corot, Claire

    2016-01-01

    Objectives The aim of this study was to evaluate Gd retention in the deep cerebellar nuclei (DCN) of linear gadolinium-based contrast agents (GBCAs) compared with a macrocyclic contrast agent. Materials and Methods The brain tissue retention of Gd of 3 linear GBCAs (gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide) and a macrocyclic GBCA (gadoterate meglumine) was compared in healthy rats (n = 8 per group) that received 20 intravenous injections of 0.6 mmol Gd/kg (4 injections per week for 5 weeks). An additional control group with saline was included. T1-weighted magnetic resonance imaging was performed before injection and once a week during the 5 weeks of injections and for another 4 additional weeks after contrast period. Total gadolinium concentration was measured with inductively coupled plasma mass spectrometry. Blinded qualitative and quantitative evaluations of the T1 signal intensity in DCN were performed, as well as a statistical analysis on quantitative data. Results At completion of the injection period, all the linear contrast agents (gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide) induced a significant increase in signal intensity in DCN, unlike the macrocyclic GBCA (gadoterate meglumine) or saline. The T1 hypersignal enhancement kinetic was fast for gadodiamide. Total Gd concentrations for the 3 linear GBCAs groups at week 10 were significantly higher in the cerebellum (1.21 ± 0.48, 1.67 ± 0.17, and 3.75 ± 0.18 nmol/g for gadobenate dimeglumine, gadopentetate dimeglumine, and gadodiamide, respectively) than with the gadoterate meglumine (0.27 ± 0.16 nmol/g, P < 0.05) and saline (0.09 ± 0.12 nmol/g, P < 0.05). No significant difference was observed between the macrocyclic agent and saline. Conclusions Repeated administrations of the linear GBCAs gadodiamide, gadobenate dimeglumine, and gadopentetate dimeglumine to healthy rats were associated with progressive and significant T1 signal hyperintensity in the

  20. Multivalent counterions inhibit DNA ejection from viral capsid

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan

    2008-03-01

    Viral DNA packaged inside a bacteriophage is tighly bent. This stored bending energy of DNA is believed to be the main driving force to eject viral DNA into host cell upon capsid binding. One can control the amount of ejected DNA by subjecting the virus to a solution of PEG8000 molecules. The molecules cannot penetrate the viral capsid, therefore, they exert an osmotic pressure on the virus preventing DNA ejection. Experiments showed that for a given osmotic pressure, the degree of ejection also depends on the concentration of small ions in solution. Interestingly, for multivalent ions (such as Mg2+, Spd3+ or HexCo3+), this dependence is non-monotonic. We propose a simple electrostatic theory to explain this non-monotonic behavior. This is based on the fact that DNA molecules can invert its net charge at high enough multivalent counterion concentration. In other words, as multivalent counterion concentration is increased from zero, charge of DNA molecules change from negative to positive. At the concentration where DNA net charge is zero, the DNA molecules experience an attraction between different segments and DNA ejected amount is reduced. At low or high counterion concentration, DNA segments are charged (negatively or positively), repel each other and DNA ejected amount is increased. Fitting the result of the theory to experimental data, we obtain a numerical value for Mg2+ mediated DNA - DNA attraction energy to be -0.008kT per base.

  1. Characterization of the in vitro HIV-1 capsid assembly pathway.

    PubMed

    Barklis, Eric; Alfadhli, Ayna; McQuaw, Carolyn; Yalamuri, Suraj; Still, Amelia; Barklis, Robin Lid; Kukull, Ben; López, Claudia S

    2009-03-27

    During the morphogenesis of mature human immunodeficiency virus-1 cores, viral capsid proteins assemble conical or tubular shells around viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which capsid proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period, followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle preexisting tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature human immunodeficiency virus-1 core assembly and avenues for antiviral inhibition. PMID:19356593

  2. Visualization of Bacteriophage T3 Capsids with DNA Incompletely Packaged In Vivo

    PubMed Central

    Fang, Ping-An; Wright, Elena T.; Weintraub, Susan T.; Hakala, Kevin; Wu, Weimin; Serwer, Philip; Jiang, Wen

    2009-01-01

    The tightly packaged dsDNA genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼10-4 of packaged DNA in all particles) and are initially detected by non-denaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy (cryo-EM) of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) forms a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings like those seen

  3. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    NASA Astrophysics Data System (ADS)

    Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

    2004-11-01

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  4. Role of dynamic capsomere supply for viral capsid self-assembly

    NASA Astrophysics Data System (ADS)

    Boettcher, Marvin A.; Klein, Heinrich C. R.; Schwarz, Ulrich S.

    2015-02-01

    Many viruses rely on the self-assembly of their capsids to protect and transport their genomic material. For many viral systems, in particular for human viruses like hepatitis B, adeno or human immunodeficiency virus, that lead to persistent infections, capsomeres are continuously produced in the cytoplasm of the host cell while completed capsids exit the cell for a new round of infection. Here we use coarse-grained Brownian dynamics simulations of a generic patchy particle model to elucidate the role of the dynamic supply of capsomeres for the reversible self-assembly of empty T1 icosahedral virus capsids. We find that for high rates of capsomere influx only a narrow range of bond strengths exists for which a steady state of continuous capsid production is possible. For bond strengths smaller and larger than this optimal value, the reaction volume becomes crowded by small and large intermediates, respectively. For lower rates of capsomere influx a broader range of bond strengths exists for which a steady state of continuous capsid production is established, although now the production rate of capsids is smaller. Thus our simulations suggest that the importance of an optimal bond strength for viral capsid assembly typical for in vitro conditions can be reduced by the dynamic influx of capsomeres in a cellular environment.

  5. Immobilization and One-Dimensional Arrangement of Virus Capsids with Nanoscale Precision Using DNA Origami

    SciTech Connect

    Stephanopoulos, Nicholas; Liu, Minghui; Tong, Gary J; Li, Zhe; Liu, Yan; Yan, Hao; Francis, Matthew B

    2010-06-24

    DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (~100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.

  6. Viral genome structures, charge, and sequences are optimal for capsid assembly

    NASA Astrophysics Data System (ADS)

    Hagan, Michael

    2014-03-01

    For many viruses, the spontaneous assembly of a capsid shell around the nu-cleic acid (NA) genome is an essential step in the viral life cycle. Capsid formation is a multicomponent, out-of-equilibrium assembly process for which kinetic effects and thermodynamic constraints compete to determine the outcome. Understand-ing how viral components drive highly efficient assembly under these constraints could promote biomedical efforts to block viral propagation, and would elucidate the factors controlling assembly in a wide range of systems containing proteins and polyelectrolytes. This talk will describe coarse-grained models of capsid proteins and NAs with which we investigate the dynamics and thermodynamics of virus assembly. In con-trast to recent theoretical models, we find that capsids spontaneously `overcharge' that is, the NA length which is kinetically and thermodynamically optimal possess-es a negative charge greater than the positive charge of the capsid. When applied to specific virus capsids, the calculated optimal NA lengths closely correspond to the natural viral genome lengths. These results suggest that the features included in this model (i.e. electrostatics, excluded volume, and NA tertiary structure) play key roles in determining assembly thermodynamics and consequently exert selec-tive pressure on viral evolution. I will then discuss mechanisms by which se-quence-specific interactions between NAs and capsid proteins promote selective encapsidation of the viral genome. This work was supported by NIH R01GM108021 and the Brandeis MRSEC NSF-MRSEC-0820492.

  7. Role of electrostatic interactions in the assembly of empty spherical viral capsids

    NASA Astrophysics Data System (ADS)

    Šiber, Antonio; Podgornik, Rudolf

    2007-12-01

    We examine the role of electrostatic interactions in the assembly of empty spherical viral capsids. The charges on the protein subunits that make the viral capsid mutually interact and are expected to yield electrostatic repulsion acting against the assembly of capsids. Thus, attractive protein-protein interactions of nonelectrostatic origin must act to enable the capsid formation. We investigate whether the interplay of repulsive electrostatic and attractive interactions between the protein subunits can result in the formation of spherical viral capsids of a preferred radius. For this to be the case, we find that the attractive interactions must depend on the angle between the neighboring protein subunits (i.e., on the mean curvature of the viral capsid) so that a particular angle(s) is (are) preferred energywise. Our results for the electrostatic contributions to energetics of viral capsids nicely correlate with recent experimental determinations of the energetics of protein-protein contacts in the hepatitis B virus [P. Ceres A. Zlotnick, Biochemistry 41, 11525 (2002)].

  8. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids

    PubMed Central

    Castle, Michael J.; Turunen, Heikki T.; Vandenberghe, Luk H.; Wolfe, John H.

    2016-01-01

    More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina. PMID:26611584

  9. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly.

    PubMed

    Kim, Yoon Sik; Seo, Hyun Wook; Jung, Guhung

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. PMID:25576869

  10. Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.

    PubMed

    Porta, Claudine; Kotecha, Abhay; Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M; Fry, Elizabeth E; Stuart, David I; Charleston, Bryan

    2013-03-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  11. Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

    PubMed Central

    York, Royce L.; Yousefi, Payam A.; Bogdanoff, Walter; Haile, Sara; Tripathi, Sarvind

    2015-01-01

    ABSTRACT Human astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies. IMPORTANCE Astroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus

  12. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

    PubMed

    Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

    2016-04-01

    The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. PMID:26810656

  13. Gadolinium-enhanced magnetic resonance angiography in brain death

    NASA Astrophysics Data System (ADS)

    Luchtmann, M.; Beuing, O.; Skalej, M.; Kohl, J.; Serowy, S.; Bernarding, J.; Firsching, R.

    2014-01-01

    Confirmatory tests for the diagnosis of brain death in addition to clinical findings may shorten observation time required in some countries and may add certainty to the diagnosis under specific circumstances. The practicability of Gadolinium-enhanced magnetic resonance angiography to confirm cerebral circulatory arrest was assessed after the diagnosis of brain death in 15 patients using a 1.5 Tesla MRI scanner. In all 15 patients extracranial blood flow distal to the external carotid arteries was undisturbed. In 14 patients no contrast medium was noted within intracerebral vessels above the proximal level of the intracerebral arteries. In one patient more distal segments of the anterior and middle cerebral arteries (A3 and M3) were filled with contrast medium. Gadolinium-enhanced MRA may be considered conclusive evidence of cerebral circulatory arrest, when major intracranial vessels fail to fill with contrast medium while extracranial vessels show normal blood flow.

  14. An organic electroluminescent device made from a gadolinium complex

    NASA Astrophysics Data System (ADS)

    Gao, D.-Q.; Huang, C.-H.; Ibrahim, K.; Liu, F.-Q.

    2002-01-01

    A gadolinium ternary complex, tris(1-phenyl-3-methyl-4-isobutyryl-5-pyrazolone) (phenanthroline) gadolinium [Gd(PMIP) 3(Phen)] was synthesized and used as a light emitting material in the organic electroluminescent (EL) devices. The triple layer device with a structure of indium tin oxide (ITO)/ N, N'-diphenyl- N, N'-bis(3-methylphenyl)-1,1'-biphenyl-4,4'-diamine (TPD) (20 nm)/Gd(PMIP) 3(Phen) (80 nm)/2, 9-dimethyl-4, 7-diphenyl-1, 10-phenanthroline (bathocuproine or BCP) (20 nm)/Mg: Ag(200 nm)/Ag(100 nm) exhibited green emission peaking at 535 nm. A maximum luminance of 230 cd/m 2 at 17 V and a peak power efficiency of 0.02 lm/w at 9 V were obtained.

  15. About a Gadolinium-doped Water Cherenkov LAGUNA Detector

    NASA Astrophysics Data System (ADS)

    Labarga, Luis

    2010-11-01

    Water Cherenkov (wC) detectors are extremely powerful apparatuses for scientific research. Nevertheless they lack of neutron tagging capabilities, which translates, mainly, into an inability to identify the anti-matter nature of the reacting incoming anti-neutrino particles. A solution was proposed by R. Beacon and M. Vagins back in 2004: by dissolving in the water a compound with nucleus with very large cross section for neutron capture like the Gadolinium, with a corresponding emission of photons of enough energy to be detected, they can tag thermal neutrons with an efficiency larger than 80%. In this talk we detail the technique and its implications in the measurement capabilities and, as well, the new backgrounds induced. We discuss the improvement on their physics program, also for the case of LAGUNA type detectors. We comment shortly the status of the pioneering R&D program of the Super-Kamiokande Collaboration towards dissolving a Gadolinium compound in its water.

  16. About a Gadolinium-doped Water Cherenkov LAGUNA Detector

    SciTech Connect

    Labarga, Luis

    2010-11-24

    Water Cherenkov (wC) detectors are extremely powerful apparatuses for scientific research. Nevertheless they lack of neutron tagging capabilities, which translates, mainly, into an inability to identify the anti-matter nature of the reacting incoming anti-neutrino particles. A solution was proposed by R. Beacon and M. Vagins back in 2004: by dissolving in the water a compound with nucleus with very large cross section for neutron capture like the Gadolinium, with a corresponding emission of photons of enough energy to be detected, they can tag thermal neutrons with an efficiency larger than 80%. In this talk we detail the technique and its implications in the measurement capabilities and, as well, the new backgrounds induced. We discuss the improvement on their physics program, also for the case of LAGUNA type detectors. We comment shortly the status of the pioneering R and D program of the Super-Kamiokande Collaboration towards dissolving a Gadolinium compound in its water.

  17. Solid-state gadolinium{endash}magnesium hydride optical switch

    SciTech Connect

    Armitage, R.; Rubin, M.; Richardson, T.; OBrien, N.; Chen, Y.

    1999-09-01

    The optical switching properties of gadolinium{endash}magnesium hydride have been demonstrated in a solid-state electrochromic device. With positive polarization of the hydride electrode, the visible reflectance approaches 35{percent} with virtually zero transmission, while with negative polarization, the visible transmission exceeds 25{percent} at 650 nm. The switching is reversible, with intermediate optical properties between the transparent and reflecting states. {copyright} {ital 1999 American Institute of Physics.}

  18. Magnons as a Bose-Einstein Condensate in Nanocrystalline Gadolinium

    SciTech Connect

    Kaul, S. N.; Mathew, S. P.

    2011-06-17

    The recent observation [S. P. Mathew et al., J. Phys. Conf. Ser. 200, 072047 (2010)] of the anomalous softening of spin-wave modes at low temperatures in nanocrystalline gadolinium is interpreted as a Bose-Einstein condensation (BEC) of magnons. A self-consistent calculation, based on the BEC picture, is shown to closely reproduce the observed temperature variations of magnetization and specific heat at constant magnetic fields.

  19. Growth and scintillation properties of gadolinium and yttrium orthovanadate crystals

    NASA Astrophysics Data System (ADS)

    Voloshina, O. V.; Baumer, V. N.; Bondar, V. G.; Kurtsev, D. A.; Gorbacheva, T. E.; Zenya, I. M.; Zhukov, A. V.; Sidletskiy, O. Ts.

    2012-02-01

    Aiming to explore the possibility of using the undoped rare-earth orthovanadates as scintillation materials, we developed the procedure for growth of gadolinium (GdVO 4) and yttrium (YVO 4) orthovanadate single crystals by Czochralski method, and determined the optimal conditions of their after-growth annealing. Optical, luminescent, and scintillation properties of YVO 4 and GdVO 4 were discussed versus known literature data. Scintillation characteristics of GdVO 4 were determined for the first time.

  20. Capsid-like Arrays in Crystals of Chimpanzee Adenovirus Hexon

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

    The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 Angstroms, b = 433.0 Angstroms, c = 159.3 Angstroms, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 Angstroms resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.

  1. Gadolinium nitride films deposited using a PEALD based process

    NASA Astrophysics Data System (ADS)

    Fang, Ziwen; Williams, Paul A.; Odedra, Rajesh; Jeon, Hyeongtag; Potter, Richard J.

    2012-01-01

    Gadolinium nitride films have been deposited on Si(100) using a plasma-enhanced ALD (PEALD) based process. The deposition was carried out using tris(methylcyclopentadienyl)gadolinium {Gd(MeCp) 3} and remote nitrogen plasma, separated by argon pulses. Films were deposited at temperatures between 150 and 300 °C and capped with tantalum nitride to prevent post-deposition oxidation. Film composition was initially assessed using EDX and selected samples were subsequently depth profiled using medium energy ion scattering (MEIS) or AES. X-ray diffraction appears to show that the films are effectively amorphous. Films deposited at 200 °C were found to have a Gd:N ratio close to 1:1 and a low oxygen incorporation (˜5%). Although the growth was affected by partial thermal decomposition of the Gd(MeCp) 3, it was still possible to obtain smooth (Ra.=˜0.7 nm) films with good thickness uniformity (97%). Less successful attempts to deposit gadolinium nitride using thermal ALD with ammonia or mono-methyl-hydrazine are also reported.

  2. Type of MRI contrast, tissue gadolinium, and fibrosis

    PubMed Central

    Do, Catherine; Barnes, Jeffrey L.; Tan, Chunyan

    2014-01-01

    It has been presupposed that the thermodynamic stability constant (Ktherm) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low-Ktherm gadodiamide with high-Ktherm gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low-Ktherm chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects. PMID:25100280

  3. Type of MRI contrast, tissue gadolinium, and fibrosis.

    PubMed

    Do, Catherine; Barnes, Jeffrey L; Tan, Chunyan; Wagner, Brent

    2014-10-01

    It has been presupposed that the thermodynamic stability constant (K(therm)) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low-K(therm) gadodiamide with high-K(therm) gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low-K(therm) chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects. PMID:25100280

  4. Characteristics of Gadolinium Oxide Nanoparticles Using Terahertz Spectroscopy (abstract)

    NASA Astrophysics Data System (ADS)

    Lee, Dongkyu; Maeng, Inhee; Oh, Seung Jae; Kim, Taekhoon; Cho, Byung Kyu; Lee, Kwangyeol; Son, Joo-Hiuk

    2009-04-01

    The penetration property of the terahertz electromagnetic (THz) wave is relevant to its use. We used the THz wave spectroscopy system which easily penetrates some materials that do not contain water, e.g., plastic and ceramics. The system has been developed for several purposes, including measuring the properties of semiconductors and bio-materials, and detecting plastic bombs and ceramic knives at airports. It is also used for medical imaging systems, such as magnetic resonance imaging (MRI), at some research institutes. It can show not only the difference in amplitude, but also the difference of the phase of each point of sample. MRI technology usually uses contrast agents to enhance the quality of the image. Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), made with a heavy metal ion, is commonly used as a clinical MRI contrast agent. Gadolinium oxide (Gd2O3) nanoparticle is a new contrast agent. It serves to equip the core of each particle with antibodies or ligands. It can freely circulate in blood vessels without amassing in the liver or lungs. This study shows the characteristics of gadolinium oxide nanoparticles to further advance terahertz medical imaging.

  5. Characteristics of Gadolinium Oxide Nanoparticles Using Terahertz Spectroscopy

    SciTech Connect

    Lee, Dongkyu; Maeng, Inhee; Son, Joo-Hiuk; Oh, Seung Jae; Kim, Taekhoon; Cho, Byung Kyu; Lee, Kwangyeol

    2009-04-19

    The penetration property of the terahertz electromagnetic (THz) wave is relevant to its use. We used the THz wave spectroscopy system which easily penetrates some materials that do not contain water, e.g., plastic and ceramics. The system has been developed for several purposes, including measuring the properties of semiconductors and bio-materials, and detecting plastic bombs and ceramic knives at airports. It is also used for medical imaging systems, such as magnetic resonance imaging (MRI), at some research institutes. It can show not only the difference in amplitude, but also the difference of the phase of each point of sample. MRI technology usually uses contrast agents to enhance the quality of the image. Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), made with a heavy metal ion, is commonly used as a clinical MRI contrast agent. Gadolinium oxide (Gd{sub 2}O{sub 3}) nanoparticle is a new contrast agent. It serves to equip the core of each particle with antibodies or ligands. It can freely circulate in blood vessels without amassing in the liver or lungs. This study shows the characteristics of gadolinium oxide nanoparticles to further advance terahertz medical imaging.

  6. Dynamic allostery governs cyclophilin A–HIV capsid interplay

    PubMed Central

    Lu, Manman; Hou, Guangjin; Zhang, Huilan; Suiter, Christopher L.; Ahn, Jinwoo; Byeon, In-Ja L.; Langmead, Christopher J.; Hung, Ivan; Gor'kov, Peter L.; Gan, Zhehong; Brey, William; Aiken, Christopher; Zhang, Peijun; Schulten, Klaus; Gronenborn, Angela M.; Polenova, Tatyana

    2015-01-01

    Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone 1H-15N and 1H-13C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence. PMID:26553990

  7. Bioinformatics analysis of the epitope regions for norovirus capsid protein

    PubMed Central

    2013-01-01

    Background Norovirus is the major cause of nonbacterial epidemic gastroenteritis, being highly prevalent in both developing and developed countries. Despite of the available monoclonal antibodies (MAbs) for different sub-genogroups, a comprehensive epitope analysis based on various bioinformatics technology is highly desired for future potential antibody development in clinical diagonosis and treatment. Methods A total of 18 full-length human norovirus capsid protein sequences were downloaded from GenBank. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the protein antigen spatial epitope prediction webserver (SEPPA) for predicting the possible spatial epitopes with the default threshold. The results were processed using the Biosoftware. Results Compared with GI, we found that the GII genogroup had four deletions and two special insertions in the VP1 region. The predicted conformational epitope regions mainly concentrated on N-terminal (1~96), Middle Part (298~305, 355~375) and C-terminal (560~570). We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. Conclusions The predicted conformational epitope regions of norovirus VP1 mainly concentrated on N-terminal, Middle Part and C-terminal. We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. The overlapping with experimental epitopes indicates the important role of latest computational technologies. With the fast development of computational immunology tools, the bioinformatics pipeline will be more and more critical to vaccine design. PMID:23514273

  8. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    SciTech Connect

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  9. Gadolinium-based contrast agents: did we miss something in the last 25 years?

    PubMed

    Beomonte Zobel, Bruno; Quattrocchi, Carlo Cosimo; Errante, Yuri; Grasso, Rosario Francesco

    2016-06-01

    In the last 24 months, several clinical and experimental studies, suggested first and demonstrated later, a progressive concentration of Gadolinium in the brain of normal renal function patients, following repeated injections of some of the commercially approved Gadolinium-Based Contrast Agents. Although, till now, Gadolinium brain deposits have not been associated to any kind of neurological signs or symptoms, they oblige the radiology community to modify the actual approach in using Gadolinium contrast media in daily practice, to reduce unknown possible risks for patients. PMID:26706453

  10. Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

    NASA Astrophysics Data System (ADS)

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

  11. Magic-angle spinning NMR of intact bacteriophages: insights into the capsid, DNA and their interface.

    PubMed

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses. PMID:25797007

  12. Pt, Co-Pt and Fe-Pt alloy nanoclusters encapsulated in virus capsids

    NASA Astrophysics Data System (ADS)

    Okuda, M.; Eloi, J.-C.; Jones, S. E. Ward; Verwegen, M.; Cornelissen, J. J. L. M.; Schwarzacher, W.

    2016-03-01

    Nanostructured Pt-based alloys show great promise, not only for catalysis but also in medical and magnetic applications. To extend the properties of this class of materials, we have developed a means of synthesizing Pt and Pt-based alloy nanoclusters in the capsid of a virus. Pure Pt and Pt-alloy nanoclusters are formed through the chemical reduction of [PtCl4]- by NaBH4 with/without additional metal ions (Co or Fe). The opening and closing of the ion channels in the virus capsid were controlled by changing the pH and ionic strength of the solution. The size of the nanoclusters is limited to 18 nm by the internal diameter of the capsid. Their magnetic properties suggest potential applications in hyperthermia for the Co-Pt and Fe-Pt magnetic alloy nanoclusters. This study introduces a new way to fabricate size-restricted nanoclusters using virus capsid.

  13. Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway.

    PubMed

    Yang, Joo-Sung; Ramanathan, Mathura P; Muthumani, Karuppiah; Choo, Andrew Y; Jin, Sung-Ha; Yu, Qian-Chun; Hwang, Daniel S; Choo, Daniel K; Lee, Mark D; Dang, Kesen; Tang, Waixing; Kim, J Joseph; Weiner, David B

    2002-12-01

    West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade. PMID:12498651

  14. Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion.

    PubMed

    Bosse, Jens B; Hogue, Ian B; Feric, Marina; Thiberge, Stephan Y; Sodeik, Beate; Brangwynne, Clifford P; Enquist, Lynn W

    2015-10-20

    The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids. PMID:26438852

  15. Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion

    PubMed Central

    Bosse, Jens B.; Hogue, Ian B.; Feric, Marina; Thiberge, Stephan Y.; Sodeik, Beate; Brangwynne, Clifford P.; Enquist, Lynn W.

    2015-01-01

    The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids. PMID:26438852

  16. Molecular evolution of the capsid gene in human norovirus genogroup II

    PubMed Central

    Kobayashi, Miho; Matsushima, Yuki; Motoya, Takumi; Sakon, Naomi; Shigemoto, Naoki; Okamoto-Nakagawa, Reiko; Nishimura, Koichi; Yamashita, Yasutaka; Kuroda, Makoto; Saruki, Nobuhiro; Ryo, Akihide; Saraya, Takeshi; Morita, Yukio; Shirabe, Komei; Ishikawa, Mariko; Takahashi, Tomoko; Shinomiya, Hiroto; Okabe, Nobuhiko; Nagasawa, Koo; Suzuki, Yoshiyuki; Katayama, Kazuhiko; Kimura, Hirokazu

    2016-01-01

    Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10−3 substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>102) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans. PMID:27384324

  17. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6

    PubMed Central

    Bhattacharya, Akash; Alam, Steven L.; Fricke, Thomas; Zadrozny, Kaneil; Sedzicki, Jaroslaw; Taylor, Alexander B.; Demeler, Borries; Pornillos, Owen; Ganser-Pornillos, Barbie K.; Diaz-Griffero, Felipe; Ivanov, Dmitri N.; Yeager, Mark

    2014-01-01

    Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host–virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD–CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD–CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD–CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus. PMID:25518861

  18. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6

    SciTech Connect

    Bhattacharya, Akash; Alam, Steven L.; Fricke, Thomas; Zadrozny, Kaneil; Sedzicki, Jaroslaw; Taylor, Alexander B.; Demeler, Borries; Pornillos, Owen; Ganser-Pornillos, Barbie K.; Diaz-Griffero, Felipe; Ivanov, Dmitri N.; Yeager, Mark

    2014-12-17

    Upon infection of susceptible cells by HIV-1, the conical capsid formed by ~250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. In this study, the capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host–virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD–CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD–CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD–CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.

  19. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6

    DOE PAGESBeta

    Bhattacharya, Akash; Alam, Steven L.; Fricke, Thomas; Zadrozny, Kaneil; Sedzicki, Jaroslaw; Taylor, Alexander B.; Demeler, Borries; Pornillos, Owen; Ganser-Pornillos, Barbie K.; Diaz-Griffero, Felipe; et al

    2014-12-17

    Upon infection of susceptible cells by HIV-1, the conical capsid formed by ~250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. In this study, the capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host–virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD–CTD interface is criticalmore » for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD–CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD–CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.« less

  20. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

  1. In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

    PubMed

    Lilavivat, Seth; Sardar, Debosmita; Jana, Subrata; Thomas, Geoffrey C; Woycechowsky, Kenneth J

    2012-08-15

    In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems. PMID:22827162

  2. An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

    SciTech Connect

    Zheng, Yan Kielian, Margaret

    2015-10-15

    Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particles had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.

  3. Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

    PubMed Central

    Yang, Haitao; Ji, Xiaoyun; Zhao, Gongpu; Ning, Jiying; Zhao, Qi; Aiken, Christopher; Gronenborn, Angela M.; Zhang, Peijun; Xiong, Yong

    2012-01-01

    Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction. PMID:23091002

  4. Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

    PubMed Central

    Shi, Jiong; Zhou, Jing; Halambage, Upul D.; Shah, Vaibhav B.; Burse, Mallori J.; Wu, Hua; Blair, Wade S.; Butler, Scott L.

    2014-01-01

    ABSTRACT The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes. IMPORTANCE The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral

  5. Gadolinium-based contrast agent toxicity: a review of known and proposed mechanisms.

    PubMed

    Rogosnitzky, Moshe; Branch, Stacy

    2016-06-01

    Gadolinium chelates are widely used as contrast media for magnetic resonance imaging. The approved gadolinium-based contrast agents (GBCAs) have historically been considered safe and well tolerated when used at recommended dosing levels. However, for nearly a decade, an association between GBCA administration and the development of nephrogenic systemic fibrosis (NSF) has been recognized in patients with severe renal impairment. This has led to modifications in clinical practices aimed at reducing the potential and incidence of NSF development. Newer reports have emerged regarding the accumulation of gadolinium in various tissues of patients who do not have renal impairment, including bone, brain, and kidneys. Despite the observations of gadolinium accumulation in tissues regardless of renal function, very limited clinical data regarding the potential for and mechanisms of toxicity is available. This significant gap in knowledge warrants retrospective cohort study efforts, as well as prospective studies that involve gadolinium ion (Gd(3+)) testing in patients exposed to GBCA. This review examines the potential biochemical and molecular basis of gadolinium toxicity, possible clinical significance of gadolinium tissue retention and accumulation, and methods that can limit gadolinium body burden. PMID:27053146

  6. Use of Gadolinium as a Primary Criticality Control in Disposing Waste Containing Plutonium at SRS

    SciTech Connect

    Andrew, Vincent

    2005-01-04

    Use of gadolinium as a neutron poison has been proposed for disposing of several metric tons of excess plutonium at the Savannah River Site (SRS). The plutonium will first be dissolved in nitric acid in small batches. Gadolinium nitrate will then be added to the plutonium nitrate solution. The Gd-poisoned plutonium solution will be neutralized and transferred to large under-ground tanks. The pH of small batches of neutralized plutonium solution will be adjusted prior to addition of the glass frit for eventual production as glass logs. The use of gadolinium as the neutron poison would minimize the number of glass logs generated from this disposition. The primary criticality safety concerns regarding the disposal process are: (1) maintaining neutron moderation under all processing conditions since gadolinium has a very large absorption cross section for thermal neutrons, (2) characteristics of plutonium and gadolinium precipitation during the neutralization process, (3) mixing characteristics of the precipitate to ensure that plutonium would remain homogeneously mixed with gadolinium, and (4) potential separation of plutonium and gadolinium during nitric and formic acids addition. A number of experiments were conducted at the Savannah River National Laboratory to study the behavior of plutonium and gadolinium at various stages of the disposition process.

  7. Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

    PubMed Central

    Bauer, D. W.; Li, D.; Huffman, J.; Homa, F. L.; Wilson, K.; Leavitt, J. C.; Casjens, S. R.; Baines, J.

    2015-01-01

    ABSTRACT We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages λ and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals. IMPORTANCE A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage λ) as an experimental model system, we

  8. The diffusive way out: Herpesviruses remodel the host nucleus, enabling capsids to access the inner nuclear membrane.

    PubMed

    Bosse, Jens B; Enquist, Lynn W

    2016-01-01

    Herpesviruses are large DNA viruses that utilize the host nucleus for genome replication as well as capsid assembly. After maturation, these 125 nm large capsid assemblies must cross the nucleoplasm to engage the nuclear envelope and bud into the cytoplasm. Here we summarize our recent findings how this motility is facilitated. We suggest that herpesvirus induced nuclear remodeling allows capsids to move by diffusion in the nucleus and not by motor-dependent transport. PMID:26889771

  9. Gadolinium-based nanoparticles for theranostic MRI-radiosensitization.

    PubMed

    Lux, François; Sancey, Lucie; Bianchi, Andrea; Crémillieux, Yannick; Roux, Stéphane; Tillement, Olivier

    2015-01-01

    A rapid development of gadolinium-based nanoparticles is observed due to their attractive properties as MRI-positive contrast agents. Indeed, they display high relaxivity, adapted biodistribution and passive uptake in the tumor thanks to enhanced permeability and retention effect. In addition to these imaging properties, it has been recently shown that they can act as effective radiosensitizers under different types of irradiation (radiotherapy, neutron therapy or hadron therapy). These new therapeutic modalities pave the way to therapy guided by imaging and to personalized medicine. PMID:25715316

  10. Magnetoresistance of polycrystalline gadolinium with varying grain size

    SciTech Connect

    Chakravorty, Manotosh Raychaudhuri, A. K.

    2015-01-21

    In this paper, we report a study of evolution of low field magnetoresistance (MR) of Gadolinium as the grain size in the sample is changed from few microns (∼4 μm) to the nanoscopic regime (∼35 nm). The low field MR has a clear effect on varying grain size. In large grain sample (few μm), the magnetic domains are controlled by local anisotropy field determined mainly by the magnetocrystalline anisotropy. The low field MR clearly reflects the temperature dependence of the magnetocrystalline anisotropy. For decreasing gain size, the contribution of spin disorder at the grain boundary increases and enhances the local anisotropy field.

  11. Studies of MRI relaxivities of gadolinium-labeled dendrons

    NASA Astrophysics Data System (ADS)

    Pan, Hongmu; Daniel, Marie-Christine

    2011-05-01

    In cancer detection, imaging techniques have a great importance in early diagnosis. The more sensitive the imaging technique and the earlier the tumor can be detected. Contrast agents have the capability to increase the sensitivity in imaging techniques such as magnetic resonance imaging (MRI). Until now, gadolinium-based contrast agents are mainly used for MRI, and show good enhancement. But improvement is needed for detection of smaller tumors at the earliest stage possible. The dendrons complexed with Gd(DOTA) were synthesized and evaluated as a new MRI contrast agent. The longitudinal and transverse relaxation effects were tested and compared with commercial drug Magnevist, Gd(DTPA).

  12. Electrical and optical properties of gadolinium doped bismuth ferrite nanoparticles

    SciTech Connect

    Mukherjee, A. Banerjee, M. Basu, S.; Pal, M.

    2014-04-24

    Multiferroic bismuth ferrite (BFO) and gadolinium (Gd) doped bismuth ferrite had been synthesized by a sol-gel method. Particle size had been estimated by Transmission electron microscopy (TEM) and found to decrease with Gd doping. We studied the temperature and frequency dependence of impedance and electric modulus and calculated the grain and grain boundary resistance and capacitance of the investigated samples. We observed that electrical activation energy increases for all the doped samples. Optical band gap also increases for the doped samples which can be used in photocatalytic application of BFO.

  13. Dual-Energy X-Radiography With Gadolinium Filter

    NASA Technical Reports Server (NTRS)

    Rutt, Brian

    1987-01-01

    Image resolution increased, and cost reduced. Proposed dual-energy x-ray imaging system, continuous bremsstrahlung spectrum from x-ray tube filtered by foil of nonradioactive gadolinium or another rare-earth metal to form two-peaked energy spectrum. After passing through patient or object under examination, filtered radiation detected by array of energy-discriminating, photon-counting detectors. Detector outputs processed to form x-ray image for each peak and possibly enhanced image based on data taken at both peaks.

  14. Ascorbate and endocytosed Motexafin gadolinium induce lysosomal rupture.

    PubMed

    Berndt, Carsten; Kurz, Tino; Bannenberg, Sarah; Jacob, Ralf; Holmgren, Arne; Brunk, Ulf T

    2011-08-28

    Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials. PMID:21492999

  15. Gadolinium Thin Foils in a Plasma Panel Sensor as an Alternative to 3He

    SciTech Connect

    Varner Jr, Robert L; Beene, James R; Friedman, Dr. Peter S.

    2010-01-01

    Gadolinium has long been investigated as a detector for neutrons. It has a thermal neutron capture cross-section that is unparalleled among stable elements, because of the isotopes $^{155,157}$Gd. As a replacement for $^3$He, gadolinium has a significant defect, it produces many gamma-rays with an energy sum of 8 MeV. It also produces conversion electrons, mostly 29 keV in energy. The key to replacing $^3$He with gadolinium is using a gamma-blind electron detector to detect the conversion electrons. We suggest that coupling a layer of gadolinium to a Plasma Panel Sensor (PPS) can provide highly efficient, nearly gamma-blind detection of the conversion. The PPS is a proposed detector under development as a dense array of avalanche counters based on plasma display technology. We will present simulations of the response of prototypes of this detector and considerations of the use of gadolinium in the PPS.

  16. Hydroxyproline in the major capsid protein VP1 of polyomavirus

    SciTech Connect

    Ludlow, J.W.; Consigli, R.A.

    1989-06-01

    Amino acid analysis of (/sup 3/H)proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of /sup 3/H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from (/sup 35/S)methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

  17. Modeling capsid self-assembly: design and analysis

    NASA Astrophysics Data System (ADS)

    Rapaport, D. C.

    2010-12-01

    A series of simulations aimed at elucidating the self-assembly dynamics of spherical virus capsids is described. This little-understood phenomenon is a fascinating example of the complex processes that occur in the simplest of organisms. The fact that different viruses adopt similar structural forms is an indication of a common underlying design, motivating the use of simplified, low-resolution models in exploring the assembly process. Several versions of a molecular dynamics approach are described. Polyhedral shells of different sizes are involved, the assembly pathways are either irreversible or reversible and an explicit solvent is optionally included. Model design, simulation methodology and analysis techniques are discussed. The analysis focuses on the growth pathways and the nature of the intermediate states, properties that are hard to access experimentally. Among the key observations are that efficient growth proceeds by means of a cascade of highly reversible stages, and that while there are a large variety of possible partial assemblies, only a relatively small number of strongly bonded configurations are actually encountered.

  18. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  19. Bioluminescent enzyme immunoassay for the detection of norovirus capsid antigen.

    PubMed

    Sakamaki, Nozomi; Ohiro, Yoshiyuki; Ito, Mitsuki; Makinodan, Mitsuru; Ohta, Tsubasa; Suzuki, Wataru; Takayasu, Susumu; Tsuge, Harufumi

    2012-12-01

    An ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y = 0.66x - 3.21, r = 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 10(5) to 10(6) copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis. PMID:23081816

  20. Understanding Dengue Virus Capsid Protein Interaction with Key Biological Targets

    PubMed Central

    Faustino, André F.; Martins, Ivo C.; Carvalho, Filomena A.; Castanho, Miguel A. R. B.; Maurer-Stroh, Sebastian; Santos, Nuno C.

    2015-01-01

    Dengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α-helix 4 (APOEα4) and PLIN3 α-helix 5 (PLIN3α5) sequences, which are also highly superimposable structurally. Interestingly, APOE α-helical N-terminal sequence and structure superimposes with DENV C α-helices α1 and α2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα4 and PLIN3α5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α-helical structures. PMID:26161501

  1. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  2. The preparation of organic infrared semiconductor phthalocyanine gadolinium (III) and its optical and structural characterizations

    NASA Astrophysics Data System (ADS)

    Tang, Li-bin; Ji, Rong-bin; Song, Li-yuan; Chen, Xue-mei; Ma, Yu; Wang, Yi-feng; Qian, Ming; Song, Lei; Su, Hai-ying; Zhuang, Ji-sheng; Yang, Rui-yu

    2009-07-01

    In order to increase the species of organic infrared semiconductor, we synthesized organic infrared semiconductor phthalocyanine gadolinium by using o-phthalodinitrile and GdCl3 as reactants, ammonium molybdate as catalyzer. Under light and dark field modes of microscope, the translucency emerald-like powder of phthalocyanine gadolinium has been observed, the size of the small grain for the sample is around 5μm in diameter, the size of larger grain may reach to several tens of microns. The main vibrational peaks in FT-IR spectrum and Raman spectrum have been assigned. Elementary analysis shows that the experimental data of phthalocyanine gadolinium in the main agree with those of calculated data. The UV-Vis absorption spectrum of the sample indicates the sandwich-like structure of phthalocyanine gadolinium. The organic infrared semiconductor phthalocyanine gadolinium thin film on quartz substrate has been prepared with our synthesized powdered sample by using solution method. The characterizations of XRD and UV-Vis-NIR absorption have been carried out for the phthalocyanine gadolinium thin film on quartz substrate, XRD shows that phthalocyanine gadolinium diffractions occur at 2θ=6.851,8.290 and 8.820 degrees, the corresponding plane spacings (d) for the diffraction peaks are 12.8921, 10.6570, and 10.0176Å.The diffraction peaks locate at low diffraction angle, suggesting that the molecular size of the phthalocyanine gadolinium is big that causes the large spacing of crystal planes. The UV-Vis-NIR absorption of phthalocyanine gadolinium thin film on quartz substrate implies that within near infrared band there is a absorption in the 1.3~2.0μm wavelength range peaked at ca. 1.75μm, indicating the important potential application value of phthalocyanine gadolinium in the field of organic infrared optoelectronics.

  3. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    SciTech Connect

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  4. Transient contacts on the exterior of the HK97 procapsid that are essential for capsid assembly

    PubMed Central

    Tso, Dan-ju; Hendrix, Roger W.; Duda, Robert L.

    2014-01-01

    The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~400 G-loops interacts with Lysine 178 of the E-loop (Extended Loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly, and in some cases induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral or hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge, but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles. PMID:24657766

  5. Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Infection by Virus Capsid Destabilization▿

    PubMed Central

    Shi, Jiong; Zhou, Jing; Shah, Vaibhav B.; Aiken, Christopher; Whitby, Kevin

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or “uncoating,” in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention. PMID:20962083

  6. The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

    PubMed Central

    Martinez, Ruben; Schellenberger, Pascale; Vasishtan, Daven; Aknin, Cindy; Austin, Sisley; Dacheux, Denis; Rayne, Fabienne; Siebert, Alistair; Ruzsics, Zsolt; Gruenewald, Kay

    2014-01-01

    ABSTRACT Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. PMID:25473051

  7. Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

    PubMed Central

    Halder, Sujata; Nam, Hyun-Joo; Govindasamy, Lakshmanan; Vogel, Michèle; Dinsart, Christiane; Salomé, Nathalie; McKenna, Robert

    2013-01-01

    The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. PMID:23449783

  8. Small-molecule inhibition of human immunodeficiency virus type 1 infection by virus capsid destabilization.

    PubMed

    Shi, Jiong; Zhou, Jing; Shah, Vaibhav B; Aiken, Christopher; Whitby, Kevin

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or "uncoating," in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention. PMID:20962083

  9. Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids.

    PubMed

    Bereszczak, Jessica Z; Rose, Rebecca J; van Duijn, Esther; Watts, Norman R; Wingfield, Paul T; Steven, Alasdair C; Heck, Albert J R

    2013-05-01

    Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions. PMID:23597076

  10. Gadolinium-hydrogen ion exchange of zirconium phosphate

    NASA Technical Reports Server (NTRS)

    Liu, D. C.; Power, J. L.

    1972-01-01

    The Gd(+3)/H(+) ion exchange on a commercial zirconium phosphate ion exchanger was investigated in chloride, sulfate, and phosphate solutions of Gd(+3) at gadolinium concentrations of 0.001 to 1 millimole per cc and in the pH range of 0 to 3.5. Relatively low Gd(+3) capacities, in the range of 0.01 to 0.1 millimole per g of ion exchanger were found at room temperature. A significant difference in Gd(+3) sorption was observed, depending on whether the ion exchanger was converted from initial conditions of greater or lesser Gd(+3) sorption than the specific final conditions. Correlations were found between decrease in Gd(+3) capacity and loss of exchanger phosphate groups due to hydrolysis during washing and between increase in capacity and treatment with H3PO4. Fitting of the experimental data to ideal ion exchange equilibrium expressions indicated that each Gd(+3) ion is sorbed on only one site of the ion exchanger. The selectivity quotient was determined to be 2.5 + or - 0.4 at room temperature on gadolinium desorption in chloride solutions.

  11. Magnetic Surfactants and Polymers with Gadolinium Counterions for Protein Separations.

    PubMed

    Brown, Paul; Bromberg, Lev; Rial-Hermida, M Isabel; Wasbrough, Matthew; Hatton, T Alan; Alvarez-Lorenzo, Carmen

    2016-01-26

    New magnetic surfactants, (cationic hexadecyltrimethlyammonium bromotrichlorogadolinate (CTAG), decyltrimethylammonium bromotrichlorogadolinate (DTAG), and a magnetic polymer (poly(3-acrylamidopropyl)trimethylammonium tetrachlorogadolinate (APTAG)) have been synthesized by the simple mixing of the corresponding surfactants and polymer with gadolinium metal ions. A magnetic anionic surfactant, gadolinium tri(1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate) (Gd(AOT)3), was synthesized via metathesis. Both routes enable facile preparation of magnetically responsive magnetic polymers and surfactants without the need to rely on nanocomposites or organic frameworks with polyradicals. Electrical conductivity, surface tensiometry, SQUID magnetometry, and small-angle neutron scattering (SANS) demonstrate surface activity and self-aggregation behavior of the magnetic surfactants similar to their magnetically inert parent analogues but with added magnetic properties. The binding of the magnetic surfactants to proteins enables efficient separations under low-strength (0.33 T) magnetic fields in a new, nanoparticle-free approach to magnetophoretic protein separations and extractions. Importantly, the toxicity of the magnetic surfactants and polymers is, in some cases, lower than that of their halide analogues. PMID:26725503

  12. Dielectric and magnetic properties of some gadolinium silica nanoceramics

    SciTech Connect

    Coroiu, I. Pascuta, P. Bosca, M. Culea, E.

    2013-11-13

    Some nanostructure gadolinium silica glass-ceramics were obtained undergoing a sol gel method and a heat-treatment at 1000°C about two hours. The magnetic and dielectric properties of these samples were studied. The magnetic properties were evidenced performing susceptibility measurements in the 80-300K temperature range. A Curie-Weiss behavior has acquired. The values estimated for paramagnetic Curie temperature being small and positive suggest the presence of weak ferromagnetic interactions between Gd{sup 3+} ions. The dielectric properties were evaluated from dielectric permittivity (ε{sub r}) and dielectric loss (tanδ) measurements at the frequency 1 kHz, 10 kHz and 100 kHz, in the 25-225°C temperature range and dielectric dispersion at room temperature for 79.5 kHz - 1GHz frequency area. The dielectric properties suggest that the main polarization mechanism corresponds to interfacial polarization, characteristic for polycrystalline-structured dielectrics. The polycrystalline structure of the samples is due to the polymorphous transformations of the nanostructure silica crystallites in the presence of gadolinium oxide. They were highlighted by SEM micrographs.

  13. The relationship between thermodynamics and the toxicity of gadolinium complexes.

    PubMed

    Cacheris, W P; Quay, S C; Rocklage, S M

    1990-01-01

    The suitability of gadolinium complexes as magnetic resonance imaging contrast agents depends on a number of factors. A thermodynamic relationship to toxicity exists if one assumes that the chemotoxicity of the intact complex is minimal but that the toxicity of the components of the complex (free metal and uncomplexed ligands) is substantial. Release of Gd3+ from the complex is responsible for the toxicity associated with gadolinium complexes; this release appears to be a consequence of Zn2+, Cu2+, and Ca2+ transmetallation in vivo. This hypothesis is supported by acute toxicity experiments, which demonstrate that despite a 50-fold range of LD50 values for four Gd complexes, all become lethally toxic when they release precisely the same quantity of Gd3+, and by subchronic rodent toxicity experiments, which demonstrate a set of gross and microscopic findings similar to those known to be caused by Zn2+ deficiency. Finally, this hypothesis predicts that subtle changes in formulation can further enhance the intrinsic safety of these complexes. PMID:2118207

  14. Mitochondrial dysfunction induced by different concentrations of gadolinium ion.

    PubMed

    Zhao, Jie; Zhou, Zhi-Qiang; Jin, Jian-Cheng; Yuan, Lian; He, Huan; Jiang, Feng-Lei; Yang, Xiao-Gang; Dai, Jie; Liu, Yi

    2014-04-01

    Gadolinium-based compounds are the most widely used paramagnetic contrast agents in magnetic resonance imaging on the world. But the tricationic gadolinium ion (Gd(3+)) could induce cell apoptosis probably because of its effects on mitochondria. Until now, the mechanism about how Gd(3+) interacts with mitochondria is not well elucidated. In this work, mitochondrial swelling, collapsed transmembrane potential and decreased membrane fluidity were observed to be important factors for mitochondrial permeability transition pore (mtPTP) opening induced by Gd(3+). The protection effect of CsA (Cyclosporin A) could confirm high concentration of Gd(3+) (500 μM) would trigger mtPTP opening. Moreover, mitochondrial outer membrane breakdown and volume expansion observed clearly by transmission electron microscopy and the release of Cyt c (Cytochrome c) could explain the mtPTP opening from another aspect. In addition, MBM(+) (monobromobimane(+)) and DTT (dithiothreitol) could protect thiol (-SH) groups from oxidation so that the toxicity of Gd(3+) might be resulted from the chelation of -SH of membrane proteins by free Gd(3+). Gd(3+) could inhibit the initiation of mitochondrial membrane lipid peroxidation, so it might interact with anionic lipids too. These findings will highly contribute to the safe applications of Gd-based agents. PMID:24321333

  15. Hepatobiliary MR Imaging with Gadolinium Based Contrast Agents

    PubMed Central

    Frydrychowicz, Alex; Lubner, Meghan G.; Brown, Jeffrey J.; Merkle, Elmar M.; Nagle, Scott K.; Rofsky, Neil M.; Reeder, Scott B.

    2011-01-01

    The advent of gadolinium-based “hepatobiliary” contrast agents offers new opportunities for diagnostic MRI and has triggered a great interest for innovative imaging approaches to the liver and bile ducts. In this review article we will discuss the imaging properties of the two gadolinium-based hepatobiliary contrast agents currently available in the USA, gadobenate dimeglumine and gadoxetic acid, as well as important pharmacokinetic differences that affect their diagnostic performance. We will review potential applications, protocol optimization strategies, as well as diagnostic pitfalls. A variety of illustrative case examples will be used to demonstrate the role of these agents in detection and characterization of liver lesions as well as for imaging the biliary system. Changes in MR protocols geared towards optimizing workflow and imaging quality will also be discussed. It is our aim that the information provided in this article will facilitate the optimal utilization of these agents, and will stimulate the reader‘s pursuit of new applications for future benefit. PMID:22334493

  16. Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy.

    PubMed Central

    Baker, T S; Newcomb, W W; Booy, F P; Brown, J C; Steven, A C

    1990-01-01

    Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, rising approximately 5 nm above a midlayer of density. The innermost layer, or "floor," is a 4-nm-thick sheet of virtually continuous density except for the orifices of the channels that traverse each capsomer. Hexon protrusions are distinctly hexagonal in shape, and penton protrusions are pentagonal. The structures of the three kinds of hexons (distinguished according to their positions on the surface lattice) are closely similar but differ somewhat in their respective orientations and in the shapes of their channels. The most prominent features of the midlayer are threefold nodules ("triplexes") at the trigonal lattice points. By analogy with other viral capsids, the triplexes may represent trimers of another capsid protein, possibly VP23 (36 kilodaltons [kDa]) or VP26 (12 kDa). Intermediate capsids differ from light capsids, which are empty, in having one or more internal components. In individual images from which the shell structure has been filtered away, these components are seen to have dimensions of 20 to 30 nm but to lack a visible substructure. This material--which is smeared out in the reconstruction, implying that its distribution is not icosahedrally symmetric or necessarily consistent from particle to particle--consists of aggregates of VP22 (46 kDa). From several lines of evidence, we conclude that this protein is located entirely within the capsid shell. These

  17. The Structure of Human Parechovirus 1 Reveals an Association of the RNA Genome with the Capsid

    PubMed Central

    Kalynych, Sergei; Pálková, Lenka

    2015-01-01

    ABSTRACT Parechoviruses are human pathogens that cause diseases ranging from gastrointestinal disorders to encephalitis. Unlike those of most picornaviruses, parechovirus capsids are composed of only three subunits: VP0, VP1, and VP3. Here, we present the structure of a human parechovirus 1 (HPeV-1) virion determined to a resolution of 3.1 Å. We found that interactions among pentamers in the HPeV-1 capsid are mediated by the N termini of VP0s, which correspond to the capsid protein VP4 and the N-terminal part of the capsid protein VP2 of other picornaviruses. In order to facilitate delivery of the virus genome into the cytoplasm, the N termini of VP0s have to be released from contacts between pentamers and exposed at the particle surface, resulting in capsid disruption. A hydrophobic pocket, which can be targeted by capsid-binding antiviral compounds in many other picornaviruses, is not present in HPeV-1. However, we found that interactions between the HPeV-1 single-stranded RNA genome and subunits VP1 and VP3 in the virion impose a partial icosahedral ordering on the genome. The residues involved in RNA binding are conserved among all parechoviruses, suggesting a putative role of the genome in virion stability or assembly. Therefore, putative small molecules that could disrupt HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors. IMPORTANCE Human parechoviruses (HPeVs) are pathogens that cause diseases ranging from respiratory and gastrointestinal disorders to encephalitis. Recently, there have been outbreaks of HPeV infections in Western Europe and North America. We present the first atomic structure of parechovirus HPeV-1 determined by X-ray crystallography. The structure explains why HPeVs cannot be targeted by antiviral compounds that are effective against other picornaviruses. Furthermore, we found that the interactions of the HPeV-1 genome with the capsid resulted in a partial icosahedral ordering of the genome. The residues

  18. Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

    PubMed Central

    Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

    1996-01-01

    The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

  19. A molecular thermodynamic model for the stability of hepatitis B capsids.

    PubMed

    Kim, Jehoon; Wu, Jianzhong

    2014-06-21

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments. PMID:24952568

  20. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    PubMed Central

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-01-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. PMID:26940118

  1. A Novel Pyridazinone Derivative Inhibits Hepatitis B Virus Replication by Inducing Genome-Free Capsid Formation

    PubMed Central

    Wang, Ya-Juan; Lu, Dong; Xu, Yi-Bin; Xing, Wei-Qiang; Tong, Xian-Kun; Wang, Gui-Feng; Feng, Chun-Lan; He, Pei-Lan; Zuo, Jian-Ping

    2015-01-01

    Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection. PMID:26349829

  2. A molecular thermodynamic model for the stability of hepatitis B capsids

    SciTech Connect

    Kim, Jehoon; Wu, Jianzhong

    2014-06-21

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  3. Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

    PubMed Central

    Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

    2013-01-01

    Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

  4. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site.

    PubMed

    Liu, Chuang; Perilla, Juan R; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A; Zhao, Gongpu; Bedwell, Gregory J; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M; Prevelige, Peter E; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-01-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. PMID:26940118

  5. A molecular thermodynamic model for the stability of hepatitis B capsids

    NASA Astrophysics Data System (ADS)

    Kim, Jehoon; Wu, Jianzhong

    2014-06-01

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  6. Effect of capsid confinement on the chromatin organization of the SV40 minichromosome

    PubMed Central

    Saper, Gadiel; Kler, Stanislav; Asor, Roi; Oppenheim, Ariella; Raviv, Uri; Harries, Daniel

    2013-01-01

    Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a ‘molten droplet’ state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement. PMID:23258701

  7. X-Ray Structures of the Hexameric Building Block of the HIV Capsid

    SciTech Connect

    Pornillos, Owen; Ganser-Pornillos, Barbie K.; Kelly, Brian N.; Hua, Yuanzi; Whitby, Frank G.; Stout, C. David; Sundquist, Wesley I.; Hill, Christopher P.; Yeager, Mark

    2009-09-11

    The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.

  8. X-ray Structures of the Hexameric Building Block of the HIV Capsid

    PubMed Central

    Pornillos, Owen; Ganser-Pornillos, Barbie K.; Kelly, Brian N.; Hua, Yuanzi; Whitby, Frank G.; Stout, C. David; Sundquist, Wesley I.; Hill, Christopher P.; Yeager, Mark

    2010-01-01

    SUMMARY The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to forming quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition, and should facilitate structure-based drug design strategies. PMID:19523676

  9. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  10. Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids

    PubMed Central

    Cardone, Giovanni; Duda, Robert L.; Cheng, Naiqian; You, Lili; Conway, James F.; Hendrix, Roger W.

    2014-01-01

    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. PMID:25389177

  11. Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting.

    PubMed

    ElSohly, Adel M; Netirojjanakul, Chawita; Aanei, Ioana L; Jager, Astraea; Bendall, Sean C; Farkas, Michelle E; Nolan, Garry P; Francis, Matthew B

    2015-08-19

    The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics. PMID:26076186

  12. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-03-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

  13. Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

    PubMed Central

    Kharkwal, Himanshu; Furgiuele, Sara Shanda; Smith, Caitlin G.

    2015-01-01

    ABSTRACT UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCE Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to

  14. Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface.

    PubMed Central

    Jiang, J; Abu-Shilbayeh, L; Rao, V B

    1997-01-01

    The exterior of bacteriophage T4 capsid is coated with two outer capsid proteins, Hoc (highly antigenic outer capsid protein; molecular mass, 40 kDa) and Soc (small outer capsid protein; molecular mass, 9 kDa), at symmetrical positions on the icosahedron (160 copies of Hoc and 960 copies of Soc per capsid particle). Both these proteins are nonessential for phage infectivity and viability and assemble onto the capsid surface after completion of capsid assembly. We developed a phage display system which allowed in-frame fusions of foreign DNA at a unique cloning site in the 5' end of hoc or soc. A DNA fragment corresponding to the 36-amino-acid PorA peptide from Neisseria meningitidis was cloned into the display vectors to generate fusions at the N terminus of Hoc or Soc. The PorA-Hoc and PorA-Soc fusion proteins retained the ability to bind to the capsid surface, and the bound peptide was displayed in an accessible form as shown by its reactivity with specific monoclonal antibodies in an enzyme-linked immunosorbent assay. By employing T4 genetic strategies, we show that more than one subtype-specific PorA peptide can be displayed on the capsid surface and that the peptide can also be displayed on a DNA-free empty capsid. Both the PorA-Hoc and PorA-Soc recombinant phages are highly immunogenic in mice and elicit strong antipeptide antibody titers even with a weak adjuvant such as Alhydrogel or no adjuvant at all. The data suggest that the phage T4 hoc-soc system is an attractive system for display of peptides on an icosahedral capsid surface and may emerge as a powerful system for construction of the next generation multicomponent vaccines. PMID:9353063

  15. Extreme Genetic Fragility of the HIV-1 Capsid

    PubMed Central

    Rihn, Suzannah J.; Wilson, Sam J.; Loman, Nick J.; Alim, Mudathir; Bakker, Saskia E.; Bhella, David; Gifford, Robert J.; Rixon, Frazer J.; Bieniasz, Paul D.

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

  16. Extreme genetic fragility of the HIV-1 capsid.

    PubMed

    Rihn, Suzannah J; Wilson, Sam J; Loman, Nick J; Alim, Mudathir; Bakker, Saskia E; Bhella, David; Gifford, Robert J; Rixon, Frazer J; Bieniasz, Paul D

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

  17. Growth Control in Colon Epithelial Cells: Gadolinium Enhances Calcium-Mediated Growth Regulation

    PubMed Central

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K.

    2013-01-01

    Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1–5 µM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet. PMID:23008064

  18. Gadolinium nanoparticles and contrast agent as radiation sensitizers.

    PubMed

    Taupin, Florence; Flaender, Mélanie; Delorme, Rachel; Brochard, Thierry; Mayol, Jean-François; Arnaud, Josiane; Perriat, Pascal; Sancey, Lucie; Lux, François; Barth, Rolf F; Carrière, Marie; Ravanat, Jean-Luc; Elleaume, Hélène

    2015-06-01

    The goal of the present study was to evaluate and compare the radiosensitizing properties of gadolinium nanoparticles (NPs) with the gadolinium contrast agent (GdCA) Magnevist(®) in order to better understand the mechanisms by which they act as radiation sensitizers. This was determined following either low energy synchrotron irradiation or high energy gamma irradiation of F98 rat glioma cells exposed to ultrasmall gadolinium NPs (GdNPs, hydrodynamic diameter of 3 nm) or GdCA. Clonogenic assays were used to quantify cell survival after irradiation in the presence of Gd using monochromatic x-rays with energies in the 25 keV-80 keV range from a synchrotron and 1.25 MeV gamma photons from a cobalt-60 source. Radiosensitization was demonstrated with both agents in combination with X-irradiation. At the same concentration (2.1 mg mL(-1)), GdNPS had a greater effect than GdCA. The maximum sensitization-enhancement ratio at 4 Gy (SER4Gy) was observed at an energy of 65 keV for both the nanoparticles and the contrast agent (2.44   ±   0.33 and 1.50   ±   0.20, for GdNPs and GdCA, respectively). At a higher energy (1.25 MeV), radiosensitization only was observed with GdNPs (1.66   ±   0.17 and 1.01   ±   0.11, for GdNPs and GdCA, respectively). The radiation dose enhancements were highly 'energy dependent' for both agents. Secondary-electron-emission generated after photoelectric events appeared to be the primary mechanism by which Gd contrast agents functioned as radiosensitizers. On the other hand, other biological mechanisms, such as alterations in the cell cycle may explain the enhanced radiosensitizing properties of GdNPs. PMID:25988839

  19. Gadolinium nanoparticles and contrast agent as radiation sensitizers

    NASA Astrophysics Data System (ADS)

    Taupin, Florence; Flaender, Mélanie; Delorme, Rachel; Brochard, Thierry; Mayol, Jean-François; Arnaud, Josiane; Perriat, Pascal; Sancey, Lucie; Lux, François; Barth, Rolf F.; Carrière, Marie; Ravanat, Jean-Luc; Elleaume, Hélène

    2015-06-01

    The goal of the present study was to evaluate and compare the radiosensitizing properties of gadolinium nanoparticles (NPs) with the gadolinium contrast agent (GdCA) Magnevist® in order to better understand the mechanisms by which they act as radiation sensitizers. This was determined following either low energy synchrotron irradiation or high energy gamma irradiation of F98 rat glioma cells exposed to ultrasmall gadolinium NPs (GdNPs, hydrodynamic diameter of 3 nm) or GdCA. Clonogenic assays were used to quantify cell survival after irradiation in the presence of Gd using monochromatic x-rays with energies in the 25 keV-80 keV range from a synchrotron and 1.25 MeV gamma photons from a cobalt-60 source. Radiosensitization was demonstrated with both agents in combination with X-irradiation. At the same concentration (2.1 mg mL-1), GdNPS had a greater effect than GdCA. The maximum sensitization-enhancement ratio at 4 Gy (SER4Gy) was observed at an energy of 65 keV for both the nanoparticles and the contrast agent (2.44   ±   0.33 and 1.50   ±   0.20, for GdNPs and GdCA, respectively). At a higher energy (1.25 MeV), radiosensitization only was observed with GdNPs (1.66   ±   0.17 and 1.01   ±   0.11, for GdNPs and GdCA, respectively). The radiation dose enhancements were highly ‘energy dependent’ for both agents. Secondary-electron-emission generated after photoelectric events appeared to be the primary mechanism by which Gd contrast agents functioned as radiosensitizers. On the other hand, other biological mechanisms, such as alterations in the cell cycle may explain the enhanced radiosensitizing properties of GdNPs.

  20. STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

    PubMed

    Obal, G; Trajtenberg, F; Carrión, F; Tomé, L; Larrieux, N; Zhang, X; Pritsch, O; Buschiazzo, A

    2015-07-01

    Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology. PMID:26044299

  1. Spontaneous curvature as a regulator of the size of virus capsids

    NASA Astrophysics Data System (ADS)

    Šiber, Antonio; Majdandžić, Antonio

    2009-08-01

    We investigate the physical reasons underlying the high monodispersity of empty virus capsids assembled in thermodynamical equilibrium in conditions of favorable pH and ionic strength. We propose that the high fidelity of the assembly results from the effective spontaneous curvature of the viral protein assemblies and the corresponding bending rigidity that penalizes curvatures which are larger and smaller from the spontaneous one. On the example of hepatitis B virus, which has been thoroughly studied experimentally in the context of interest to us, we estimate the magnitude of bending rigidity that is needed to suppress the appearance of aberrant capsid structures (˜60kBT) . Our approach also demonstrates that the aberrant capsids that can be classified within the Caspar-Klug framework are in most circumstances likely to be smaller from the regular ones, in agreement with the experimental findings.

  2. A statistical approach to close packing of elastic rods and to DNA packaging in viral capsids

    PubMed Central

    Katzav, E.; Adda-Bedia, M.; Boudaoud, A.

    2006-01-01

    We propose a statistical approach for studying the close packing of elastic rods. This phenomenon belongs to the class of problems of confinement of low dimensional objects, such as DNA packaging in viral capsids. The method developed is based on Edwards' approach, which was successfully applied to polymer physics and to granular matter. We show that the confinement induces a configurational phase transition from a disordered (isotropic) phase to an ordered (nematic) phase. In each phase, we derive the pressure exerted by the rod (DNA) on the container (capsid) and the force necessary to inject (eject) the rod into (out of) the container. Finally, we discuss the relevance of the present results with respect to physical and biological problems. Regarding DNA packaging in viral capsids, these results establish the existence of ordered configurations, a hypothesis upon which previous calculations were built. They also show that such ordering can result from simple mechanical constraints. PMID:17146049

  3. The Impact of Capsid Proteins on Virus Removal and Inactivation During Water Treatment Processes

    PubMed Central

    Mayer, Brooke K; Yang, Yu; Gerrity, Daniel W; Abbaszadegan, Morteza

    2015-01-01

    This study examined the effect of the amino acid composition of protein capsids on virus inactivation using ultraviolet (UV) irradiation and titanium dioxide photocatalysis, and physical removal via enhanced coagulation using ferric chloride. Although genomic damage is likely more extensive than protein damage for viruses treated using UV, proteins are still substantially degraded. All amino acids demonstrated significant correlations with UV susceptibility. The hydroxyl radicals produced during photocatalysis are considered nonspecific, but they likely cause greater overall damage to virus capsid proteins relative to the genome. Oxidizing chemicals, including hydroxyl radicals, preferentially degrade amino acids over nucleotides, and the amino acid tyrosine appears to strongly influence virus inactivation. Capsid composition did not correlate strongly to virus removal during physicochemical treatment, nor did virus size. Isoelectric point may play a role in virus removal, but additional factors are likely to contribute. PMID:26604779

  4. In vitro assembly of polymorphic virus-like particles from the capsid protein of a nodavirus.

    PubMed

    Bajaj, Saumya; Banerjee, Manidipa

    2016-09-01

    Viral capsid proteins are programmed to assemble into homogeneous structures in native environments; but the molecular details of these assembly pathways are seldom clearly understood. In order to define the chain of events in the construction of a minimal system, we attempted controlled assembly of the capsid protein of a small insect nodavirus, Flock House Virus (FHV). Bacterial expression of the FHV capsid protein, and subsequent in vitro assembly, generated a heterogeneous population of closed particles. We show that in spite of the altered structure, these particles are capable of membrane disruption, like native viruses, and of incorporating and delivering foreign cargo to specific locations. The unique structure and characteristics of these particles extends our understanding of nodavirus assembly. Additionally, the establishment of a bacterial production system, and methods for in vitro assembly and packaging are of considerable benefit for biotechnological applications of FHV. PMID:27289029

  5. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    NASA Astrophysics Data System (ADS)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

    2013-10-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of protein chains, which result in the capsid stiffening. Dynamic coupling of these modes defines the extent of elasticity and reversibility of capsid mechanical deformation. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

  6. The Collagen-like Protein gp12 Is a Temperature-dependent Reversible Binder of SPP1 Viral Capsids*

    PubMed Central

    Zairi, Mohamed; Stiege, Asita C.; Nhiri, Naima; Jacquet, Eric; Tavares, Paulo

    2014-01-01

    Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface. PMID:25074929

  7. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    PubMed

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. PMID:26405031

  8. Microglia-specific targeting by novel capsid-modified AAV6 vectors

    PubMed Central

    Rosario, Awilda M; Cruz, Pedro E; Ceballos-Diaz, Carolina; Strickland, Michael R; Siemienski, Zoe; Pardo, Meghan; Schob, Keri-Lyn; Li, Andrew; Aslanidi, George V; Srivastava, Arun; Golde, Todd E; Chakrabarty, Paramita

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies. PMID:27308302

  9. Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

    NASA Astrophysics Data System (ADS)

    Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

    2016-07-01

    We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

  10. In Silico Studies of Medicinal Compounds Against Hepatitis C Capsid Protein from North India

    PubMed Central

    Mathew, Shilu; Faheem, Muhammad; Archunan, Govindaraju; Ilyas, Muhammad; Begum, Nargis; Jahangir, Syed; Qadri, Ishtiaq; Qahtani, Mohammad Al; Mathew, Shiny

    2014-01-01

    Hepatitis viral infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Over one million people are estimated to be persistently infected with hepatitis C virus (HCV) worldwide. As capsid core protein is the key element in spreading HCV; hence, it is considered to be the superlative target of antiviral compounds. Novel drug inhibitors of HCV are in need to complement or replace the current treatments such as pegylated interferon’s and ribavirin as they are partially booming and beset with various side effects. Our study was conducted to predict 3D structure of capsid core protein of HCV from northern part of India. Core, the capsid protein of HCV, handles the assembly and packaging of HCV RNA genome and is the least variable of all the ten HCV proteins among the six HCV genotypes. Therefore, we screened four phytochemicals inhibitors that are known to disrupt the interactions of core and other HCV proteins such as (a) epigallocatechin gallate (EGCG), (b) ladanein, (c) naringenin, and (d) silybin extracted from medicinal plants; targeted against active site of residues of HCV-genotype 3 (G3) (Q68867) and its subtypes 3b (Q68861) and 3g (Q68865) from north India. To study the inhibitory activity of the recruited flavonoids, we conducted a quantitative structure–activity relationship (QSAR). Furthermore, docking interaction suggests that EGCG showed a maximum number of hydrogen bond (H-bond) interactions with all the three modeled capsid proteins with high interaction energy followed by naringenin and silybin. Thus, our results strongly correlate the inhibitory activity of the selected bioflavonoid. Finally, the dynamic predicted capsid protein molecule of HCV virion provides a general avenue to target structure-based antiviral compounds that support the hypothesis that the screened inhibitors for viral capsid might constitute new class of potent agents but further confirmation is necessary using in vitro and in vivo

  11. Defect induced mobility enhancement: Gadolinium oxide (100) on Si(100)

    SciTech Connect

    Sitaputra, W.; Tsu, R.

    2012-11-26

    Growth of predominantly single crystal (100)-oriented gadolinium oxide (Gd{sub 2}O{sub 3}) on a p-type Si(100) and growth of a polycrystal with a predominant Gd{sub 2}O{sub 3}(100) crystallite on a n-type Si(100) was performed using molecular beam epitaxy. Despite a poorer crystal structure than Gd{sub 2}O{sub 3}(110), an enhancement in carrier mobility can be found only from the Gd{sub 2}O{sub 3}(100)/n-type Si(100) interface. The mobility of 1715-1780 cm{sup 2}/V {center_dot} s was observed at room temperature, for carrier concentration >10{sup 20} cm{sup -3}. This accumulation of the electrons and the mobility enhancement may arise from the two-dimensional confinement due to charge transfer across the interface similar to transfer doping.

  12. The structural response of gadolinium phosphate to pressure

    DOE PAGESBeta

    Heffernan, Karina M.; Ross, Nancy L.; Spencer, Elinor C.; Boatner, Lynn A.

    2016-06-16

    In this study, accurate elastic constants for gadolinium phosphate (GdPO4) have been measured by single-crystal high-pressure diffraction methods. The bulk modulus of GdPO4 determined under hydrostatic conditions, 128.1(8) GPa (K'=5.8(2)), is markedly different from that obtained with GdPO4 under non-hydrostatic conditions (160(2) GPa), which indicates the importance of shear stresses on the elastic response of this phosphate. Finally, high pressure Raman and diffraction analysis indicate that the PO4 tetrahedra behave as rigid units in response to pressure and that contraction of the GdPO4 structure is facilitated by bending/twisting of the Gd–O–P links that result in increased distortion in the GdO9more » polyhedra.« less

  13. Extracellular gadolinium-based contrast media: an overview.

    PubMed

    Bellin, Marie-France; Van Der Molen, Aart J

    2008-05-01

    Increasing use is made of extracellular MRI contrast agents that alter the image contrast following intravenous administration; they predominantly shorten the T1 relaxation time of tissues. The degree and location of these changes provide substantial diagnostic information. However gadolinium-based contrast agents (Gd-CA) are not inert drugs. They may cause acute non-renal adverse reactions (e.g. anaphylactoid reactions), acute renal adverse reactions (e.g. contrast induced nephropathy), delayed adverse reactions (nephrogenic systemic fibrosis) and problems at the site of injection (e.g. local necrosis). This review describes the current status of Gd-CA, their mechanism of action, chemical structure, pharmacokinetics, dosage, elimination, nephrotoxicity and adverse events. PMID:18358659

  14. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

  15. Strategies for the preparation of bifunctional gadolinium(III) chelators

    PubMed Central

    Frullano, Luca; Caravan, Peter

    2012-01-01

    The development of gadolinium chelators that can be easily and readily linked to various substrates is of primary importance for the development high relaxation efficiency and/or targeted magnetic resonance imaging (MRI) contrast agents. Over the last 25 years a large number of bifunctional chelators have been prepared. For the most part, these compounds are based on ligands that are already used in clinically approved contrast agents. More recently, new bifunctional chelators have been reported based on complexes that show a more potent relaxation effect, faster complexation kinetics and in some cases simpler synthetic procedures. This review provides an overview of the synthetic strategies used for the preparation of bifunctional chelators for MRI applications. PMID:22375102

  16. The structural response of gadolinium phosphate to pressure

    NASA Astrophysics Data System (ADS)

    Heffernan, Karina M.; Ross, Nancy L.; Spencer, Elinor C.; Boatner, Lynn A.

    2016-09-01

    Accurate elastic constants for gadolinium phosphate (GdPO4) have been measured by single-crystal high-pressure diffraction methods. The bulk modulus of GdPO4 determined under hydrostatic conditions, 128.1(8) GPa (Kʹ=5.8(2)), is markedly different from that obtained with GdPO4 under non-hydrostatic conditions (160(2) GPa), which indicates the importance of shear stresses on the elastic response of this phosphate. High pressure Raman and diffraction analysis indicate that the PO4 tetrahedra behave as rigid units in response to pressure and that contraction of the GdPO4 structure is facilitated by bending/twisting of the Gd-O-P links that result in increased distortion in the GdO9 polyhedra.

  17. Sintering and mechanical properties of gadolinium-doped ceria ceramics

    NASA Astrophysics Data System (ADS)

    Yasuda, K.; Uemura, K.; Shiota, T.

    2012-01-01

    Gadolinium-doped ceria (GDC) ceramics were made by sintering at various temperatures from 1000°C to 1400°C in air. The true density and apparent density were measured to calculate the relative density of GDC ceramics. The change in relative density revealed that densification of GDC ceramics increased up to 1200°C, and thereafter turned downward. It was suggested that pores were formed at 1300°C and 1400°C due to non-stoichiometry of ceria. JIS-type specimens were cut from the sintered body and tested by 4-point bending. Young's modulus and bending strength decreased with increasing the sintering temperature from 1200°C to 1400°C, corresponding to the change in the relative density.

  18. An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation

    PubMed Central

    Orabi, Ahmed; Bieringer, Maria; Geerlof, Arie

    2015-01-01

    ABSTRACT The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain [MD]) in the large surface protein L and a narrow region (matrix binding domain [MBD]) including isoleucine 126 on the capsid surface have been mapped, in which point mutations such as core I126A specifically blocked nucleocapsid envelopment. It is possible that the two domains interact with each other during virion morphogenesis. By the systematic evolution of ligands by exponential enrichment (SELEX) method, we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications, 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50% of the clones. The main aptamer, AO-01 (13 clones, 18%), showed the lowest dissociation constant (Kd) of 180 ± 82 nM for capsid binding among the four molecules. Its Kd for I126A capsids was 1,306 ± 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47% inhibition of virion production at 3 days posttransfection, but there was no inhibition by cotransfection of an aptamer with a random sequence. The half-life of AO-01 in cells was 2 h, which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction with a low-molecular-weight substance might represent a new approach for an antiviral therapy. IMPORTANCE Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55-base-long single-stranded DNA

  19. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  20. Vibrating virus capsids and interactions with short light pulses -- picking up good vibrations

    NASA Astrophysics Data System (ADS)

    Sankey, Otto; Benson, Daryn

    2009-10-01

    Viruses are the simplest ``life'' form. They reproduce by borrowing the machinery of their host cell. Viruses consist of an outer coat (capsid) that protects its genomic material inside. They are pathogenic to plants, bacteria, animals, and of course humans. Experimental studies at ASU by Tsen et al. have discovered that ultra-short laser pulses are capable of ``inactivating'' viruses. One potential mechanism is the coupling of light to the soft dynamical modes of the capsid. We describe theoretical modeling of this effect.

  1. Group theory of icosahedral virus capsid vibrations: a top-down approach.

    PubMed

    Peeters, Kasper; Taormina, Anne

    2009-02-21

    We explore the use of a top-down approach to analyse the dynamics of icosahedral virus capsids and complement the information obtained from bottom-up studies of viral vibrations available in the literature. A normal mode analysis based on protein association energies is used to study the frequency spectrum, in which we reveal a universal plateau of low-frequency modes shared by a large class of Caspar-Klug capsids. These modes break icosahedral symmetry and are potentially relevant to the genome release mechanism. We comment on the role of viral tiling theory in such dynamical considerations. PMID:19014954

  2. The impact of non-uniform capsid charge density on virus assembly

    NASA Astrophysics Data System (ADS)

    Li, Siyu; Erdemci-Tandogan, Gonca; Wagner, Jef; Zandi, Roya

    Many spherical viruses efficiently encapsulate their genome into shells (capsids) with icosahedral symmetry. Under many circumstances, this process is spontaneous and is primarily driven by the electrostatic interaction between positively charged capsid proteins and negatively charged genome. Through the free energy minimization of a generic potential, we calculate the optimal encapsulated genome length. In this talk, I will present our results due to a non-uniform charge distribution on the shell and its impact on the optimal size of encapsulated genome. This work was supported by the National Science Foundation through Grant No. DMR-13-10687.

  3. Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

    NASA Astrophysics Data System (ADS)

    Cohen, Brian A.

    The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin

  4. Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

    PubMed

    Machara, Aleš; Lux, Vanda; Kožíšek, Milan; Grantz Šašková, Klára; Štěpánek, Ondřej; Kotora, Martin; Parkan, Kamil; Pávová, Marcela; Glass, Bärbel; Sehr, Peter; Lewis, Joe; Müller, Barbara; Kräusslich, Hans-Georg; Konvalinka, Jan

    2016-01-28

    Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations. PMID:26685880

  5. Safety profiles of gadolinium chelates in juvenile rats differ according to the risk of dissociation.

    PubMed

    Fretellier, Nathalie; Maazouz, Meryam; Luseau, Alexandrine; Baudimont, Fannie; Jestin-Mayer, Gaëlle; Bourgery, Simon; Rasschaert, Marlène; Bruneval, Patrick; Factor, Cécile; Mecieb, Fatiha; Idée, Jean-Marc; Corot, Claire

    2014-12-01

    This study was designed to compare the safety of two gadolinium chelates (GCs), used as contrast agents for magnetic resonance imaging, in juvenile rats. Juvenile rats received five intravenous administrations (between postnatal day [PND] 4 and 18) of gadoteric acid (macrocyclic ionic GC), gadodiamide (linear nonionic GC) or saline, and sacrificed at PND 25. Gadodiamide induced mortality, alopecia and hyperpigmentation of dorsal skin. Two gadodiamide-treated rats presented severe epidermal and dermal lesions. No abnormal signs were detected following administration of gadoteric acid. Higher tissue gadolinium concentrations were found in the gadodiamide group compared to the gadoteric acid group. Dissociation of gadodiamide was observed in skin and liver, with the presence of dissociated and soluble gadolinium. In conclusion, repeated administration of gadoteric acid was well tolerated by juvenile rats. In contrast, gadodiamide induced significant toxicity and more marked tissue gadolinium retention (at least partly in the dissociated and soluble form). PMID:25462783

  6. Insights into the use of gadolinium and gadolinium/boron-based agents in imaging-guided neutron capture therapy applications.

    PubMed

    Deagostino, Annamaria; Protti, Nicoletta; Alberti, Diego; Boggio, Paolo; Bortolussi, Silva; Altieri, Saverio; Crich, Simonetta Geninatti

    2016-05-01

    Gadolinium neutron capture therapy (Gd-NCT) is currently under development as an alternative approach for cancer therapy. All of the clinical experience to date with NCT is done with (10)B, known as boron neutron capture therapy (BNCT), a binary treatment combining neutron irradiation with the delivery of boron-containing compounds to tumors. Currently, the use of Gd for NCT has been getting more attention because of its highest neutron cross-section. Although Gd-NCT was first proposed many years ago, its development has suffered due to lack of appropriate tumor-selective Gd agents. This review aims to highlight the recent advances for the design, synthesis and biological testing of new Gd- and B-Gd-containing compounds with the task of finding the best systems able to improve the NCT clinical outcome. PMID:27195428

  7. Density of Gadolinium Nitrate Solutions for the High Flux Isotope Reactor

    SciTech Connect

    Taylor, Paul Allen; Lee, Denise L

    2009-05-01

    In late 1992, the High Flux Isotope Reactor (HFIR) was planning to switch the solution contained in the poison injection tank from cadmium nitrate to gadolinium nitrate. The poison injection system is an emergency system used to shut down the reactor by adding a neutron poison to the cooling water. This system must be able to supply a minimum of 69 pounds of gadolinium to the reactor coolant system in order to guarantee that the reactor would become subcritical. A graph of the density of gadolinium nitrate solutions over a concentration range of 5 to 30 wt% and a temperature range of 15 to 40{sup o}C was prepared. Routine density measurements of the solution in the poison injection tank are made by HFIR personnel, and an adaptation of the original graph is used to determine the gadolinium nitrate concentration. In late 2008, HFIR personnel decided that the heat tracing that was present on the piping for the poison injection system could be removed without any danger of freezing the solution; however, the gadolinium nitrate solution might get as cold as 5{sup o}C. This was outside the range of the current density-concentration correlation, so the range needed to be expanded. This report supplies a new density-concentration correlation that covers the extended temperature range. The correlation is given in new units, which greatly simplifies the calculation that is required to determine the pounds of gadolinium in the tank solution. The procedure for calculating the amount of gadolinium in the HFIR poison injection system is as follows: (1) Calculate the usable volume in the system; (2) Measure the density of the solution; (3) Calculate the gadolinium concentration using the following equation: Gd(lb/ft{sup 3}) = measured density (g/mL) x 34.681 - 34.785; (4) Calculate the amount of gadolinium in the system using the following equation: Amount of Gd(lb) = Gd concentration (lb/ft{sup 3}) x usable volume (ft{sup 3}). The equation in step 3 is exact for a temperature of

  8. Gadolinium Use in Spine Pain Management Procedures for Patients with Contrast Allergies: Results in 527 Procedures

    SciTech Connect

    Safriel, Yair Ang, Roberto; Ali, Muhammed

    2008-03-15

    Introduction. To review the safety and efficacy of gadolinium in spine pain management procedures in patients at high risk for a contrast reaction and who are not suitable candidates for the use of standard non-ionic contrast. Methods. We reviewed records over a 61-month period of all image-guided spinal pain management procedures where patients had allergies making them unsuitable candidates for standard non-ionic contrast and where gadolinium was used to confirm needle tip placement prior to injection of medication. Results. Three hundred and four outpatients underwent 527 procedures. A spinal needle was used in all but 41 procedures. Gadolinium was visualized using portable C-arm fluoroscopy in vivo allowing for confirmation of needle tip location. The gadolinium dose ranged from 0.2 to 10 ml per level. The highest dose received by one patient was 15.83 ml intradiscally during a three-level discogram. Three hundred and one patients were discharged without complication or known delayed complications. One patient had documented intrathecal injection but without sequelae and 2 patients who underwent cervical procedures experienced seizures requiring admission to the intensive care unit. Both the latter patients were discharged without any further complications. Conclusion. Based on our experience we recommend using gadolinium judiciously for needle tip confirmation. We feel more confident using gadolinium in the lumbar spine and in cervical nerve blocks. Gadolinium should probably not be used as an injectate volume expander. The indications for gadolinium use in cervical needle-guided spine procedures are less clear and use of a blunt-tipped needle should be considered.

  9. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    SciTech Connect

    Walia, Rupali; Dardari, Rkia Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  10. Elastic properties and mechanical stability of chiral and filled viral capsids

    NASA Astrophysics Data System (ADS)

    Buenemann, Mathias; Lenz, Peter

    2008-11-01

    The elasticity and mechanical stability of empty and filled viral capsids under external force loading are studied in a combined analytical and numerical approach. We analyze the influence of capsid structure and chirality on the mechanical properties. We find that generally skew shells have lower stretching energy. For large Föppl-von Kármán numbers γ (γ≈105) , skew structures are stiffer in their elastic response than nonchiral ones. The discrete structure of the capsules not only leads to buckling for large γ but also influences the breakage behavior of capsules below the buckling threshold: the rupture force shows a γ1/4 scaling rather than a γ1/2 scaling as expected from our analytical results for continuous shells. Filled viral capsids are exposed to internal anisotropic pressure distributions arising from regularly packaged DNA coils. We analyze their influence on the elastic properties and rupture behavior and we discuss possible experimental consequences. Finally, we numerically investigate specific sets of parameters corresponding to specific phages such as ϕ29 and cowpea chlorotic mottle virus (CCMV). From the experimentally measured spring constants we make predictions about specific material parameters (such as bending rigidity and Young’s modulus) for both empty and filled capsids.

  11. Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids

    PubMed Central

    Glasgow, Jeff E.; Capehart, Stacy L.; Francis, Matthew B.; Tullman-Ercek, Danielle

    2012-01-01

    The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments. PMID:22953696

  12. Molecular evolution of the capsid gene in human norovirus genogroup II.

    PubMed

    Kobayashi, Miho; Matsushima, Yuki; Motoya, Takumi; Sakon, Naomi; Shigemoto, Naoki; Okamoto-Nakagawa, Reiko; Nishimura, Koichi; Yamashita, Yasutaka; Kuroda, Makoto; Saruki, Nobuhiro; Ryo, Akihide; Saraya, Takeshi; Morita, Yukio; Shirabe, Komei; Ishikawa, Mariko; Takahashi, Tomoko; Shinomiya, Hiroto; Okabe, Nobuhiko; Nagasawa, Koo; Suzuki, Yoshiyuki; Katayama, Kazuhiko; Kimura, Hirokazu

    2016-01-01

    Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans. PMID:27384324

  13. Physical Ingredients Controlling Stability and Structural Selection of Empty Viral Capsids.

    PubMed

    Aznar, María; Reguera, David

    2016-07-01

    One of the crucial steps in the viral replication cycle is the self-assembly of its protein shell. Typically, each native virus adopts a unique architecture, but the coat proteins of many viruses have the capability to self-assemble in vitro into different structures by changing the assembly conditions. However, the mechanisms determining which of the possible capsid shapes and structures is selected by a virus are still not well-known. We present a coarse-grained model to analyze and understand the physical mechanisms controlling the size and structure selection in the assembly of empty viral capsids. Using this model and Monte Carlo simulations, we have characterized the phase diagram and stability of T = 1,3,4,7 and snub cube shells. In addition, we have studied the tolerance of different shells to changes in physical parameters related to ambient conditions, identifying possible strategies to induce misassembly or failure. Finally, we discuss the factors that select the shape of a capsid as spherical, faceted, elongated, or decapsidated. Our model sheds important light on the ingredients that control the assembly and stability of viral shells. This knowledge is essential to get capsids with well-defined size and structure that could be used for promising applications in medicine or bionanotechnology. PMID:27114062

  14. Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids.

    PubMed Central

    Rhee, S S; Hunter, E

    1987-01-01

    The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release. Images PMID:3493352

  15. Probing the biophysical interplay between a viral genome and its capsid

    NASA Astrophysics Data System (ADS)

    Snijder, J.; Uetrecht, C.; Rose, R. J.; Sanchez-Eugenia, R.; Marti, G. A.; Agirre, J.; Guérin, D. M. A.; Wuite, G. J. L.; Heck, A. J. R.; Roos, W. H.

    2013-06-01

    The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

  16. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify re...

  17. Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of ...

  18. Nanoindentation of 35 Virus Capsids in a Molecular Model: Relating Mechanical Properties to Structure

    PubMed Central

    2013-01-01

    A coarse-grained model is used to study the mechanical response of 35 virus capsids of symmetries T = 1, T = 2, T = 3, pseudo T = 3, T = 4, and T = 7. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the C atoms associated with each amino acid. The number of these atoms ranges between 8 460 (for SPMV – satellite panicum mosaic virus) and 135 780 (for NBV – nudaureli virus). Nanoindentation by a broad AFM tip is modeled as compression between two planes: either both flat or one flat and one curved. Plots of the compressive force versus plate separation show a variety of behaviors, but in each case there is an elastic region which extends to a characteristic force . Crossing results in a drop in the force and irreversible damage. Across the 35 capsids studied, both and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number , defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with , where 6 is the minimum coordination for elastic stability in three dimensions. PMID:23785395

  19. Nanoindentation of 35 virus capsids in a molecular model: relating mechanical properties to structure.

    PubMed

    Cieplak, Marek; Robbins, Mark O

    2013-01-01

    A coarse-grained model is used to study the mechanical response of 35 virus capsids of symmetries T = 1, T = 2, T = 3, pseudo T = 3, T = 4, and T = 7. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the C[Formula: see text] atoms associated with each amino acid. The number of these atoms ranges between 8 460 (for SPMV - satellite panicum mosaic virus) and 135 780 (for NBV - nudaureli virus). Nanoindentation by a broad AFM tip is modeled as compression between two planes: either both flat or one flat and one curved. Plots of the compressive force versus plate separation show a variety of behaviors, but in each case there is an elastic region which extends to a characteristic force [Formula: see text]. Crossing [Formula: see text] results in a drop in the force and irreversible damage. Across the 35 capsids studied, both [Formula: see text] and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number [Formula: see text], defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with [Formula: see text], where 6 is the minimum coordination for elastic stability in three dimensions. PMID:23785395

  20. Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, res...

  1. Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles

    PubMed Central

    Wilson, David P.

    2016-01-01

    Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as

  2. Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles.

    PubMed

    Wilson, David P

    2016-01-01

    Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as

  3. Location of the Bacteriophage P22 Coat Protein C-terminus Provides Opportunities for the Design of Capsid Based Materials

    PubMed Central

    Servid, Amy; Jordan, Paul; O’Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-01-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends towards the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the exterior of the P22 capsid. Capsids displaying a 6xHis tag appended to the CP C-terminus bind to a Ni affinity column, and the addition of positively or negatively charged coiled coil peptides to the capsid results in association of these capsids upon mixing. Additionally, a single cysteine appended to the CP C-terminus results in the formation of intercapsid disulfide bonds and can serve as a site for chemical modifications. Thus, the C-terminus is a powerful location for multivalent display of peptides that facilitate nanoscale assembly and capsid modification. PMID:23957641

  4. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    SciTech Connect

    Sathish, Narayanan; Yuan Yan

    2010-11-25

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-{Delta}65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  5. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  6. A hydrophobic domain within the small capsid protein of Kaposi’s sarcoma-associated herpesvirus is required for assembly

    PubMed Central

    Capuano, Christopher M.; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N.; Desai, Keshal V.; Coombs, Gavin; McCaffery, J. Michael

    2014-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP–GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present – indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP–GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP–His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP–GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP–GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP–MCP interaction. PMID:24824860

  7. A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

    PubMed

    Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N; Desai, Keshal V; Coombs, Gavin; McCaffery, J Michael; Desai, Prashant J

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction. PMID:24824860

  8. Human parvovirus B19 VP2 empty capsids bind to human villous trophoblast cells in vitro via the globoside receptor.

    PubMed Central

    Wegner, Carole C; Jordan, Jeanne A

    2004-01-01

    BACKGROUND: Pregnant women acutely infected with human parvovirus B19 (B19) may transmit the virus to the developing fetus. The mechanism whereby the virus interacts with the placenta is unknown. It is known that globoside receptor is required for successful infection of the target cells, which are the highly undifferentiated, actively dividing colony and burst-form units of the erythroid series. Globoside is present on trophoblast cells which have intimate contact with maternal blood, and may therefore serve as a potential route for B19 transmission into the fetal compartment. OBJECTIVES: The purpose of this study was to determine whether B19 VP2 capsids could bind to villous trophoblast cells in vitro and whether globoside was involved. METHODS: Binding of B19 VP2 empty capsid to first-trimester villous trophoblast cells was assessed by multiple approaches, including ICC using either biotinylated B19 VP2 empty capsid or unlabeled B19 VP2 empty capsid. Quantification of viral binding involved I125-labeled B19 VP2 empty capsid. Competition studies included excess unlabeled empty capsids or pretreatment with globoside-specific IgM antibody. RESULTS: Linear binding of B19 VP2 capsid to purified villous trophoblast cells in vitro was clearly demonstrated (R2= 0.9524). Competition studies revealed specificity of I125-labeled B19 VP2 capsid binding to villous trophoblast cells when pretreatment with either 60-fold excess unlabeled B19 capsid or globoside-specific IgM antibody took place. The results illustrated B19's ability to bind in a specific manner to globoside-containing villous trophoblast cells. CONCLUSION: We speculate that the globoside present on trophoblast cells may play a role in viral binding in vivo, which may facilitate B19 transmission across the maternal-fetal interface. PMID:15739820

  9. Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

    PubMed Central

    Bocanegra, Rebeca; Nevot, María; Doménech, Rosa; López, Inmaculada; Abián, Olga; Rodríguez-Huete, Alicia; Cavasotto, Claudio N.; Velázquez-Campoy, Adrián; Gómez, Javier; Martínez, Miguel Ángel; Neira, José Luis; Mateu, Mauricio G.

    2011-01-01

    Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly

  10. The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

    PubMed Central

    2013-01-01

    Background Lentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits. Results We provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells. Conclusions Our results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability. PMID:23497318

  11. Optimization of an Elastic Network Augmented Coarse Grained Model to Study CCMV Capsid Deformation

    PubMed Central

    Globisch, Christoph; Krishnamani, Venkatramanan; Deserno, Markus; Peter, Christine

    2013-01-01

    The major protective coat of most viruses is a highly symmetric protein capsid that forms spontaneously from many copies of identical proteins. Structural and mechanical properties of such capsids, as well as their self-assembly process, have been studied experimentally and theoretically, including modeling efforts by computer simulations on various scales. Atomistic models include specific details of local protein binding but are limited in system size and accessible time, while coarse grained (CG) models do get access to longer time and length scales but often lack the specific local interactions. Multi-scale models aim at bridging this gap by systematically connecting different levels of resolution. Here, a CG model for CCMV (Cowpea Chlorotic Mottle Virus), a virus with an icosahedral shell of 180 identical protein monomers, is developed, where parameters are derived from atomistic simulations of capsid protein dimers in aqueous solution. In particular, a new method is introduced to combine the MARTINI CG model with a supportive elastic network based on structural fluctuations of individual monomers. In the parametrization process, both network connectivity and strength are optimized. This elastic-network optimized CG model, which solely relies on atomistic data of small units (dimers), is able to correctly predict inter-protein conformational flexibility and properties of larger capsid fragments of 20 and more subunits. Furthermore, it is shown that this CG model reproduces experimental (Atomic Force Microscopy) indentation measurements of the entire viral capsid. Thus it is shown that one obvious goal for hierarchical modeling, namely predicting mechanical properties of larger protein complexes from models that are carefully parametrized on elastic properties of smaller units, is achievable. PMID:23613730

  12. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion

    PubMed Central

    Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

    2015-01-01

    The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1–147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1–147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex. PMID

  13. Rhesus TRIM5α Disrupts the HIV-1 Capsid at the Inter­Hexamer Interfaces

    PubMed Central

    Zhao, Gongpu; Ke, Danxia; Vu, Thomas; Ahn, Jinwoo; Shah, Vaibhav B.; Yang, Ruifeng; Aiken, Christopher; Charlton, Lisa M.; Gronenborn, Angela M.; Zhang, Peijun

    2011-01-01

    TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. PMID:21455494

  14. Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

    PubMed Central

    Horowitz, Eric D.; Rahman, K. Shefaet; Bower, Brian D.; Dismuke, David J.; Falvo, Michael R.; Griffith, Jack D.

    2013-01-01

    We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release. PMID:23269804

  15. Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces.

    PubMed

    Zhao, Gongpu; Ke, Danxia; Vu, Thomas; Ahn, Jinwoo; Shah, Vaibhav B; Yang, Ruifeng; Aiken, Christopher; Charlton, Lisa M; Gronenborn, Angela M; Zhang, Peijun

    2011-03-01

    TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5α(rh)) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5α(rh) containing coiled-coil and SPRY domains (CC-SPRY(rh)). Concentration-dependent binding of CC-SPRY(rh) to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRY(rh), but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5α(rh) on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. PMID:21455494

  16. XAFS study of gadolinium and samarium bisporphyrinate complexes.

    PubMed

    Agondanou, J H; Spyroulias, G A; Purans, J; Tsikalas, G; Souleau, C; Coutsolelos, A G; Bénazeth, S

    2001-11-19

    The comparative X-ray absorption spectroscopy study of gadolinium and samarium bisporphyrinate complexes represented by the formulas Gd(III)H(oep)(tpp), Gd(III)(oep)(2), Gd(III)H(tpp)(2) and Sm(III)H(oep)(tpp), Sm(III)(oep)(2), Sm(III)H(tpp)(2) is reported. The XAFS spectra are recorded on the LURE-DCI storage ring (Orsay, France) in transmission mode on the microcrystalline samples at the Gd and Sm L(3) edges. The local environment for Ln(3+) ions has been reconstructed applying one-shell and two-shell XAFS analysis procedures. The protonated and nonprotonated bisporphyrinate complexes present different XAFS features. After our analysis on the title derivatives, the gadolinium ion (at 80 K) is found to be bonded: (i) to eight nitrogen atoms at R(Gd-N) 2.50 A, for Gd(III)(oep)(2) [Debye-Waller (DW) factor 0.004 A(2)]; (ii) to seven nitrogen atoms at R(Gd-N) 2.49 A, for Gd(III)H(oep)(tpp) [DW factor 0.005 A(2)] and one nitrogen at long distance; and (iii) to six nitrogen atoms at R(Gd-N) 2.50 A [DW factor 0.006 A(2)] and two nitrogen atoms at long distance for Gd(III)H(tpp)(2). A similar coordination sphere has been detected for the corresponding Sm derivatives. So, the samarium ion (at room temperature) is bonded: (i) to eight nitrogen atoms at R(Sm-N) 2.53 A, for Sm(III)(oep)(2) [DW factor 0.006 A(2)]; (ii) to seven nitrogen atoms at R(Sm-N) 2.53 A, for Sm(III)H(oep)(tpp) [DW factor 0.006 A(2)] and one nitrogen at long distance; and (iii) to six nitrogen atoms at R(Sm-N) 2.50 A, for Sm(III)H(tpp)(2) [DW factor 0.006 A(2)] and two nitrogen atoms at long distance. As far as concerns Ln(III)(oep)(2) complexes, the increase of Ln-N distance in the series Gd(3+) < Eu(3+) < Sm(3+) reflects an increase in the ionic radii, which are in good agreement with previously published XRD data on Eu(III)(oep)(2). Moreover, the protonated Ln(III)H(oep)(tpp) and Ln(III)H(tpp)(2) complexes possess systematically shorter distances of about 0.02 A between the XAFS and XRD data. This

  17. Impedance spectroscopic characterization of gadolinium substituted cobalt ferrite ceramics

    SciTech Connect

    Rahman, Md. T. Ramana, C. V.

    2014-10-28

    Gadolinium (Gd) substituted cobalt ferrites (CoFe{sub 2−x}Gd{sub x}O{sub 4}, referred to CFGO) with variable Gd content (x = 0.0–0.4) have been synthesized by solid state ceramic method. The crystal structure and impedance properties of CFGO compounds have been evaluated. X-ray diffraction measurements indicate that CFGO crystallize in the inverse spinel phase. The CFGO compounds exhibit lattice expansion due to substitution of larger Gd ions into the crystal lattice. Impedance spectroscopy analysis was performed under a wide range of frequency (f = 20 Hz–1 MHz) and temperature (T = 303–573 K). Electrical properties of Gd incorporated Co ferrite ceramics are enhanced compared to pure CoFe{sub 2}O{sub 4} due to the lattice distortion. Impedance spectroscopic analysis illustrates the variation of bulk grain and grain-boundary contributions towards the electrical resistance and capacitance of CFGO materials with temperature. A two-layer heterogeneous model consisting of moderately conducting grain interior (ferrite-phase) regions separated by insulating grain boundaries (resistive-phase) accurately account for the observed temperature and frequency dependent electrical characteristic of CFGO ceramics.

  18. Gadolinium Doped Water Cherenkov Detector for use as Neutron Detector

    NASA Astrophysics Data System (ADS)

    Davis, Patrick; Woltman, Brian; Mei, Dongming; Sun, Yongchen; Thomas, Keenan; Perevozchikov, Oleg

    2010-11-01

    Background characterization is imperative to the success of rare event physics research such as neutrinoless double-beta decay and dark matter searches. There are a number of different ways to measure backgrounds from muon-induced processes and other forms of high energy events. In our current research, we are constructing a research and development project for the feasibility of a Gadolinium doped water Cherenkov detector as a neutron detector. We are constructing a 46 liter acrylic housing for the Gd-doped water consisting of two acrylic cone sections connected to a middle acrylic cylinder to increase volume while still using 5 inch photo multiplier tubes (PMTs) on either end. I will present the challenges of a Gd-doped water detector and the reasons why our design should be much more successful than past metal housed detectors. I will also discuss our current progress and future goals of our detector including its use in characterizing the background in the future underground laboratory in the Sanford Lab, soon to be DUSEL.

  19. Gadolinium oxide nanoparticles as potential multimodal imaging and therapeutic agents.

    PubMed

    Kim, Tae Jeong; Chae, Kwon Seok; Chang, Yongmin; Lee, Gang Ho

    2013-01-01

    Potentials of hydrophilic and biocompatible ligand coated gadolinium oxide nanoparticles as multimodal imaging agents, drug carriers, and therapeutic agents are reviewed. First of all, they can be used as advanced T1 magnetic resonance imaging (MRI) contrast agents because they have r1 larger than those of Gd(III)-chelates due to a high density of Gd(III) per nanoparticle. They can be further functionalized by conjugating other imaging agents such as fluorescent imaging (FI), X-ray computed tomography (CT), positron emission tomography (PET), and single photon emission tomography (SPECT) agents. They can be also useful for drug carriers through morphology modifications. They themselves are also potential CT and ultrasound imaging (USI) contrast and thermal neutron capture therapeutic (NCT) agents, which are superior to commercial iodine compounds, air-filled albumin microspheres, and boron ((10)B) compounds, respectively. They, when conjugated with targeting agents such as antibodies and peptides, will provide enhanced images and be also very useful for diagnosis and therapy of diseases (so called theragnosis). PMID:23432005

  20. Cationic gadolinium chelate for magnetic resonance imaging of cartilaginous defects.

    PubMed

    Nwe, Kido; Huang, Ching-Hui; Qu, Feini; Warden-Rothman, Robert; Zhang, Clare Y; Mauck, Robert L; Tsourkas, Andrew

    2016-05-01

    The ability to detect meniscus defects by magnetic resonance arthrography (MRA) can be highly variable. To improve the delineation of fine tears, we synthesized a cationic gadolinium complex, (Gd-DOTA-AM4 )(2+) , that can electrostatically interact with Glycosaminoglycans (GAGs). The complex has a longitudinal relaxivity (r1) of 4.2 mM(-1) s(-1) and is highly stable in serum. Its efficacy in highlighting soft tissue tears was evaluated in comparison to a clinically employed contrast agent (Magnevist) using explants obtained from adult bovine menisci. In all cases, Gd-DOTA-AM4 appeared to improve the ability to detect the soft tissue defect by providing increased signal intensity along the length of the tear. Magnevist shows a strong signal near the liquid-meniscus interface, but much less contrast is observed within the defect at greater depths. This provides initial evidence that cationic contrast agents can be used to improve the diagnostic accuracy of MRA. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26853708

  1. LASERS: Efficient neodymium-doped gadolinium gallium garnet crystal laser

    NASA Astrophysics Data System (ADS)

    Doroshenko, Maxim E.; Osiko, Vyacheslav V.; Sigachev, V. B.; Timoshechkin, M. I.

    1991-07-01

    An investigation was made of the stimulated emission parameters of a laser utilizing a gadolinium gallium garnet crystal doped with neodymium ions (YAG:Nd) at the 1.062 μm wavelength. The free-running efficiency was the highest so far achieved for flashlamp-pumped lasers utilizing unsensitized garnets. For an active element 8 mm in diameter and 120 mm long the absolute efficiency was 5.4% and the differential efficiency was 5.9%. The average free-running power was 170 W. A comparison was made of the optical powers of thermal lenses in cylindrical GGG:Nd and YAG:Nd active elements and this was found to be 2.4 times higher for a GGG:Nd crystal at the same pump powers. It was shown that by using traditional methods of compensating for the thermal lens in cylindrical active elements, it is possible to develop pulsed GGG:Nd crystal lasers having an average output power higher than 100 W, an efficiency of ~ 4%, and an angular divergence of less than 10 mrad.

  2. Susceptibility-Based Analysis Of Dynamic Gadolinium Bolus Perfusion MRI

    PubMed Central

    Bonekamp, David; Barker, Peter B.; Leigh, Richard; van Zijl, Peter C.M.; Li, Xu

    2014-01-01

    Purpose An algorithm is developed for the reconstruction of dynamic, gadolinium (Gd) bolus MR perfusion images of the human brain, based on quantitative susceptibility mapping (QSM). Methods The method is evaluated in 5 perfusion scans obtained from 4 different patients scanned at 3T, and compared to the conventional analysis based on changes in the transverse relaxation rate ΔR2* and to theoretical predictions. QSM images were referenced to ventricular CSF for each dynamic of the perfusion sequence. Results Images of cerebral blood flow and blood volume were successfully reconstructed from the QSM-analysis, and were comparable to those reconstructed using ΔR2*. The magnitudes of the Gd-associated susceptibility effects in gray and white matter were consistent with theoretical predictions. Conclusion QSM-based analysis may have some theoretical advantages compared to ΔR2*, including a simpler relationship between signal change and Gd concentration. However, disadvantages are its much lower contrast-to-noise ratio, artifacts due to respiration and other effects, and more complicated reconstruction methods. More work is required to optimize data acquisition protocols for QSM-based perfusion imaging. PMID:24604343

  3. Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

    PubMed Central

    Cappadona, Ilaria; Villinger, Clarissa; Schutzius, Gabi; Mertens, Thomas

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for

  4. Flexibility in Surface-Exposed Loops in a Virus Capsid Mediates Escape from Antibody Neutralization

    PubMed Central

    Kolawole, Abimbola O.; Li, Ming; Xia, Chunsheng; Fischer, Audrey E.; Giacobbi, Nicholas S.; Rippinger, Christine M.; Proescher, Jody B. G.; Wu, Susan K.; Bessling, Seneca L.; Gamez, Monica; Yu, Chenchen; Zhang, Rebecca; Mehoke, Thomas S.; Pipas, James M.; Wolfe, Joshua T.; Lin, Jeffrey S.; Feldman, Andrew B.

    2014-01-01

    ABSTRACT New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A′B′ and E′F′ loops. Six mutations in the E′F′ loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of

  5. Pattern formation in icosahedral virus capsids: the papova viruses and Nudaurelia capensis beta virus.

    PubMed Central

    Marzec, C J; Day, L A

    1993-01-01

    The capsids of the spherical viruses all show underlying icosahedral symmetry, yet they differ markedly in capsomere shape and in capsomere position and orientation. The capsid patterns presented by the capsomere shapes, positions, and orientations of three viruses (papilloma, SV40, and N beta V) have been generated dynamically through a bottom-up procedure which provides a basis for understanding the patterns. A capsomere shape is represented in two-dimensional cross-section by a mass or charge density on the surface of a sphere, given by an expansion in spherical harmonics, and referred to herein as a morphological unit (MU). A capsid pattern is represented by an icosahedrally symmetrical superposition of such densities, determined by the positions and orientations of its MUs on the spherical surface. The fitness of an arrangement of MUs is measured by an interaction integral through which all capsid elements interact with each other via an arbitrary function of distance. A capsid pattern is generated by allowing the correct number of approximately shaped MUs to move dynamically on the sphere, positioning themselves until an extremum of the fitness function is attained. The resulting patterns are largely independent of the details of both the capsomere representation and the interaction function; thus the patterns produced are generic. The simplest useful fitness function is sigma 2, the average square of the mass (or charge) density, a minimum of which corresponds to a "uniformly spaced" MU distribution; to good approximation, the electrostatic free energy of charged capsomeres, calculated from the linearized Poisson-Boltzmann equation, is proportional to sigma 2. With disks as MUs, the model generates the coordinated lattices familiar from the quasi-equivalence theory, indexed by triangulation numbers. Using fivefold MUs, the model generates the patterns observed at different radii within the T = 7 capsid of papilloma and at the surface of SV40; threefold MUs

  6. Structure and Dynamics of Full Length HIV-1 Capsid Protein in Solution

    PubMed Central

    Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L.; Clore, G. Marius

    2013-01-01

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144–231) and crystal (residues 146–231) structures by ~30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time-scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  7. The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination

    PubMed Central

    Dearborn, Altaira D.; Spilman, Michael S.; Damle, Priyadarshan K.; Chang, Jenny R.; Monroe, Eric B.; Saad, Jamil S.; Christie, Gail E.; Dokland, Terje

    2011-01-01

    Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome, but can become mobilized by “helper” bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage, but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix-loop-helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-EM reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination. PMID:21821042

  8. Hybrid Calcium Phosphate-Polymeric Micelles Incorporating Gadolinium Chelates for Imaging-Guided Gadolinium Neutron Capture Tumor Therapy.

    PubMed

    Mi, Peng; Dewi, Novriana; Yanagie, Hironobu; Kokuryo, Daisuke; Suzuki, Minoru; Sakurai, Yoshinori; Li, Yanmin; Aoki, Ichio; Ono, Koji; Takahashi, Hiroyuki; Cabral, Horacio; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2015-06-23

    Gadolinium (Gd) chelates-loaded nanocarriers have high potential for achieving magnetic resonance imaging (MRI)-guided Gd neutron capture therapy (GdNCT) of tumors. Herein, we developed calcium phosphate micelles hybridized with PEG-polyanion block copolymers, and incorporated with the clinical MRI contrast agent Gd-diethylenetriaminepentaacetic acid (Gd-DTPA/CaP). The Gd-DTPA/CaP were nontoxic to cancer cells at the concentration of 100 μM based on Gd-DTPA, while over 50% of the cancer cells were killed by thermal neutron irradiation at this concentration. Moreover, the Gd-DTPA/CaP showed a dramatically increased accumulation of Gd-DTPA in tumors, leading to the selective contrast enhancement of tumor tissues for precise tumor location by MRI. The enhanced tumor-to-blood distribution ratio of Gd-DTPA/CaP resulted in the effective suppression of tumor growth without loss of body weight, indicating the potential of Gd-DTPA/CaP for safe cancer treatment. PMID:26033034

  9. CHARACTERIZATION OF AN ADVANCED GADOLINIUM NEUTRON ABSORBER ALLOY BY MEANS OF NEUTRON TRANSMISSION

    SciTech Connect

    Gregg W. Wachs

    2007-09-01

    Neutron transmission experiments were performed on samples of an advanced nickel-chromium-molybdenum-gadolinium (Ni-Cr-Mo-Gd) neutron absorber alloy. The primary purpose of the experiments was to demonstrate the thermal neutron absorbing capability of the alloy at specific gadolinium dopant levels. The new alloy is to be deployed for criticality control of highly enriched DOE SNF. For the transmission experiments, alloy test samples were fabricated with 0.0, 1.58 and 2.1 wt% natural gadolinium dispersed in a Ni-Cr-Mo base alloy. The transmission experiments were successfully carried out at the Los Alamos Neutron Science Center (LANSCE). Measured data from the neutron transmission experiments were compared to calculated results derived from a simple exponential transmission formula using only radiative capture cross sections. Excellent agreement between the measured and calculated results demonstrated the expected strong thermal absorption capability of the gadolinium poison and in addition, verified the measured elemental composition of the alloy test samples. The good agreement also indirectly confirmed that the gadolinium was dispersed fairly uniformly in the alloy and the ENDF VII radiative capture cross section data were accurate.

  10. The use of iodinated and gadolinium contrast media during pregnancy and lactation.

    PubMed

    Webb, Judith A W; Thomsen, Henrik S; Morcos, Sameh K

    2005-06-01

    The use of iodinated or gadolinium-based contrast media in pregnant or lactating women often causes concerns in the radiology department because of the principle of not exposing a fetus or neonate to any drugs. Because of the uncertainty about the use of contrast media during pregnancy and lactation, the Contrast Media Safety Committee of the European Society of Urogenital Radiology decided to review the literature and draw up guidelines. An extensive literature search was carried out and summarized in a report. Based on the limited information available, simple guidelines have been drawn up. The report and guidelines were discussed at the 11th European Symposium on Urogenital Radiology in Santiago de Compostela, Spain. Mutagenic and teratogenic effects have not been described after administration of gadolinium or iodinated contrast media. Free iodide in radiographic contrast medium given to the mother has the potential to depress fetal/neonatal thyroid function. Neonatal thyroid function should be checked during the 1st week if iodinated contrast media have been given during pregnancy. No effect on the fetus has been seen after gadolinium contrast media. Only tiny amounts of iodinated or gadolinium-based contrast medium given to a lactating mother reach the milk, and only a minute proportion entering the baby's gut is absorbed. The very small potential risk associated with absorption of contrast medium may be considered insufficient to warrant stopping breast-feeding for 24 h following either iodinated or gadolinium contrast agents. PMID:15609057

  11. Gadolinium-containing MRI contrast agents: important variations on a theme for NSF.

    PubMed

    Kuo, Phillip H

    2008-01-01

    Millions of doses of gadolinium-based contrast agents (GBCAs) are administered annually to improve the clinical utility of magnetic resonance imaging. All the approved agents incorporate one atom of the rare earth metal gadolinium into a chelate to improve the safety of the ordinarily toxic free gadolinium. The undeniable epidemiologic link between GBCAs and nephrogenic systemic fibrosis (NSF) has prompted renewed investigation into the different chemical properties of the GBCAs despite their clinical interchangeability. Gadolinium-based contrast agents can be divided into different categories: linear versus macrocyclic structure, ionic versus nonionic, and non-protein-binding versus protein-binding agents. The GBCAs differ significantly with respect to transmetallation and kinetic and thermodynamic stability and therefore their propensity to release free gadolinium, which is hypothesized to induce NSF. That gadodiamide, with its susceptibility to transmetallation and relatively low thermodynamic and kinetic stability, is associated with the most cases of NSF supports this hypothesis. On the other hand, the greater stability of a macrocyclic agent hypothetically would confer a greater safety margin with regard to NSF. Because few published data on an experimental model of NSF exist, continuing vigilance is necessary to report new cases of NSF, especially with regard to the agents with small market share. PMID:18180006

  12. Extraction of gadolinium from high flux isotope reactor control plates. [Alternative method

    SciTech Connect

    Kohring, M.W.

    1987-04-01

    Gadolinium-153 is an important radioisotope used in the diagnosis of various bone disorders. Recent medical and technical developments in the detection and cure of osteoporosis, a bone disease affecting an estimated 50 million people, have greatly increased the demand for this isotope. The Oak Ridge National Laboratory (ORNL) has produced /sup 153/Gd since 1980 primarily through the irradiation of a natural europium-oxide powder followed by the chemical separation of the gadolinium fraction from the europium material. Due to the higher demand for /sup 153/Gd, an alternative production method to supplement this process has been investigated. This process involves the extraction of gadolinium from the europium-bearing region of highly radioactive, spent control plates used at the High Flux Isotope Reactor (HFIR) with a subsequent re-irradiation of the extracted material for the production of the /sup 153/Gd. Based on the results of experimental and calculational analyses, up to 25 grams of valuable gadolinium (greater than or equal to60% enriched in /sup 152/Gd) resides in the europium-bearing region of the HFIR control components of which 70% is recoverable. At a specific activity yield of 40 curies of /sup 153/Gd for each gram of gadolinium re-irradiated, 700 one-curie sources can be produced from each control plate assayed.

  13. Tumor-induced lymph node alterations detected by MRI lymphography using gadolinium nanoparticles

    PubMed Central

    Partridge, S. C.; Kurland, B. F.; Liu, C.-L.; Ho, R. J. Y.; Ruddell, A.

    2015-01-01

    Contrast-enhanced MRI lymphography shows potential to identify alterations in lymph drainage through lymph nodes (LNs) in cancer and other diseases. MRI studies have typically used low molecular weight gadolinium contrast agents, however larger gadolinium-loaded nanoparticles possess characteristics that could improve the specificity and sensitivity of lymphography. The performance of three gadolinium contrast agents with different sizes and properties was compared by 3T MRI after subcutaneous injection. Mice bearing B16-F10 melanoma footpad tumors were imaged to assess tumor-induced alterations in lymph drainage through tumor-draining popliteal and inguinal LNs versus contralateral uninvolved drainage. Gadolinium lipid nanoparticles were able to identify tumor-induced alterations in contrast agent drainage into the popliteal LN, while lower molecular weight or albumin-binding gadolinium agents were less effective. All of the contrast agents distributed in foci around the cortex and medulla of tumor-draining popliteal LNs, while they were restricted to the cortex of non-draining LNs. Surprisingly, second-tier tumor-draining inguinal LNs exhibited reduced uptake, indicating that tumors can also divert LN drainage. These characteristics of tumor-induced lymph drainage could be useful for diagnosis of LN pathology in cancer and other diseases. The preferential uptake of nanoparticle contrasts into tumor-draining LNs could also allow selective targeting of therapies to tumor-draining LNs. PMID:26497382

  14. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    PubMed

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  15. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    PubMed

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  16. Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy.

    PubMed

    Liu, An-An; Zhang, Zhenfeng; Sun, En-Ze; Zheng, Zhenhua; Zhang, Zhi-Ling; Hu, Qinxue; Wang, Hanzhong; Pang, Dai-Wen

    2016-01-26

    Real-time, long-term, single-particle tracking (SPT) provides us an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, we have established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technology. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus. PMID:26720596

  17. GADOLINIUM OXALATE SOLUBILITY MEASUREMENTS IN NITRIC ACID SOLUTIONS

    SciTech Connect

    Pierce, R. A.

    2012-03-12

    HB-Line will begin processing Pu solutions during FY2012 that will involve the recovery of Pu using oxalate precipitation and filtration. After the precipitation and filtration processes, the filtrate solution will be transferred from HB-Line to H-Canyon. The presence of excess oxalate and unfiltered Pu oxalate solids in these solutions create a criticality safety issue if they are sent to H-Canyon without controls in H-Canyon. One approach involves H-Canyon receiving the filtrate solution into a tank that is poisoned with soluble gadolinium (Gd). Decomposition of the oxalate will occur within a subsequent H-Canyon vessel. The receipt of excess oxalate into the H-Canyon receipt tanks has the potential to precipitate a portion of the Gd poison in the receipt tanks. Because the amount of Gd in solution determines the maximum amount of Pu solids that H-Canyon can receive, H-Canyon Engineering requested that SRNL determine the solubility of Gd in aqueous solutions of 4-10 M nitric acid (HNO{sub 3}), 4-12 g/L Gd, and 0.15-0.25 M oxalic acid (H{sub 2}C{sub 2}O{sub 4}) at 25 °C. The target soluble Gd concentration is 6 g/L. The data indicate that the target can be achieved above 6 M HNO{sub 3} and below 0.25 M H{sub 2}C{sub 2}O{sub 4}. At 25 °C, for 6 M HNO{sub 3}, 11 g/L and 7 g/L Gd are soluble in 0.15 M and 0.25 M H{sub 2}C{sub 2}O{sub 4}, respectively. In 4 M HNO{sub 3}, the Gd solubility drops significantly to 2.5 g/L and 0.8 g/L in 0.15 M and 0.25 M H{sub 2}C{sub 2}O{sub 4}, respectively. The solubility of Gd at 8-10 M HNO{sub 3} exceeds the solubility at 6 M HNO{sub 3}. The data for 4 M HNO{sub 3} showed good agreement with data in the literature. To achieve a target of 6 g/L soluble Gd in solution in the presence of 0.15-0.25 M oxalate, the HNO{sub 3} concentration must be maintained at or above 6 M HNO{sub 3}. The solubility of Gd in 4 M HNO{sub 3} with 0.15 M oxalate at 10 °C is about 1.5 g/L. For 6 M HNO{sub 3} with 0.15 M oxalate, the solubility of Gd at 10

  18. Chronic hepatitis B infection and HBV DNA-containing capsids: Modeling and analysis

    NASA Astrophysics Data System (ADS)

    Manna, Kalyan; Chakrabarty, Siddhartha P.

    2015-05-01

    We analyze the dynamics of chronic HBV infection taking into account both uninfected and infected hepatocytes along with the intracellular HBV DNA-containing capsids and the virions. While previous HBV models have included either the uninfected hepatocytes or the intracellular HBV DNA-containing capsids, our model accounts for both these two populations. We prove the conditions for local and global stability of both the uninfected and infected steady states in terms of the basic reproduction number. Further, we incorporate a time lag in the model to encompass the intracellular delay in the production of the infected hepatocytes and find that this delay does not affect the overall dynamics of the system. The results for the model and the delay model are finally numerically illustrated.

  19. Simulation studies of a phenomenological model for elongated virus capsid formation

    NASA Astrophysics Data System (ADS)

    Chen, Ting; Glotzer, Sharon C.

    2007-05-01

    We study a phenomenological model in which the simulated packing of hard, attractive spheres on a prolate spheroid surface with convexity constraints produces structures identical to those of prolate virus capsid structures. Our simulation approach combines the traditional Monte Carlo method with a modified method of random sampling on an ellipsoidal surface and a convex hull searching algorithm. Using this approach we identify the minimum physical requirements for nonicosahedral, elongated virus capsids, such as two aberrant flock house virus particles and the prolate prohead of bacteriophage ϕ29 , and discuss the implication of our simulation results in the context of recent experimental findings. Our predicted structures may also be experimentally realized by the evaporation-driven assembly of colloidal spheres under appropriate conditions.

  20. Shepherd model for knot-limited polymer ejection from a capsid.

    PubMed

    Antal, Tibor; Krapivsky, P L; Redner, S

    2009-12-01

    We construct a tractable model to describe the rate at which a knotted polymer is ejected from a spherical capsid via a small pore. Knots are too large to fit through the pore and must reptate to the end of the polymer for ejection to occur. The reptation of knots is described by symmetric exclusion on the line, with the internal capsid pressure represented by an additional biased particle that drives knots to the end of the chain. We compute the exact ejection speed for a finite number of knots L and find that it scales as 1/L. We establish a mapping to the solvable zero-range process. We also construct a continuum theory for many knots that matches the exact discrete theory for large L. PMID:19703473

  1. Virus capsid expansion driven by the capture of mobile surface loops

    PubMed Central

    Lee, Kelly K.; Gan, Lu; Tsuruta, Hiro; Moyer, Crystal; Conway, James F.; Duda, Robert L.; Hendrix, Roger W.; Steven, Alasdair C.; Johnson, John E.

    2008-01-01

    Summary The capsids of tailed DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking occurring during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in crosslink pattern accompanies conversion of a semi-mature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the Balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching. PMID:18940605

  2. Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

    NASA Astrophysics Data System (ADS)

    Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

    1995-01-01

    Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

  3. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

    SciTech Connect

    Liu Yingjie; Wu Jinlu; Chen Hu; Hew, Choy Leong; Yan Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  4. Virus Capsid Expansion Driven By the Capture of Mobile Surface Loops

    SciTech Connect

    Lee, K.K.; Gan, L.; Tsuruta, H.; Moyer, C.; Conway, J.F.; Duda, R.L.; Hendrix, R.W.; Steven, A.C.; Johnson, J.E.

    2009-05-20

    The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching.

  5. Patterns of late gadolinium enhancement in Duchenne muscular dystrophy carriers

    PubMed Central

    2014-01-01

    Background This study was designed to assess whether cardiovascular magnetic resonance imaging (CMR) in Duchenne muscular dystrophy carriers (DMDc) may index any cell milieu elements of LV dysfunction and whether this cardiac phenotype may be related to genotype. The null hypothesis was that myocardial fibrosis, assessed by late gadolinium enhancement (LGE), might be similarly accounted for in DMDc and gender and age-matched controls. Methods Thirty DMDc patients had CMR and genotyping with 37 gender and age-matched controls. Systolic and diastolic LV function was assessed by 2D-echocardiography. Results Absolute and percent LGE were higher in muscular symptomatic (sym) than asymptomatic (asy) DMDc (1.77 ± 0.27 vs 0.76 ± 0.17 ml; F = 19.6, p < 0.0001 and 1.86 ± 0.26% vs 0.68 ± 0.17%, F = 22.1, p < 0.0001, respectively). There was no correlation between LGE and age. LGE was seen most frequently in segments 5 and 6; segment 5 was involved in all asy-DMDc. Subepicardial LGE predominated, compared to the mid-myocardial one (11 out of 14 DMDc). LGE was absent in the subendocardium. No correlations were seen between genotyping (type of mutation, gene region and protein domain), confined to the exon’s study, and cardiac phenotype. Conclusions A typical myocardial LGE-pattern location (LV segments 5 and 6) was a common finding in DMDc. LGE was more frequently subepicardial plus midmyocardial in sym-DMDc, with normal LV systolic and diastolic function. No genotype-phenothype correlation was found. PMID:25008475

  6. Utility of late gadolinium enhancement in pediatric cardiac MRI.

    PubMed

    Etesami, Maryam; Gilkeson, Robert C; Rajiah, Prabhakar

    2016-07-01

    Late gadolinium enhancement (LGE) cardiac magnetic resonance (MR) imaging sequence is increasingly used in the evaluation of pediatric cardiovascular disorders, and although LGE might be a normal feature at the sites of previous surgeries, it is pathologically seen as a result of extracellular space expansion, either from acute cell damage or chronic scarring or fibrosis. LGE is broadly divided into ischemic and non-ischemic patterns. LGE caused by myocardial infarction occurs in a vascular distribution and always involves the subendocardial portion, progressively involving the outer regions in a waveform pattern. Non-ischemic cardiomyopathies can have a mid-myocardial (either linear or patchy), subepicardial or diffuse subendocardial distribution. Idiopathic dilated cardiomyopathy can have a linear mid-myocardial pattern, while hypertrophic cardiomyopathy can have fine, patchy enhancement in hypertrophied and non-hypertrophied segments as well as right ventricular insertion points. Myocarditis and sarcoidosis have a mid-myocardial or subepicardial pattern of LGE. Fabry disease typically affects the basal inferolateral segment while Danon disease typically spares the septum. Pericarditis is characterized by diffuse or focal pericardial thickening and enhancement. Thrombus, the most common non-neoplastic cardiac mass, is characterized by absence of enhancement in all sequences, while neoplastic masses show at least some contrast enhancement, depending on the pathology. Regardless of the etiology, presence of LGE is associated with a poor prognosis. In this review, we describe the technical modifications required for performing LGE cardiac MR sequence in children, review and illustrate the patterns of LGE in children, and discuss their clinical significance. PMID:26718199

  7. Gadolinium block of calcium channels: influence of bicarbonate.

    PubMed

    Boland, L M; Brown, T A; Dingledine, R

    1991-11-01

    The selectivity of block of voltage-activated barium (Ba2+) currents by lanthanide ions was studied in a rat dorsal root ganglion (DRG) cell line (F11-B9), rat and frog peripheral neurons, and rat cardiac myocytes using the whole-cell patch clamp technique. Gadolinium (Gd3+) produced a dose-dependent and complete inhibition of whole-cell Ba2+ current in all cells studied, including cells expressing identified dihydropyridine-sensitive L-type currents and omega-conotoxin-sensitive N-type currents. Like Gd3+, lutetium (Lu3+) and lanthanum (La3+) blocked all Ba2+ current with little selectivity for different components of the whole-cell current. Gd3+ block of Ba2+ currents was incomplete, however, when sodium bicarbonate (5-22.6 mM) was added to the standard HEPES-buffered external Ba2+ solution. In rat DRG neurons and F11-B9 cells, a fraction of the whole-cell Ba2+ current recorded in the presence of bicarbonate was resistant to block by saturating concentrations of Gd3+ (50-100 microM). The resistant current inactivated more rapidly than the original current giving the appearance that, under these conditions, Gd3+ block is more selective for the slowly inactivating component of the whole-cell current. Bicarbonate modification of Gd3+ block occurred both before and after omega-conotoxin block of N-type currents in rat DRG neurons, suggesting that even in the presence of bicarbonate, Gd3+ block was not selective for N-type currents. PMID:1786527

  8. Status of the Super-Kamiokande gadolinium project

    NASA Astrophysics Data System (ADS)

    Mori, Takaaki

    2013-12-01

    The diffuse flux of neutrinos released from all past supernova explosions in the universe is known as the supernova relic neutrinos (SRN). Super-Kamiokande (SK) has conducted searches for these SRN events via their inverse beta decay interactions in the detector, in the process establishing the world's best limits on this still unobserved flux. These limits are within about a factor of two of the theoretically predicted fluxes. But these searches are background limited, and without some major improvement further progress will be difficult. The addition of gadolinium (Gd) compound into the SK detector was proposed to reduce background. Gd has the largest thermal neutron capture cross-section among all stable nuclei and emits an 8 MeV γ cascade following the capture. By coincidental tagging of positrons with the γ rays from Gd neutron capture, we can identify the dominant SRN signal in SK: inverse beta decay. This Gd-loading technique should allow SK to make the world's first observation of a SRN signal. We will demonstrate the principle of a Gd-doped water Cherenkov detector (transparency of the Gd-doped water, Gd-doped water circulation method, neutron capture efficiency, etc) with a dedicated test facility named EGADS. EGADS consists of a 200 ton water Cherenkov detector, a Gd dissolving pre-treatment device, a Gd-capable water circulation system, and a custom-built water transparency measurement device. We have evaluated Gd-doped water circulation using the main EGADS water system since 2012. The evaluation of the overall performance of EGADS will start in 2013 after PMT installation.

  9. Reprogramming Immune Response With Capsid-Optimized AAV6 Vectors for Immunotherapy of Cancer.

    PubMed

    Pandya, Munjal; Britt, Kellee; Hoffman, Brad; Ling, Chen; Aslanidi, George V

    2015-09-01

    In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors. PMID:26261893

  10. Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

    PubMed Central

    Ke, Danxia; Ning, Jiying; DeLucia, Maria; Ahn, Jinwoo; Gronenborn, Angela M.; Aiken, Christopher; Zhang, Peijun

    2012-01-01

    During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles. PMID:22927821

  11. TRIM5α Disrupts the Structure of Assembled HIV-1 Capsid Complexes In Vitro▿

    PubMed Central

    Black, Lesa R.; Aiken, Christopher

    2010-01-01

    The host restriction factor TRIM5α provides intrinsic defense against retroviral infections in mammalian cells. TRIM5α blocks infection by targeting the viral capsid after entry but prior to completion of reverse transcription, but whether this interaction directly alters the structure of the viral capsid is unknown. A previous study reported that rhesus macaque TRIM5α protein stably associates with cylindrical complexes formed by assembly of recombinant HIV-1 CA-NC protein in vitro and that restriction leads to accelerated HIV-1 uncoating in target cells. To gain further insight into the mechanism of TRIM5α-dependent restriction, we examined the structural effects of TRIM5 proteins on preassembled CA-NC complexes by electron microscopy. Incubation of assembled complexes with lysate of cells expressing the restrictive rhesus TRIM5α protein resulted in marked disruption of the normal cylindrical structure of the complexes. In contrast, incubation with lysate of control cells or cells expressing comparable levels of the nonrestrictive human TRIM5α protein had little effect on the complexes. Incubation with lysate of cells expressing the TRIMCyp restriction factor also disrupted the cylinders. The effect of TRIMCyp was prevented by the addition of cyclosporine, which inhibits binding of TRIMCyp to the HIV-1 capsid. Thus, disruption of CA-NC cylinders by TRIM5α and TRIMCyp was correlated with the specificity of restriction. Collectively, these results suggest that TRIM5α-dependent restriction of HIV-1 infection results from structural perturbation of the viral capsid leading to aberrant HIV-1 uncoating in target cells. PMID:20410272

  12. TRIM5alpha disrupts the structure of assembled HIV-1 capsid complexes in vitro.

    PubMed

    Black, Lesa R; Aiken, Christopher

    2010-07-01

    The host restriction factor TRIM5alpha provides intrinsic defense against retroviral infections in mammalian cells. TRIM5alpha blocks infection by targeting the viral capsid after entry but prior to completion of reverse transcription, but whether this interaction directly alters the structure of the viral capsid is unknown. A previous study reported that rhesus macaque TRIM5alpha protein stably associates with cylindrical complexes formed by assembly of recombinant HIV-1 CA-NC protein in vitro and that restriction leads to accelerated HIV-1 uncoating in target cells. To gain further insight into the mechanism of TRIM5alpha-dependent restriction, we examined the structural effects of TRIM5 proteins on preassembled CA-NC complexes by electron microscopy. Incubation of assembled complexes with lysate of cells expressing the restrictive rhesus TRIM5alpha protein resulted in marked disruption of the normal cylindrical structure of the complexes. In contrast, incubation with lysate of control cells or cells expressing comparable levels of the nonrestrictive human TRIM5alpha protein had little effect on the complexes. Incubation with lysate of cells expressing the TRIMCyp restriction factor also disrupted the cylinders. The effect of TRIMCyp was prevented by the addition of cyclosporine, which inhibits binding of TRIMCyp to the HIV-1 capsid. Thus, disruption of CA-NC cylinders by TRIM5alpha and TRIMCyp was correlated with the specificity of restriction. Collectively, these results suggest that TRIM5alpha-dependent restriction of HIV-1 infection results from structural perturbation of the viral capsid leading to aberrant HIV-1 uncoating in target cells. PMID:20410272

  13. Magnetocaloric effect of an Fe-based metallic glass compared to benchmark gadolinium

    NASA Astrophysics Data System (ADS)

    Waske, A.; Hermann, H.; Mattern, N.; Skokov, K.; Gutfleisch, O.; Eckert, J.

    2012-12-01

    We report on the magnetocaloric effect in an Fe-based metallic glass (Fe80B12Nb8) as compared to the benchmark material gadolinium. From temperature-dependent magnetization measurements, the magnetic entropy change was calculated using the thermodynamic Maxwell relations. The adiabatic temperature change was directly measured for both materials using a dedicated setup. An analysis of the magnetic transition in amorphous Fe80B12Nb8 and crystalline gadolinium using a mean field and a phenomenological model was carried out. It was shown that both materials, in particular crystalline gadolinium, which does not possess structural disorder but merely a fluctuation of the exchange integral, can be described using the Handrich model for the magnetic transition of disordered materials. Furthermore, the Landau theory of second-order phase transitions quantitatively describes the magnetic entropy change and its dependence on the applied field very well for both materials with different definitions of disorder.

  14. Effect of gadolinium on the properties of Pr-Dy-Fe-Co-B materials

    NASA Astrophysics Data System (ADS)

    Kablov, E. N.; Ospennikova, O. G.; Piskorskii, V. P.; Rezchikova, I. I.; Valeev, R. A.; Korolev, D. V.

    2015-11-01

    Sintered (Pr1- x- y Dy x Gd y )13-14(Fe1- z Co z )balB6-7 materials ( x = 0.18-0.58, y = 0.05-0.33, z = 0.2-0.36) have been studied. The magnetic moments of gadolinium ions and those of the sublattice formed by Fe and Co ions are shown to be ordered antiferromagnetically. It is noted that an increase in the content of gadolinium, which substitutes for dysprosium, leads to an increase in residual induction B r , a decrease in coercive force H cJ , and an increase in the absolute value of the temperature coefficient of induction. The opposite effect takes place in the case of substitution of gadolinium for praseodymium in materials with a fixed dysprosium content.

  15. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors

    NASA Astrophysics Data System (ADS)

    Dufort, Sandrine; Le Duc, Géraldine; Salomé, Murielle; Bentivegna, Valerie; Sancey, Lucie; Bräuer-Krisch, Elke; Requardt, Herwig; Lux, François; Coll, Jean-Luc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

    2016-07-01

    We recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps.

  16. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors

    PubMed Central

    Dufort, Sandrine; Le Duc, Géraldine; Salomé, Murielle; Bentivegna, Valerie; Sancey, Lucie; Bräuer-Krisch, Elke; Requardt, Herwig; Lux, François; Coll, Jean-Luc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

    2016-01-01

    We recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps. PMID:27411781

  17. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors.

    PubMed

    Dufort, Sandrine; Le Duc, Géraldine; Salomé, Murielle; Bentivegna, Valerie; Sancey, Lucie; Bräuer-Krisch, Elke; Requardt, Herwig; Lux, François; Coll, Jean-Luc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

    2016-01-01

    We recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps. PMID:27411781

  18. Discovery of dual inhibitors targeting both HIV-1 capsid and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid.

    PubMed

    Li, Jiebo; Tan, Zhiwu; Tang, Shixing; Hewlett, Indira; Pang, Ruifang; He, Meizi; He, Shanshan; Tian, Baohe; Chen, Kan; Yang, Ming

    2009-04-15

    HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid (CA) and human cyclophilin A (CypA) play essential roles in these processes. We designed and synthesized a series of thiourea compounds as HIV-1 assembly and disassembly dual inhibitors targeting both HIV-1 CA protein and human CypA. The SIV-induced syncytium antiviral evaluation indicated that all of the inhibitors displayed antiviral activities in SIV-infected CEM cells at the concentration of 0.6-15.8 microM for 50% of maximum effective rate. Their abilities to bind CA and CypA were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity and PPIase inhibition assay. Assembly studies in vitro demonstrated that the compounds could potently disrupt CA assembly with a dose-dependent manner. All of these molecules could bind CypA with binding affinities (Kd values) of 51.0-512.8 microM. Fifteen of the CypA binding compounds showed potent PPIase inhibitory activities (IC(50) values<1 microM) while they could not bind either to HIV-1 Protease or to HIV-1 Integrase in the enzyme assays. These results suggested that 15 compounds could block HIV-1 replication by inhibiting the PPIase activity of CypA to interfere with capsid disassembly and disrupting CA assembly. PMID:19328002

  19. Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages▿

    PubMed Central

    Hanson, Laura K.; Slater, Jacquelyn S.; Cavanaugh, Victoria J.; Newcomb, William W.; Bolin, Lisa L.; Nelson, Christine N.; Fetters, Lisa D.; Tang, Qiyi; Brown, Jay C.; Maul, Gerd G.; Campbell, Ann E.

    2009-01-01

    Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins. PMID:19458005

  20. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis.

    PubMed

    Jacques, David A; McEwan, William A; Hilditch, Laura; Price, Amanda J; Towers, Greg J; James, Leo C

    2016-08-18

    During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a 'molecular iris' formed by the amino-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target. PMID:27509857

  1. Hierarchical Assembly of Plasmonic Nanostructures using Virus Capsid Scaffolds on DNA Origami Tiles

    SciTech Connect

    Wang, Debin; Capehart, Stacy L.; Pal, Suchetan; Liu, Minghui; Zhang, Lei; Schuck, P. J.; Liu, Yan; Yan, Hao; Francis, Matthew B.; De Yoreo, James J.

    2014-07-07

    Plasmonic nanoarchitectures using biological scaffolds have shown the potential to attain controllable plasmonic fluorescence via precise spatial arrangement of fluorophores and plasmonic antennae. However, previous studies report a predominance of fluorescence quenching for small metal nanoparticles (less than ~60 nm) due to their small scattering cross-sections. In this work, we report the design and performance of fluorescent plasmonic structures composed of fluorophore-modified virus capsids and gold nanoparticles (AuNPs) assembled on DNA origami tiles. The virus capsid creates a scaffold for control over the three dimensional arrangement of the fluorophores, whereas the DNA origami tile provides precise control over the distance between the capsid and the AuNP. Using finite-difference time-domain (FDTD) numerical simulations and multimodal single-particle imaging measurements, we show that the judicial design of these structures places the dye molecules near the hot spot of the AuNP. This effectively increases the fluorescence intensity in the quenching regime of the AuNP, with an enhancement factor that increases with increasing AuNP size. This strategy of using biological scaffolds to control fluorescence paves the way for exploring the parameters that determine plasmonic fluorescence. It may lead to a better understanding of the antenna effects of photon absorption and emission, enabling the construction of multicomponent plasmonic systems.

  2. Directed assembly of hierarchical light-harvesting complexes using virus capsid scaffolds and DNA origami tiles

    NASA Astrophysics Data System (ADS)

    Wang, Debin; Capehart, Stacy; Pal, Suchetan; Liu, Minghui; Lau, Jolene; Yan, Hao; Francis, Matthew; Deyoreo, Jim; LBNL Team; UCB Team; ASU Team

    2013-03-01

    Directed assembly of nanostructures with molecular precision is of great importance to develop an insightful understanding of assembly pathways and dynamics as well as to derive new functionalities. In this work, we explore the use of virus capsids and DNA origami tiles as 3D scaffolds and 2D templates for directed assembly of light-harvesting molecules and plasmonic gold nanoparticles to achieve tunable photoemission. Bacteriophage MS2 virus capsids with well-defined spherical macromolecular structures are genetically modified to provide predictable steric arrangements of light-harvesting molecules. DNA origami tiles act as programmable planar templates to provide higher-order organization of oligonucleotide-functionalized light-harvesting capsids and plasmonic gold nanoparticles. The direct observation of distance dependent photoluminescence emission is carried out by our correlative approach combining atomic force microscopy and confocal fluorescence microscopy, which is in good agreement with our numerical simulation and theoretical calculation. This work will facilitate the construction of multicomponent biological-metal hybrid plasmonic nanostructures for nanophotonics and biosensing applications.

  3. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

    PubMed Central

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

  4. Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis

    PubMed Central

    Chintakuntlawar, Ashish V.; Zhou, Xiaohong; Rajaiya, Jaya; Chodosh, James

    2010-01-01

    Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins. PMID:20419141

  5. Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7*♦

    PubMed Central

    Ionel, Alina; Velázquez-Muriel, Javier A.; Luque, Daniel; Cuervo, Ana; Castón, José R.; Valpuesta, José M.; Martín-Benito, Jaime; Carrascosa, José L.

    2011-01-01

    Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses. PMID:20962334

  6. Conversion of a dodecahedral protein capsid into pentamers via minimal point mutations.

    PubMed

    Chen, Hsiao-Nung; Woycechowsky, Kenneth J

    2012-06-12

    Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis. PMID:22606973

  7. Derivation of a triple mosaic adenovirus based on modification of the minor capsid protein IX

    SciTech Connect

    Tang Yizhe; Le, Long P.; Matthews, Qiana L.; Han Tie; Wu Hongju; Curiel, David T.

    2008-08-01

    Adenoviral capsid protein IX (pIX) has been shown to be a potential locale to insert targeting, imaging-related and therapeutic modalities by genetic modification. Recent evidences suggested that capsid protein mosaicism could be a promising strategy for improving the utility of Ad vector. In this study, we explored a method to genetically generate triple pIX mosaic Ad serotype 5 (Ad5) displaying three types of pIX on a single virion. pIXs were modified at their carboxy termini with a Flag sequence, a hexahistidine sequence (His{sub 6}) or a monomeric red fluorescent protein (mRFP1), respectively. Western blotting analysis and fluorescence microscopy of the purified recombinant viruses indicated that all three modified pIXs were incorporated into the viral particles. Immuno-gold electron microscopy (EM) further confirmed that three types of pIX indeed co-existed on an individual virion. These results firstly validated a triple mosaic capsid configuration on pIX, and demonstrated the possibility of further radical design.

  8. Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.

    PubMed

    Gil-Carton, David; Jaakkola, Salla T; Charro, Diego; Peralta, Bibiana; Castaño-Díez, Daniel; Oksanen, Hanna M; Bamford, Dennis H; Abrescia, Nicola G A

    2015-10-01

    Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly. PMID:26320579

  9. Sequence Conservation, Radial Distance and Packing Density in Spherical Viral Capsids

    PubMed Central

    Lee, Chi-Wen; Huang, Tsun-Tsao; Shih, Chung-Shiuan; Hwang, Jenn-Kang

    2015-01-01

    The conservation level of a residue is a useful measure about the importance of that residue in protein structure and function. Much information about sequence conservation comes from aligning homologous sequences. Profiles showing the variation of the conservation level along the sequence are usually interpreted in evolutionary terms and dictated by site similarities of a proper set of homologous sequences. Here, we report that, of the viral icosahedral capsids, the sequence conservation profile can be determined by variations in the distances between residues and the centroid of the capsid – with a direct inverse proportionality between the conservation level and the centroid distance – as well as by the spatial variations in local packing density. Examining both the centroid and the packing density models against a dataset of 51 crystal structures of nonhomologous icosahedral capsids, we found that many global patterns and minor features derived from the viral structures are consistent with those present in the sequence conservation profiles. The quantitative link between the level of conservation and structural features like centroid-distance or packing density allows us to look at residue conservation from a structural viewpoint as well as from an evolutionary viewpoint. PMID:26132081

  10. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    PubMed

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. PMID:26414293

  11. Nature's favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold.

    PubMed

    Suhanovsky, Margaret M; Teschke, Carolyn M

    2015-05-01

    For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. PMID:25864106

  12. Unclosed HIV-1 capsids suggest a curled sheet model of assembly.

    PubMed

    Yu, Zhiheng; Dobro, Megan J; Woodward, Cora L; Levandovsky, Artem; Danielson, Cindy M; Sandrin, Virginie; Shi, Jiong; Aiken, Christopher; Zandi, Roya; Hope, Thomas J; Jensen, Grant J

    2013-01-01

    The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms. PMID:23079241

  13. Stability of Norwalk Virus Capsid Protein Interfaces Evaluated by in Silico Nanoindentation

    PubMed Central

    Boyd, Kevin J.; Bansal, Prakhar; Feng, Jun; May, Eric R.

    2015-01-01

    Norwalk virus causes severe gastroenteritis for which there is currently no specific anti-viral therapy. A stage of the infection process is uncoating of the protein capsid to expose the viral genome and allow for viral replication. A mechanical characterization of the Norwalk virus may provide important information relating to the mechanism of uncoating. The mechanical strength of the Norwalk virus has previously been investigated using atomic force microscopy (AFM) nanoindentation experiments. Those experiments cannot resolve specific molecular interactions, and therefore, we have employed a molecular modeling approach to gain insights into the potential uncoating mechanism of the Norwalk capsid. In this study, we perform simulated nanoindentation using a coarse-grained structure-based model, which provides an estimate of the spring constant in good agreement with the experimentally determined value. We further analyze the fracture mechanisms and determine weak interfaces in the capsid structure, which are potential sites to inhibit uncoating by stabilization of these weak interfaces. We conclude by identifying potential target sites at the junction of a weak protein–protein interface. PMID:26284238

  14. A Beta-Herpesvirus with Fluorescent Capsids to Study Transport in Living Cells

    PubMed Central

    Bosse, Jens B.; Bauerfeind, Rudolf; Popilka, Leonhard; Marcinowski, Lisa; Taeglich, Martina; Jung, Christophe; Striebinger, Hannah; von Einem, Jens; Gaul, Ulrike; Walther, Paul; Koszinowski, Ulrich H.; Ruzsics, Zsolt

    2012-01-01

    Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion. PMID:22792376

  15. Unclosed HIV-1 Capsids Suggest a Curled Sheet Model of Assembly

    PubMed Central

    Yu, Zhiheng; Dobro, Megan J.; Woodward, Cora L.; Levandovsky, Artem; Danielson, Cindy M.; Sandrin, Virginie; Shi, Jiong; Aiken, Christopher; Zandi, Roya; Hope, Thomas J.; Jensen, Grant J.

    2012-01-01

    The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ∼25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms. PMID:23079241

  16. Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure.

    PubMed

    Seth, P

    1994-12-15

    Adenoviruses have been previously shown to enhance the delivery of many ligands including proteins and plasmid DNAs to the cells. The key biochemical step during this process is the ability of adenovirus to disrupt (lyse) the endosome membrane releasing the co-internalized virus and the other ligands into the cytosol (Seth et al, 1986, In: Adenovirus attachment and entry into cells, pp 191-195, American Society for Microbiology, Washington, D.C.). To understand the role of the adenovirus proteins involved in the endosome lysis, it is further shown here that empty capsids of adenovirus also possess this membrane vesicle lytic activity; though the activity is about 5-times lower than the adenovirus. Incubation of adenovirus with low concentration of ionic detergent or brief exposure to 45 degrees C destroyed this lytic activity without affecting the adenovirus binding to cell surface receptor, suggesting the lytic activity of adenovirus to be of enzymatic nature. However, exposing adenovirus to conditions that can disrupt adenovirus capsid structure such as heating at 65 degrees C, treating with 0.5% SDS, treating with different proteases, dialyzing against no glycerol buffer, treating with 6 M urea or with 10% pyridine, and sonication destroyed the adenovirus-associated lytic activity. Results suggest the requirement of an intact capsid structure for adenovirus-mediated lysis of the endosome. PMID:7802664

  17. Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

    SciTech Connect

    Sae-Ueng, Udom; Li, Dong; Zuo, Xiaobing; Huffman, Jamie B.; Homa, Fred L.; Rau, Donald; Evilevitch, Alex

    2014-10-01

    DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

  18. Contrasting behaviour of anthropogenic gadolinium and natural rare earth elements in estuaries and the gadolinium input into the North Sea

    NASA Astrophysics Data System (ADS)

    Kulaksız, Serkan; Bau, Michael

    2007-08-01

    All major rivers in northwestern Germany that flow into the North Sea, including the Weser River, display rare earth element (REE) patterns with large positive gadolinium (Gd) anomalies that indicate the presence of anthropogenic Gd derived from contrast agents used in magnetic resonance imaging. This microcontaminant cannot be removed by common sewage treatment technology and enters rivers and lakes with the discharge from waste water treatment plants. As elsewhere, a large fraction of the natural dissolved REE in the Weser River are associated with colloids. These colloids aggregate during mixing of freshwater and seawater in the low-salinity part of the Weser Estuary and the dissolved REE are partially removed from the river water together with the colloids. In marked contrast to the natural REE, the anthropogenic Gd behaves conservatively during this estuarine mixing and transits through the Weser Estuary almost unaffected. This indicates that the speciation of anthropogenic Gd is different from that of natural Gd and suggests a long environmental half-life of the anthropogenic Gd complexes used as contrast agents. The amount of anthropogenic Gd introduced into seawater via rivers is significant and produces anthropogenic positive Gd anomalies in coastal seawater. This is observed in the southwestern North Sea, off the coast of the East Frisian Islands, where anthropogenic Gd is mostly derived from the rivers Rhine and Thames. Its long environmental half-life and conservative estuarine behaviour suggest that anthropogenic Gd might be utilized as a pseudo-natural far-field tracer for truly dissolved riverine REE input into seawater and for discharge from waste water treatment plants and for sewage in river, ground and drinking water. The widespread distribution of anthropogenic Gd is an example of how the increasing use of "exotic" (ultra)trace elements in high-tech processes will in the future significantly hamper studies of the distribution and geochemical

  19. Structural and optical properties of Nd3+ doped gadolinium oxide 1D nanorods

    NASA Astrophysics Data System (ADS)

    Boopathi, G.; Raj, S. Gokul; Kumar, G. Ramesh; Mohan, R.

    2014-04-01

    Neodymium doped gadolinium hydroxide [Nd:Gd(OH)3] nanorods were successfully synthesized at 60 °C through co-precipitation method. The dopant percentage was maintained at 5% and calcination was done at 750 °C temperature for 1 hour to form the respective neodymium doped gadolinium oxide [Nd:Gd2O3] nanorods. The as-formed and annealed products were investigated in detail by using powder X-ray diffraction (XRD) pattern, scanning electron microscopy (SEM) with an energy dispersive X-ray spectrum (EDX), high-resolution transmission electron microscopy (HRTEM) and photoluminescence (PL) spectrophotometry.

  20. Gadolinium-Conjugated Gold Nanoshells for Multimodal Diagnostic Imaging and Photothermal Cancer Therapy

    PubMed Central

    Coughlin, Andrew J.; Ananta, Jeyarama S.; Deng, Nanfu; Larina, Irina V.; Decuzzi, Paolo

    2014-01-01

    Multimodal imaging offers the potential to improve diagnosis and enhance the specificity of photothermal cancer therapy. Toward this goal, we have engineered gadolinium-conjugated gold nanoshells and demonstrated that they enhance contrast for magnetic resonance imaging, X-Ray, optical coherence tomography, reflectance confocal microscopy, and two-photon luminescence. Additionally, these particles effectively convert near-infrared light to heat, which can be used to ablate cancer cells. Ultimately, these studies demonstrate the potential of gadolinium-nanoshells for image-guided photothermal ablation. PMID:24115690

  1. Structural, morphological and optical investigations on Sm{sup 3+} doped gadolinium oxide nanorods

    SciTech Connect

    Boopathi, G.; Mohan, R.; Raj, S. Gokul; Kumar, G. Ramesh

    2014-04-24

    One dimensional uniform Sm{sup 3+} doped gadolinium hydroxide nanorods have been prepared via simple co– precipitation technique at 60 °C temperature for 1 hour. The samples were calcinated at 750 °C to obtain Sm{sup 3+} doped gadolinium oxide nanorods. The 1D nanorods were then subjected to different characterization techniques to ascertain its structural stability and its morphology were investigated using high–resolution transmission electron microscopy. Photoluminescence (PL) spectrophotometry was investigated and the obtained results were discussed in detail.

  2. Structural and optical properties of Nd{sup 3+} doped gadolinium oxide 1D nanorods

    SciTech Connect

    Boopathi, G. Mohan, R.; Raj, S. Gokul; Kumar, G. Ramesh

    2014-04-24

    Neodymium doped gadolinium hydroxide [Nd:Gd(OH)3] nanorods were successfully synthesized at 60 °C through co-precipitation method. The dopant percentage was maintained at 5% and calcination was done at 750 °C temperature for 1 hour to form the respective neodymium doped gadolinium oxide [Nd:Gd{sub 2}O{sub 3}] nanorods. The as-formed and annealed products were investigated in detail by using powder X-ray diffraction (XRD) pattern, scanning electron microscopy (SEM) with an energy dispersive X-ray spectrum (EDX), high-resolution transmission electron microscopy (HRTEM) and photoluminescence (PL) spectrophotometry.

  3. Misconnections in the Critically Ill: Injection of High-Dose Gadolinium into an External Ventricular Drain.

    PubMed

    Singh, Sumit; Rejai, Sepehr; Antongiorgi, Zarah; Gonzalez, Nestor; Stelzner, Matthias

    2016-03-01

    We report an unfortunate case of accidental administration of intrathecal gadolinium through an external ventricular drain in a postcraniotomy patient during magnetic resonance imaging of the brain. The incident occurred after the venous contrast line was connected mistakenly to the ventricular drainage catheter. The patient subsequently developed confusion, aphasia, and right facial droop with new computed tomography evidence of diffuse cerebral edema and stroke. Review of the magnetic resonance image revealed the inappropriate presence of subarachnoid gadolinium. Despite all interventions, the patient developed irreversible neurologic disability. We address the clinical sequelae, management strategies, and factors contributing to the catheter misconnection that led to this event. PMID:26462163

  4. Gadolinium-phthalein complexone as a contrast agent for hepatobiliary MR imaging

    SciTech Connect

    Kawamura, Y.; Endo, K.; Koizumi, M.; Watanabe, Y.; Saga, T.; Konishi, J.; Horiuchi, K.; Yokoyama, A.

    1989-01-01

    Gadolinium-phthalein complexone (Gd-PC) was developed as a hepatobiliary magnetic resonance (MR) contrast agent. Phthalein complexone is one of the iminodiacetic acid derivatives and a structural analogue of bromosulfophthalein. Gadolinium-PC substantially enhanced signal intensity of normal functioning livers on T1-weighted MR images. Contrast enhancement of rabbit liver and gradual accumulation of high intensity bile in the gallbladder were observed after intravenous injection of 0.05 and 0.1 mmol/kg Gd-PC. However, 0.1 mmol/kg of Gd-DTPA caused little effect on liver MR.

  5. Molecular Dynamics Simulations to Determine the Structure and Dynamics of Hepatitis B Virus Capsid Bound to a Novel Anti-viral Drug.

    PubMed

    Watanabe, Go; Sato, Shunsuke; Iwadate, Mitsuo; Umeyama, Hideaki; Hayakawa, Michiyo; Murakami, Yoshiki; Yoneda, Shigetaka

    2016-01-01

    Hepatitis B virus (HBV) chronically infects millions of people worldwide and is a major cause of serious liver diseases, including liver cirrhosis and liver cancer. In our previous study, in silico screening was used to isolate new anti-viral compounds predicted to bind to the HBV capsid. Four of the isolated compounds have been reported to suppress the cellular multiplication of HBV experimentally. In the present study, molecular dynamics simulations of the HBV capsid were performed under rotational symmetry boundary conditions, to clarify how the structure and dynamics of the capsid are affected at the atomic level by the binding of one of the isolated compounds, C13. Two simulations of the free HBV capsid, two further simulations of the capsid-C13 complex, and one simulation of the capsid-AT-130 complex were performed. For statistical confidence, each set of simulations was repeated by five times, changing the simulation conditions. C13 continued to bind at the predicted binding site during the simulations, supporting the hypothesis that C13 is a capsid-binding compound. The structure and dynamics of the HBV capsid were greatly influenced by the binding and release of C13, and these effects were essentially identical to those seen for AT-130, indicating that C13 likely inhibits the function of the HBV capsid. PMID:27581644

  6. Late Gadolinium Enhancement Among Survivors of Sudden Cardiac Arrest

    PubMed Central

    Neilan, Tomas G.; Farhad, Hoshang; Mayrhofer, Thomas; Shah, Ravi V.; Dodson, John A.; Abbasi, Siddique A.; Danik, Stephan B.; Verdini, Daniel J.; Tokuda, Michifumi; Tedrow, Usha B.; Jerosch-Herold, Michael; Hoffmann, Udo; Ghoshhajra, Brian B.; Stevenson, William G.; Kwong, Raymond Y.

    2016-01-01

    OBJECTIVES The aim of this study was to describe the role of contrast-enhanced cardiac magnetic resonance (CMR) in the workup of patients with aborted sudden cardiac arrest (SCA) and in the prediction of long-term outcomes. BACKGROUND Myocardial fibrosis is a key substrate for SCA, and late gadolinium enhancement (LGE) on a CMR study is a robust technique for imaging of myocardial fibrosis. METHODS We performed a retrospective review of all survivors of SCA who were referred for CMR studies and performed follow-up for the subsequent occurrence of an adverse event (death and appropriate defibrillator therapy). RESULTS After a workup that included a clinical history, electrocardiogram, echocardiography, and coronary angiogram, 137 patients underwent CMR for workup of aborted SCA (66% male; mean age 56 ± 11 years; left ventricular ejection fraction 43 ± 12%). The presenting arrhythmias were ventricular fibrillation (n = 105 [77%]) and ventricular tachycardia (n = 32 [23%]). Overall, LGE was found in 98 patients (71%), with an average extent of 9.9 ± 5% of the left ventricular myocardium. CMR imaging provided a diagnosis or an arrhythmic substrate in 104 patients (76%), including the presence of an infarct-pattern LGE in 60 patients (44%), noninfarct LGE in 21 (15%), active myocarditis in 14 (10%), hypertrophic cardiomyopathy in 3 (2%), sarcoidosis in 3, and arrhythmogenic cardiomyopathy in 3. In a median follow-up of 29 months (range 18 to 43 months), there were 63 events. In a multivariable analysis, the strongest predictors of recurrent events were the presence of LGE (adjusted hazard ratio: 6.7; 95% CI: 2.38 to 18.85; p < 0.001) and the extent of LGE (hazard ratio: 1.15; 95% CI: 1.11 to 1.19; p < 0.001). CONCLUSIONS Among patients with SCA, CMR with contrast identified LGE in 71% and provided a potential arrhythmic substrate in 76%. In follow-up, both the presence and extent of LGE identified a group at markedly increased risk of future adverse events. PMID

  7. GADOLINIUM OXALATE SOLUBILITY MEASUREMENTS IN NITRIC ACID SOLUTIONS

    SciTech Connect

    Pierce, R.

    2012-02-22

    HB-Line will begin processing Pu solutions during FY2012 that will involve the recovery of Pu using oxalate precipitation and filtration. After the precipitation and filtration processes, the filtrate solution will be transferred from HB-Line to H-Canyon. The presence of excess oxalate and unfiltered Pu oxalate solids in these solutions create a criticality safety issue if they are sent to H-Canyon without controls in H-Canyon. One approach involves H-Canyon receiving the filtrate solution into a tank that is poisoned with soluble gadolinium (Gd). Decomposition of the oxalate will occur within a subsequent H-Canyon vessel. The receipt of excess oxalate into the H-Canyon receipt tanks has the potential to precipitate a portion of the Gd poison in the receipt tanks. Because the amount of Gd in solution determines the maximum amount of Pu solids that H-Canyon can receive, H-Canyon Engineering requested that SRNL determine the solubility of Gd in aqueous solutions of 4-10 M nitric acid (HNO{sub 3}), 4-12 g/L Gd, and 0.15-0.25 M oxalic acid (H{sub 2}C{sub 2}O{sub 4}) at 25 C. The target soluble Gd concentration is 6 g/L. The data indicate that the target can be achieved above 6 M HNO{sub 3} and below 0.25 M H{sub 2}C{sub 2}O{sub 4}. For 6 M HNO{sub 3}, 10.5 g/L and 7 g/L Gd are soluble in 0.15 M and 0.25 M H{sub 2}C{sub 2}O{sub 4}, respectively. In 4 M HNO{sub 3}, the Gd solubility drops significantly to 2 g/L and 0.25 g/L in 0.15 M and 0.25 M H{sub 2}C{sub 2}O{sub 4}, respectively. The solubility of Gd at 8-10 M HNO{sub 3} exceeds the solubility at 6 M HNO{sub 3}. The data for 4 M HNO{sub 3} showed good agreement with data in the literature. To achieve a target of 6 g/L soluble Gd in solution in the presence of 0.15-0.25 M oxalate, the HNO{sub 3} concentration must be maintained at or above 6 M HNO{sub 3}.

  8. A Manganese Alternative to Gadolinium for MRI Contrast.

    PubMed

    Gale, Eric M; Atanasova, Iliyana P; Blasi, Francesco; Ay, Ilknur; Caravan, Peter

    2015-12-16

    Contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used to diagnose soft tissue and vascular abnormalities. However, safety concerns limit the use of iodinated and gadolinium (Gd)-based CT and MRI contrast media in renally compromised patients. With an estimated 14% of the US population suffering from chronic kidney disease (CKD), contrast media compatible with renal impairment is sorely needed. We present the new manganese(II) complex [Mn(PyC3A)(H2O)](-) as a Gd alternative. [Mn(PyC3A)(H2O)](-) is among the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40). In the presence of 25 mol equiv of Zn at pH 6.0, 37 °C, [Mn(PyC3A)(H2O)](-) is 20-fold more resistant to dissociation than [Gd(DTPA)(H2O)](2-). Relaxivity of [Mn(PyC3A)(H2O)](-) in blood plasma is comparable to commercial Gd contrast agents. Biodistribution analysis confirms that [Mn(PyC3A)(H2O)](-) clears via a mixed renal/hepatobiliary pathway with >99% elimination by 24 h. [Mn(PyC3A)(H2O)](-) was modified to form a bifunctional chelator and 4 chelates were conjugated to a fibrin-specific peptide to give Mn-FBP. Mn-FBP binds the soluble fibrin fragment DD(E) with Kd = 110 nM. Per Mn relaxivity of Mn-FBP is 4-fold greater than [Mn(PyC3A)(H2O)](-) and increases 60% in the presence of fibrin, consistent with binding. Mn-FBP provided equivalent thrombus enhancement to the state of the art Gd analogue, EP-2104R, in a rat model of arterial thrombosis. Mn metabolite analysis reveals no evidence of dechelation and the probe was >99% eliminated after 24 h. [Mn(PyC3A)(H2O)](-) is a lead development candidate for an imaging probe that is compatible with renally compromised patients. PMID:26588204

  9. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    SciTech Connect

    Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo; Sathaliyawala, Taheri; Matyas, Gary R.; Alving, Carl R.; Leppla, Stephen H.; Rao, Venigalla B. . E-mail: rao@cua.edu

    2006-02-05

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

  10. The structure of rare earth thin films: holmium and gadolinium on yttrium

    NASA Astrophysics Data System (ADS)

    Bentall, M. J.; Cowley, R. A.; Ward, R. C. C.; Wells, M. R.; Stunault, A.

    2003-11-01

    Single-crystal holmium and gadolinium layers have been grown on yttrium substrates using the molecular beam epitaxy technique and their structures investigated using high resolution x-ray scattering. The experiments were performed using a Philips MRD diffractometer in Oxford, and with the XMaS facility at the ESRF. Holmium layers with a thickness below T_{\\mathrm {c}}'=115\

  11. A water-soluble gadolinium metallofullerenol: facile preparation, magnetic properties and magnetic resonance imaging application.

    PubMed

    Li, Jie; Wang, Taishan; Feng, Yongqiang; Zhang, Ying; Zhen, Mingming; Shu, Chunying; Jiang, Li; Wang, Yuqing; Wang, Chunru

    2016-06-01

    A new water-soluble gadolinium metallofullerenol was prepared through a solid-liquid reaction. It was characterized to have an enhanced effective magnetic moment, and improved T1-weighted relaxivity and magnetic resonance imaging performance in the liver. This material prepared by a facile method has wide application as a contrast agent and biological medicine. PMID:27064096

  12. A gadolinium(iii) complex that shows room-temperature phosphorescence in the crystalline state.

    PubMed

    Nakai, Hidetaka; Kitagawa, Kazuhiro; Seo, Juncheol; Matsumoto, Takahiro; Ogo, Seiji

    2016-07-19

    This paper presents a gadolinium(iii) complex that shows blue phosphorescence in the crystalline state at room temperature under air atmosphere; color of the crystals can be changed to pale-green from blue by doping of 1-naphthol. PMID:27221814

  13. Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor.

    PubMed Central

    Beaudet-Miller, M; Zhang, R; Durkin, J; Gibson, W; Kwong, A D; Hong, Z

    1996-01-01

    We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein

  14. Effect of internal cleavage site mutations in human immunodeficiency virus type 1 capsid protein on its structure and function.

    PubMed

    Tóth, Ferenc; Kádas, János; Mótyán, János András; Tőzsér, József

    2016-08-01

    The capsid protein of the human immunodeficiency virus type 1 has been found to be a substrate of the retroviral protease in vitro, and its processing was predicted to be strongly dependent on a pH-induced conformational change. Several protease cleavage sites have been identified within the capsid protein, but the importance of its cleavage by the viral protease at the early phase of infection is controversial. To confirm the relevance of this process, we aimed to design, produce, and characterize mutant capsid proteins, in which the protein susceptibility toward HIV-1 protease is altered without affecting other steps of the viral life cycle. Our results indicate that while the introduced mutations changed the cleavage rate at the mutated sites of the capsid protein by HIV-1 protease, some of them caused only negligible or moderate structural changes (A78V, L189F, and L189I). However, the effects of other mutations (W23A, A77P, and L189P) were dramatic, as assessed by secondary structure determination or cyclophilin A-binding assay. Based on our observations, the L189F mutant capsid remains structurally and functionally unchanged and may therefore be the best candidate for use in studies aimed at better understanding the role of the protease in the early postentry events of viral infection or retrovirus-mediated gene transduction. PMID:27516963

  15. Microplate-Based Assay for Identifying Small Molecules That Bind a Specific Intersubunit Interface within the Assembled HIV-1 Capsid

    PubMed Central

    Halambage, Upul D.; Wong, Jason P.; Melancon, Bruce J.; Lindsley, Craig W.

    2015-01-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid–targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  16. Structure of FIV capsid C-terminal domain demonstrates lentiviral evasion of genetic fragility by coevolved substitutions

    PubMed Central

    Khwaja, Aya; Galilee, Meytal; Marx, Ailie; Alian, Akram

    2016-01-01

    Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed. PMID:27102180

  17. Structure of FIV capsid C-terminal domain demonstrates lentiviral evasion of genetic fragility by coevolved substitutions.

    PubMed

    Khwaja, Aya; Galilee, Meytal; Marx, Ailie; Alian, Akram

    2016-01-01

    Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed. PMID:27102180

  18. CryoEM analysis of capsid assembly and structural changes upon interactions with a host restriction factor, TRIM5α.

    PubMed

    Zhao, Gongpu; Zhang, Peijun

    2014-01-01

    After virus fusion with a target cell, the viral core is released into the host cell cytoplasm and undergoes a controlled disassembly process, termed uncoating, before or as reverse transcription takes place. The cellular protein TRIM5α is a host cell restriction factor that blocks HIV-1 infection in rhesus macaque cells by targeting the viral capsid and inducing premature uncoating. The molecular mechanism of the interaction between capsid and TRIM5α remains unclear. Here, we describe an approach that utilizes cryo-electron microscopy (cryoEM) to examine the structural changes exerted on HIV-1 capsid (CA) assembly by TRIM5α binding. The TRIM5α interaction sites on CA assembly were further dissected by combining cryoEM with pair-wise cysteine mutations that crosslink CA either within a CA hexamer or between CA hexamers. Based on the structural information from cryoEM and crosslinking results from in vitro CA assemblies and purified intact HIV-1 cores, we demonstrate that direct binding of TRIM5α CC-SPRY domains to the viral capsid results in disruption and fragmentation of the surface lattice of HIV-1 capsid, specifically at inter-hexamer interfaces. The method described here can be easily adopted to study other important interactions in multi-protein complexes. PMID:24158810

  19. High Sensitivity of Late Gadolinium Enhancement for Predicting Microscopic Myocardial Scarring in Biopsied Specimens in Hypertrophic Cardiomyopathy

    PubMed Central

    Konno, Tetsuo; Hayashi, Kenshi; Fujino, Noboru; Nagata, Yoji; Hodatsu, Akihiko; Masuta, Eiichi; Sakata, Kenji; Nakamura, Hiroyuki; Kawashiri, Masa-aki; Yamagishi, Masakazu

    2014-01-01

    Background Myocardial scarring can be assessed by cardiac magnetic resonance imaging with late gadolinium enhancement and by endomyocardial biopsy. However, accuracy of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens remains unknown in hypertrophic cardiomyopathy. We investigated whether late gadolinium enhancement in the whole heart reflects microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. Methods and Results Twenty-one consecutive patients with hypertrophic cardiomyopathy who were examined both by cardiac magnetic resonance imaging and by endomyocardial biopsy were retrospectively studied. The right interventricular septum was the target site for endomyocardial biopsy in all patients. Late gadolinium enhancement in the ventricular septum had an excellent sensitivity (100%) with a low specificity (40%) for predicting microscopic myocardial scarring in biopsied specimens. The sensitivity of late gadolinium enhancement in the whole heart remained 100% with a specificity of 27% for predicting microscopic myocardial scarring in biopsied specimens. Quantitative assessments of fibrosis revealed that the extent of late gadolinium enhancement in the whole heart was the only independent variable related to the microscopic collagen fraction in biopsied specimens (β  =  0.59, 95% confident interval: 0.15 – 1.0, p  =  0.012). Conclusions Although there was a compromise in the specificity, the sensitivity of late gadolinium enhancement was excellent for prediction of microscopic myocardial scarring in hypertrophic cardiomyopathy. Moreover, the severity of late gadolinium enhancement was independently associated with the quantitative collagen fraction in biopsied specimens in hypertrophic cardiomyopathy. These findings indicate that late gadolinium enhancement can reflect both the presence and the extent of microscopic myocardial scarring in the small biopsied specimens in

  20. Acute adverse reactions to magnetic resonance contrast media--gadolinium chelates.

    PubMed

    Li, A; Wong, C S; Wong, M K; Lee, C M; Au Yeung, M C

    2006-05-01

    The objective of this study was to evaluate the clinical safety of intravenous gadolinium-based contrast media used in patients who underwent MRI at a single institution. Acute adverse reactions to intravenous gadolinium-based contrast media used for MRI at the Princess Margaret Hospital, Hong Kong, SAR, from January 1999 to November 2004 were recorded in an incidence log book. The medical records of patients' demographics were retrospectively reviewed and the nature, frequency and severity of the adverse reactions were investigated and documented. The incidence of acute adverse reactions to intravenous gadolinium-based contrast media was 0.48% (45 patients with 46 adverse reactions). The severity of these adverse reactions were 96% mild, 2% moderate (one patient developed shortness of breath that required oxygen supplementation and intravenous steroidal management) and 2% severe (one patient developed an anaphylactoid reaction, but successfully recovered through timely resuscitation). No patients were recorded as having contrast extravasation and none died as a result of any adverse reaction. Among the 45 patients who developed adverse reactions, three patients (6.7%) had prior adverse reactions to iodinated contrast media, three (6.7%) had prior reactions to a different gadolinium-based contrast agent, one (2%) had asthma and nine (20%) had a history of drug/food allergy. Overall, 41% of the adverse reactions were not documented in the final MRI report or the clinical medical records. Gadolinium-based contrast media are safe and well tolerated by the vast majority of patients. In our study, the adverse reaction rate (0.48%) and the incidence of severe anaphylactoid reaction (0.01%) concur with those reported in the literature. Although most of the symptoms are mild and transient, these adverse reactions must be accurately documented and managed. PMID:16632615

  1. A virus capsid component mediates virion retention and transmission by its insect vector

    PubMed Central

    Chen, Angel Y. S.; Walker, Gregory P.; Carter, David; Ng, James C. K.

    2011-01-01

    Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen–vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors. PMID:21930903

  2. Enumeration of viral capsid assembly pathways: tree orbits under permutation group action.

    PubMed

    Bóna, Miklós; Sitharam, Meera; Vince, Andrew

    2011-04-01

    This paper uses combinatorics and group theory to answer questions about the assembly of icosahedral viral shells. Although the geometric structure of the capsid (shell) is fairly well understood in terms of its constituent subunits, the assembly process is not. For the purpose of this paper, the capsid is modeled by a polyhedron whose facets represent the monomers. The assembly process is modeled by a rooted tree, the leaves representing the facets of the polyhedron, the root representing the assembled polyhedron, and the internal vertices representing intermediate stages of assembly (subsets of facets). Besides its virological motivation, the enumeration of orbits of trees under the action of a finite group is of independent mathematical interest. If G is a finite group acting on a finite set X, then there is a natural induced action of G on the set T(x) of trees whose leaves are bijectively labeled by the elements of X. If G acts simply on X, then |X|:=|X(n)|=n·|G|, where n is the number of G-orbits in X. The basic combinatorial results in this paper are (1) a formula for the number of orbits of each size in the action of G on T(x)(n), for every n, and (2) a simple algorithm to find the stabilizer of a tree τ ∈T(x) in G that runs in linear time and does not need memory in addition to its input tree. These results help to clarify the effect of symmetry on the probability and number of assembly pathways for icosahedral viral capsids, and more generally for any finite, symmetric macromolecular assembly. PMID:21174231

  3. Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

    PubMed Central

    Chen, Nai-Chi; Yoshimura, Masato; Guan, Hong-Hsiang; Wang, Ting-Yu; Misumi, Yuko; Lin, Chien-Chih; Chuankhayan, Phimonphan; Nakagawa, Atsushi; Chan, Sunney I.; Tsukihara, Tomitake; Chen, Tzong-Yueh; Chen, Chun-Jung

    2015-01-01

    Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus. PMID:26491970

  4. Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

    PubMed

    Martinez-Torrecuadrada, J L; Castón, J R; Castro, M; Carrascosa, J L; Rodriguez, J F; Casal, J I

    2000-12-20

    Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. PMID:11118356

  5. Nucleotide sequence and expression of the capsid protein gene of feline calicivirus.

    PubMed Central

    Neill, J D; Reardon, I M; Heinrikson, R L

    1991-01-01

    The sequence of the 3'-terminal 2,486 bases of the feline calicivirus (FCV) genome was determined. This region of the FCV genome, from which the 2.4-kb subgenomic RNA is derived, contained two open reading frames. The larger open reading frame, found in the 5' end of the subgenomic mRNA, contained 2,004 bases encoding a polypeptide of 73,467 Da. The smaller open reading frame, encoded in the 3' end of the mRNA, was composed of 318 bases, encoding a polypeptide of 12,185 Da. The AUG initiation codon of the second open reading frame overlapped the UGA termination codon of the first, with the sequence AUGA. The nucleotide sequence of the region containing this overlap resembles the -1 frameshift sequences of the retroviruses. The 5' end of the 2.4-kb subgenomic RNA was mapped by primer extension analysis. There were two apparent transcription initiation points, both of which were 5' to the AUG initiation codon of the large open reading frame. Transcription from these sites yielded RNA transcripts with 5' nontranslated leader regions of 17 and 18 bases. The total length of the 2.4-kb subgenomic RNA was 2,375 bases (from the 5'-most start site) excluding the poly(A) tail. Edman degradation of the purified capsid protein of FCV showed that the capsid protein was encoded by the large open reading frame. Western immunoblot analysis of FCV-infected cells using a feline anti-FCV antiserum demonstrated that translation of the capsid protein was detectable at 3 h postinfection and continued to accumulate until 8 h postinfection, the last time examined. Images PMID:1716692

  6. Bioinformatic analysis of non-VP1 capsid protein of coxsackievirus A6.

    PubMed

    Liu, Hong-Bo; Yang, Guang-Fei; Liang, Si-Jia; Lin, Jun

    2016-08-01

    This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents. PMID:27465341

  7. Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

    PubMed Central

    Green, K Y; Kapikian, A Z; Valdesuso, J; Sosnovtsev, S; Treanor, J J; Lew, J F

    1997-01-01

    The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent. PMID:9196224

  8. Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

    PubMed

    White, L J; Ball, J M; Hardy, M E; Tanaka, T N; Kitamoto, N; Estes, M K

    1996-10-01

    Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region

  9. Cellular phosphoinositides and the maturation of bluetongue virus, a non-enveloped capsid virus

    PubMed Central

    2013-01-01

    Background Bluetongue virus (BTV), a member of Orbivirus genus in the Reoviridae family is a double capsid virus enclosing a genome of 10 double-stranded RNA segments. A non-structural protein of BTV, NS3, which is associated with cellular membranes and interacts with outer capsid proteins, has been shown to be involved in virus morphogenesis in infected cells. In addition, studies have also shown that during the later stages of virus infection NS3 behaves similarly to HIV protein Gag, an enveloped viral protein. Since Gag protein is known to interact with membrane lipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and one of the known binding partners of NS3, cellular protein p11 also interacts with annexin a PI(4,5)P2 interacting protein, this study was designed to understand the role of this negatively charged membrane lipid in BTV assembly and maturation. Methods Over expression of cellular enzymes that either depleted cells of PI(4,5)P2 or altered the distribution of PI(4,5)P2, were used to analyze the effect of the lipid on BTV maturation at different times post-infection. The production of mature virus particles was monitored by plaque assay. Microscopic techniques such as confocal microscopy and electron microscopy (EM) were also undertaken to study localization of virus proteins and virus particles in cells, respectively. Results Initially, confocal microscopic analysis demonstrated that PI(4,5)P2 not only co-localized with NS3, but it also co-localized with VP5, one of the outer capsid proteins of BTV. Subsequently, experiments involving depletion of cellular PI(4,5)P2 or its relocation demonstrated an inhibitory effect on normal BTV maturation and it also led to a redistribution of BTV proteins within the cell. The data was supported further by EM visualization showing that modulation of PI(4,5)P2 in cells indeed resulted in less particle production. Conclusion This study to our knowledge, is the first report demonstrating involvement of PI(4,5)P2

  10. DNA knots reveal a chiral organization of DNA in phage capsids

    NASA Astrophysics Data System (ADS)

    Arsuaga, Javier; Vazquez, Mariel; McGuirk, Paul; Trigueros, Sonia; Sumners, De Witt; Roca, Joaquim

    2005-06-01

    Icosahedral bacteriophages pack their double-stranded DNA genomes to near-crystalline density and achieve one of the highest levels of DNA condensation found in nature. Despite numerous studies, some essential properties of the packaging geometry of the DNA inside the phage capsid are still unknown. We present a different approach to the problems of randomness and chirality of the packed DNA. We recently showed that most DNA molecules extracted from bacteriophage P4 are highly knotted because of the cyclization of the linear DNA molecule confined in the phage capsid. Here, we show that these knots provide information about the global arrangement of the DNA inside the capsid. First, we analyze the distribution of the viral DNA knots by high-resolution gel electrophoresis. Next, we perform Monte Carlo computer simulations of random knotting for freely jointed polygons confined to spherical volumes. Comparison of the knot distributions obtained by both techniques produces a topological proof of nonrandom packaging of the viral DNA. Moreover, our simulations show that the scarcity of the achiral knot 41 and the predominance of the torus knot 51 over the twist knot 52 observed in the viral distribution of DNA knots cannot be obtained by confinement alone but must include writhe bias in the conformation sampling. These results indicate that the packaging geometry of the DNA inside the viral capsid is writhe-directed. Author contributions: D.W.S. and J.R. designed research; J.A. and J.R. performed research; J.A., M.V., P.M., S.T., and J.R. contributed new reagents/analytic tools; J.A., M.V., P.M., and J.R. analyzed data; and D.W.S. and J.R. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.‡Present address: Department of Mathematics and Center for Computation in the Life Sciences, San Francisco State University, San Francisco, CA 94132.

  11. Modeling capsid kinetics assembly from the steady state distribution of multi-sizes aggregates

    NASA Astrophysics Data System (ADS)

    Hozé, Nathanaël; Holcman, David

    2014-01-01

    The kinetics of aggregation for particles of various sizes depends on their diffusive arrival and fusion at a specific nucleation site. We present here a mean-field approximation and a stochastic jump model for aggregates at equilibrium. This approach is an alternative to the classical Smoluchowski equations that do not have a close form and are not solvable in general. We analyze these mean-field equations and obtain the kinetics of a cluster formation. Our approach provides a simplified theoretical framework to study the kinetics of viral capsid formation, such as HIV from the self-assembly of the structural proteins Gag.

  12. Polyvalent Display of Heme on Hepatitis B Virus Capsid Protein through Coordination to Hexahistidine Tags

    PubMed Central

    Prasuhn, Duane E.; Kuzelka, Jane; Strable, Erica; Udit, Andrew K.; Cho, So-Hye; Lander, Gabriel C.; Quispe, Joel D.; Diers, James R.; Bocian, David F.; Potter, Clint; Carragher, Bridget; Finn, M.G.

    2009-01-01

    SUMMARY The addition of a hexahistidine tag to the N terminus of the hepatitis B capsid protein gives rise to a self-assembled particle with 80 sites of high local density of histidine side chains. Iron protoporphyrin IX has been found to bind tightly at each of these sites, making a polyvalent system of well-defined spacing between metalloporphyrin complexes. The spectroscopic and redox properties of the resulting particle are consistent with the presence of 80 site-isolated bis(histidine)-bound heme centers, comprising a polyvalent b-type cytochrome mimic. PMID:18482703

  13. The parvoviral capsid controls an intracellular phase of infection essential for efficient killing of stepwise-transformed human fibroblasts.

    PubMed

    Paglino, Justin; Tattersall, Peter

    2011-07-20

    Members of the rodent subgroup of the genus Parvovirus exhibit lytic replication and spread in many human tumor cells and are therefore attractive candidates for oncolytic virotherapy. However, the significant variation in tumor tropism observed for these viruses remains largely unexplained. We report here that LuIII kills BJ-ELR 'stepwise-transformed' human fibroblasts efficiently, while MVM does not. Using viral chimeras, we mapped this property to the LuIII capsid gene, VP2, which is necessary and sufficient to confer the killer phenotype on MVM. LuIII VP2 facilitates a post-entry, pre-DNA-amplification step early in the life cycle, suggesting the existence of an intracellular moiety whose efficient interaction with the incoming capsid shell is critical to infection. Thus targeting of human cancers of different tissue-type origins will require use of parvoviruses with capsids that effectively make this critical interaction. PMID:21600623

  14. Electrostatic Interaction between RNA and Protein Capsid in CCMV Simulated by a Coarse-grain RNA model and a Monte Carlo Approach

    PubMed Central

    Zhang, Deqiang; Konecny, Robert; Baker, Nathan A.; McCammon, J. Andrew

    2008-01-01

    Although many viruses have been crystallized and the protein capsid structures have been determined by X-ray crystallography, the nucleic acids often can not be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of Cowpea Chlorotic Mottle Virus (CCMV). Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive-charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations. PMID:15386271

  15. Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach

    PubMed Central

    Mowat, FM; Gornik, KR; Dinculescu, A; Boye, SL; Hauswirth, WW; Petersen-Jones, SM; Bartoe, JT

    2013-01-01

    Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected, 4 dogs received intravitreal injections, 8 dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal), and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency, and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach. PMID:24225638

  16. Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid.

    PubMed Central

    Parker, J S; Parrish, C R

    1997-01-01

    We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range. PMID:9371580

  17. The UL6 Gene Product Forms the Portal for Entry of DNA into the Herpes Simplex Virus Capsid

    PubMed Central

    Newcomb, William W.; Juhas, Rachel M.; Thomsen, Darrell R.; Homa, Fred L.; Burch, April D.; Weller, Sandra K.; Brown, Jay C.

    2001-01-01

    During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and

  18. Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein.

    PubMed

    McCabe, Victoria J; Tarpey, Ian; Spibey, Norman

    2002-06-01

    Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma-FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma-FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat. PMID:12057600

  19. Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

    NASA Astrophysics Data System (ADS)

    Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

    2012-08-01

    Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

  20. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

  1. Megabirnavirus structure reveals a putative 120-subunit capsid formed by asymmetrical dimers with distinctive large protrusions.

    PubMed

    Miyazaki, Naoyuki; Salaipeth, Lakha; Kanematsu, Satoko; Iwasaki, Kenji; Suzuki, Nobuhiro

    2015-08-01

    Rosellinia necatrix megabirnavirus 1 (RnMBV1) W779 is a bi-segmented dsRNA virus and a strain of the type species Rosellinia necatrix megabirnavirus 1 of the family Megabirnaviridae. RnMBV1 causes severe reduction of both mycelial growth of Rosellinia necatrix in synthetic medium and fungal virulence to plant hosts, and thus has strong potential for virocontrol (biological control using viruses) of white rot. The structure of RnMBV1 was examined by cryo-electron microscopy and three-dimensional reconstruction at 15.7 Å resolution. The diameter of the RnMBV1 capsid was 520 Å, and the capsid was composed of 60 asymmetrical dimers in the T = 1 (so-called T = 2) lattice that is well conserved among dsRNA viruses. However, RnMBV1 has putatively 120 large protrusions with a width of ∼ 45 Å and a height of ∼ 50 Å on the virus surface, making it distinguishable from the other dsRNA viruses. PMID:25968130

  2. Simulation Study of the Contribution of Oligomer/Oligomer Binding to Capsid Assembly Kinetics

    PubMed Central

    Zhang, Tiequan; Schwartz, Russell

    2006-01-01

    The process by which hundreds of identical capsid proteins self-assemble into icosahedral structures is complex and poorly understood. Establishing constraints on the assembly pathways is crucial to building reliable theoretical models. For example, it is currently an open question to what degree overall assembly kinetics are dominated by one or a few most efficient pathways versus the enormous number theoretically possible. The importance of this question, however, is often overlooked due to the difficulties of addressing it in either theoretical or experimental practice. We apply a computer model based on a discrete-event simulation method to evaluate the contributions of nondominant pathways to overall assembly kinetics. This is accomplished by comparing two possible assembly models: one allowing growth to proceed only by the accretion of individual assembly subunits and the other allowing the binding of sterically compatible assembly intermediates any sizes. Simulations show that the two models perform almost identically under low binding rate conditions, where growth is strongly nucleation-limited, but sharply diverge under conditions of higher association rates or coat protein concentrations. The results suggest the importance of identifying the actual binding pattern if one is to build reliable models of capsid assembly or other complex self-assembly processes. PMID:16214864

  3. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

    PubMed Central

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-01-01

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. PMID:26114473

  4. Signals for Bidirectional Nucleocytoplasmic Transport in the Duck Hepatitis B Virus Capsid Protein

    PubMed Central

    Mabit, Helene; Breiner, Klaus M.; Knaust, Andreas; Zachmann-Brand, Beate; Schaller, Heinz

    2001-01-01

    Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids. PMID:11160696

  5. Stochastic Kinetics of Viral Capsid Assembly Based on Detailed Protein Structures

    PubMed Central

    Hemberg, Martin; Yaliraki, Sophia N.; Barahona, Mauricio

    2006-01-01

    We present a generic computational framework for the simulation of viral capsid assembly which is quantitative and specific. Starting from PDB files containing atomic coordinates, the algorithm builds a coarse-grained description of protein oligomers based on graph rigidity. These reduced protein descriptions are used in an extended Gillespie algorithm to investigate the stochastic kinetics of the assembly process. The association rates are obtained from a diffusive Smoluchowski equation for rapid coagulation, modified to account for water shielding and protein structure. The dissociation rates are derived by interpreting the splitting of oligomers as a process of graph partitioning akin to the escape from a multidimensional well. This modular framework is quantitative yet computationally tractable, with a small number of physically motivated parameters. The methodology is illustrated using two different viruses which are shown to follow quantitatively different assembly pathways. We also show how in this model the quasi-stationary kinetics of assembly can be described as a Markovian cascading process, in which only a few intermediates and a small proportion of pathways are present. The observed pathways and intermediates can be related a posteriori to structural and energetic properties of the capsid oligomers. PMID:16473916

  6. A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces.

    PubMed

    Garfinkel, David J; Tucker, Jessica M; Saha, Agniva; Nishida, Yuri; Pachulska-Wieczorek, Katarzyna; Błaszczyk, Leszek; Purzycka, Katarzyna J

    2016-05-01

    Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host-parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions. PMID:26650614

  7. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    PubMed Central

    Han, Yun; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; Yang, Jun; Polenova, Tatyana

    2010-01-01

    In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly 13C,15N-labelled CA exhibit narrow lines, indicative of conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy. PMID:20092249

  8. Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens

    PubMed Central

    Klein, Sarah R.; Jiang, Hong; Hossain, Mohammad B.; Fan, Xuejun; Gumin, Joy; Dong, Andrew; Alonso, Marta M.; Gomez-Manzano, Candelaria; Fueyo, Juan

    2016-01-01

    Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins. PMID:27093696

  9. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

    PubMed Central

    Haynes, J I; Consigli, R A

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Images PMID:1318417

  10. Capsid-Modified Adenoviral Vectors for Improved Muscle-Directed Gene Therapy

    PubMed Central

    Guse, Kilian; Suzuki, Masataka; Sule, Gautam; Bertin, Terry K.; Tyynismaa, Henna; Ahola-Erkkilä, Sofia; Palmer, Donna; Suomalainen, Anu; Ng, Philip; Cerullo, Vincenzo; Hemminki, Akseli

    2012-01-01

    Abstract Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy. PMID:22888960

  11. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    SciTech Connect

    Han, Yun; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; Yang, Jun; Polenova, Tatyana E.

    2010-02-17

    In mature HIV-1 virions, the 26.6 kDa CA protein is assembled into a characteristic cone-shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this paper, we present a solid-state NMR analysis of the wild-type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly 13C,15Nlabeled CA exhibit narrow lines, indicative of the conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy.

  12. Molecular recognition in the human immunodeficiency virus capsid and antiviral design.

    PubMed

    Bocanegra, Rebeca; Rodríguez-Huete, Alicia; Fuertes, Miguel Ángel; Del Álamo, Marta; Mateu, Mauricio G

    2012-11-01

    Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds. PMID:22728445

  13. MATURATION IN ACTION: CRYOEM STUDY OF A VIRAL CAPSID CAUGHT DURING EXPANSION

    PubMed Central

    Veesler, D.; Quispe, J.; Grigorieff, N.; Potter, C.S.; Carragher, B.; Johnson, J. E.

    2012-01-01

    SUMMARY Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate prior to any crosslink formation. This form displays 6-fold symmetric hexamers, but, unexpectedly, capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that release of this coat subunit strain acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions. PMID:22748764

  14. A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3

    SciTech Connect

    Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning; Zhao, Wenran; Zhong, Zhaohua

    2012-11-25

    The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

  15. Development of Viral Capsid DNA Aptamer Conjugates as Cell-Targeted Delivery Vehicles

    NASA Astrophysics Data System (ADS)

    Tong, Gary Jen-Wei

    The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes. In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO4. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved. Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent

  16. Tracing gadolinium-based contrast agents from surface water to drinking water by means of speciation analysis.

    PubMed

    Birka, Marvin; Wehe, Christoph A; Hachmöller, Oliver; Sperling, Michael; Karst, Uwe

    2016-04-01

    In recent decades, a significant amount of anthropogenic gadolinium has been released into the environment as a result of the broad application of contrast agents for magnetic resonance imaging (MRI). Since this anthropogenic gadolinium anomaly has also been detected in drinking water, it has become necessary to investigate the possible effect of drinking water purification on these highly polar microcontaminats. Therefore, a novel highly sensitive method for speciation analysis of gadolinium is presented. For that purpose, the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma-mass spectrometry (ICP-MS) was employed. In order to enhance the detection power, sample introduction was carried out by ultrasonic nebulization. In combination with a novel HILIC method using a diol-based stationary phase, it was possible to achieve superior limits of detection for frequently applied gadolinium-based contrast agents below 20pmol/L. With this method, the contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were determined in concentrations up to 159pmol/L in samples from several waterworks in a densely populated region of Germany alongside the river Ruhr as well as from a waterworks near a catchment lake. Thereby, the direct impact of anthropogenic gadolinium species being present in the surface water on the amount of anthropogenic gadolinium in drinking water was shown. There was no evidence for the degradation of contrast agents, the release of Gd(3+) or the presence of further Gd species. PMID:26931429

  17. The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions

    PubMed Central

    Moilanen, Anni M.; Rissanen, Ilona A.; Määttä, Juha A. E.; Hytönen, Vesa P.; Ihalainen, Janne A.

    2015-01-01

    ABSTRACT Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid. IMPORTANCE The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the

  18. First measurements with new high-resolution gadolinium-GEM neutron detectors

    NASA Astrophysics Data System (ADS)

    Pfeiffer, D.; Resnati, F.; Birch, J.; Etxegarai, M.; Hall-Wilton, R.; Höglund, C.; Hultman, L.; Llamas-Jansa, I.; Oliveri, E.; Oksanen, E.; Robinson, L.; Ropelewski, L.; Schmidt, S.; Streli, C.; Thuiner, P.

    2016-05-01

    European Spallation Source instruments like the macromolecular diffractometer (NMX) require an excellent neutron detection efficiency, high-rate capabilities, time resolution, and an unprecedented spatial resolution in the order of a few hundred micrometers over a wide angular range of the incoming neutrons. For these instruments solid converters in combination with Micro Pattern Gaseous Detectors (MPGDs) are a promising option. A GEM detector with gadolinium converter was tested on a cold neutron beam at the IFE research reactor in Norway. The μTPC analysis, proven to improve the spatial resolution in the case of 10B converters, is extended to gadolinium based detectors. For the first time, a Gd-GEM was successfully operated to detect neutrons with a measured efficiency of 11.8% at a wavelength of 2 Åand a position resolution better than 250 μm.

  19. The evolution of gadolinium based contrast agents: from single-modality to multi-modality.

    PubMed

    Zhang, Li; Liu, Ruiqing; Peng, Hui; Li, Penghui; Xu, Zushun; Whittaker, Andrew K

    2016-05-19

    Gadolinium-based contrast agents are extensively used as magnetic resonance imaging (MRI) contrast agents due to their outstanding signal enhancement and ease of chemical modification. However, it is increasingly recognized that information obtained from single modal molecular imaging cannot satisfy the higher requirements on the efficiency and accuracy for clinical diagnosis and medical research, due to its limitation and default rooted in single molecular imaging technique itself. To compensate for the deficiencies of single function magnetic resonance imaging contrast agents, the combination of multi-modality imaging has turned to be the research hotpot in recent years. This review presents an overview on the recent developments of the functionalization of gadolinium-based contrast agents, and their application in biomedicine applications. PMID:27159645

  20. Gadolinium concentration analysis in brain phantom by X-ray fluorescence.

    PubMed

    Almalki, Musaed; Majid, Samir Abdul; Butler, Philip H; Reinisch, Lou

    2010-06-01

    We have measured the X-ray fluorescence from gadolinium as a function of concentration and position in tumors of different sizes and shapes in a head phantom. The gadolinium fluorescence was excited with a 36 GBq Am-241 source. The fluorescence signal was detected with a CdTe detector and a multi-channel analyzer. The fluorescence peak was clearly separated from the scattered X-rays. Concentrations of 5.62-78.63 mg/ml of Gd ion were used in 1, 2, and 3 cm diameter spherical tumors and a 2x4 cm oblate spheroid tumor. The data show trends approaching saturation for the highest concentrations, probably due to reabsorption in the tumor. A comparison of X-ray photographic imaging and densitometer measurements to determine concentration is also presented. PMID:20596811

  1. Biocompatible Polyhydroxyethylaspartamide-based Micelles with Gadolinium for MRI Contrast Agents

    NASA Astrophysics Data System (ADS)

    Jeong, Sang Young; Kim, Hyo Jeong; Kwak, Byung-Kook; Lee, Ha-Young; Seong, Hasoo; Shin, Byung Cheol; Yuk, Soon Hong; Hwang, Sung-Joo; Cho, Sun Hang

    2010-12-01

    Biocompatible poly-[ N-(2-hydroxyethyl)- d, l-aspartamide]-methoxypoly(ethyleneglycol)-hexadecylamine (PHEA-mPEG-C16) conjugated with 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid-gadolinium (DOTA-Gd) via ethylenediamine (ED) was synthesized as a magnetic resonance imaging (MRI) contrast agent. Amphiphilic PHEA-mPEG-C16-ED-DOTA-Gd forms micelle in aqueous solution. All the synthesized materials were characterized by proton nuclear magnetic resonance (1H NMR). Micelle size and shape were examined by dynamic light scattering (DLS) and atomic force microscopy (AFM). Micelles with PHEA-mPEG-C16-ED-DOTA-Gd showed higher relaxivities than the commercially available gadolinium contrast agent. Moreover, the signal intensity of a rabbit liver was effectively increased after intravenous injection of PHEA-mPEG-C16-ED-DOTA-Gd.

  2. Hypertrophic olivary degeneration with gadolinium enhancement after posterior fossa surgery in a child with medulloblastoma.

    PubMed

    Nowak, Johannes; Alkonyi, Balint; Rutkowski, Stefan; Homola, György A; Warmuth-Metz, Monika

    2014-05-01

    Hypertrophic olivary degeneration (HOD) is a rare transsynaptic form of degeneration occurring secondary to the disruption of the dentato-rubro-olivary pathway ("Guillain-Mollaret triangle"). HOD can be caused by ischemic, hemorrhagic, traumatic, or neoplastic lesions, and it can also occur following posterior fossa surgery. MRI characteristics of HOD include T2 signal increase and hypertrophy. To date, blood–brain barrier disruption has not been reported in HOD. Here, we present the first case of HOD with temporary gadolinium enhancement in a 10-year-old child 7 months after resection of a posterior fossa medulloblastoma. The recognition of gadolinium enhancement as a radiological feature of HOD may help to distinguish between this benign secondary condition and tumor recurrence. PMID:24122017

  3. Determination of stability constants and acute toxicity of potential hepatotropic gadolinium complexes.

    PubMed

    Mikiciuk-Olasik, Elzbieta; Wojewoda, Emilia; Bilichowski, Ireneusz; Witczak, Małgorzata; Karwowski, Bolesław; Wagrowska-Danilewicz, Małgorzata; Stasikowska, Olga

    2010-01-01

    Due to their high specificity for the hepatobiliary system, iminodiacetic acid derivatives are known to form a class of hepatobiliary agents. In this paper we present new hepatotropic gadolinium complexes to be used as potential MRI contrast agents. Derivatives of N-(2-phenylamine-2-oxoethyl) iminodiacetic acid are introduced as ligands into such complexes. In this way, we hope to achieve a valuable diagnostic tool for investigating of pathological changes in the liver. Stability constants of complexes were determined by potentiometric titration in 0.1 mol L(-1) NaNO3 solution at 20.0 +/- 0.1 degrees C. Stability and selectivity constants were also determined for endogenous metal ions such as Cu2+, Ca2+, and Zn2+ with the use of SUPERQUAD computer program. Acute toxicity of new gadolinium complexes was assessed in mice and histopathology examinations were carried out. PMID:20369788

  4. Gadolinium-based nanoparticles for highly efficient T1-weighted magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Lim, Eun-Kyung; Kang, Byunghoon; Choi, Yuna; Jang, Eunji; Han, Seungmin; Lee, Kwangyeol; Suh, Jin-Suck; Haam, Seungjoo; Huh, Yong-Min

    2014-06-01

    We developed Pyrene-Gadolinium (Py-Gd) nanoparticles as pH-sensitive magnetic resonance imaging (MRI) contrast agents capable of showing a high-Mr signal in cancer-specific environments, such as acidic conditions. Py-Gd nanoparticles were prepared by coating Py-Gd, which is a complex of gadolinium with pyrenyl molecules, with pyrenyl polyethyleneglycol PEG using a nano-emulsion method. These particles show better longitudinal relaxation time (T1) MR signals in acidic conditions than they do in neutral conditions. Furthermore, the particles exhibit biocompatibility and MR contrast effects in both in vitro and in vivo studies. From these results, we confirm that Py-Gd nanoparticles have the potential to be applied for accurate cancer diagnosis and therapy.

  5. Thermodynamic property evaluation and magnetic refrigeration cycle analysis for gadolinium gallium garnet

    SciTech Connect

    Murphy, R.W.

    1994-12-01

    Based on relevant material property data and previous model formulations, a magnetothermodynamic property map for gadolinium gallium garnet (Gd{sub 3}Ga{sub 5}O{sub 12}) was adapted for refrigeration cycle analysis in the temperature range 4-40 K and the magnetic field range 0-6 T. Employing methods similar to those previously developed for other materials and temperature ranges, assessments of limitations and relative performance were made for Carnot, ideal regenerative, and pseudo-constant field regenerative cycles. It was found that although Carnot cycle limitations on available temperature lift for gadolinium gallium garnet are not as severe as the limitations for materials previously examined, considerable improvement in cooling capacity and temperature lift combinations can be achieved by using regenerative cycles if serious loss mechanisms are avoided.

  6. Detection of anthropogenic gadolinium in the Brisbane River plume in Moreton Bay, Queensland, Australia.

    PubMed

    Lawrence, Michael G

    2010-07-01

    Wastewater effluent is known to contain macro and micropollutants, which may be deleterious to environmental health. One such class of micropollutants is chelated gadolinium, which are used as MRI contrast agents. As these MRI contrast agents can be assumed to behave conservatively during estuarine mixing, it is possible to calculate how much wastewater is represented in any particular sample. In this study, the percentage contribution of wastewater at specific locations in Moreton Bay, Qld, were determined by calculating the additional anthropogenic gadolinium contribution to the total rare earth element concentrations. Wastewater contributions were measured at concentrations as low as 0.2%, demonstrating the applicability of this technique for wastewater effluent plume mapping. PMID:20409563

  7. The evolution of gadolinium based contrast agents: from single-modality to multi-modality

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Liu, Ruiqing; Peng, Hui; Li, Penghui; Xu, Zushun; Whittaker, Andrew K.

    2016-05-01

    Gadolinium-based contrast agents are extensively used as magnetic resonance imaging (MRI) contrast agents due to their outstanding signal enhancement and ease of chemical modification. However, it is increasingly recognized that information obtained from single modal molecular imaging cannot satisfy the higher requirements on the efficiency and accuracy for clinical diagnosis and medical research, due to its limitation and default rooted in single molecular imaging technique itself. To compensate for the deficiencies of single function magnetic resonance imaging contrast agents, the combination of multi-modality imaging has turned to be the research hotpot in recent years. This review presents an overview on the recent developments of the functionalization of gadolinium-based contrast agents, and their application in biomedicine applications.

  8. Gadolinium-modulated 19F signals from Perfluorocarbon Nanoparticles as a New Strategy for Molecular Imaging

    PubMed Central

    Neubauer, Anne M.; Myerson, Jacob; Caruthers, Shelton D.; Hockett, Franklin D.; Winter, Patrick M.; Chen, Junjie; Gaffney, Patrick J.; Robertson, J. David; Lanza, Gregory M.; Wickline, Samuel A.

    2008-01-01

    Recent advances in the design of fluorinated nanoparticles for magnetic resonance molecular imaging have enabled specific detection of 19F nuclei, providing unique and quantifiable spectral signatures. However, a pressing need for signal enhancement exists because the total 19F in imaging voxels is often limited. By directly incorporating a relaxation agent (gadolinium) into the lipid monolayer that surrounds the perfluorocarbon, a marked augmentation of the 19F signal from 200nm nanoparticles was achieved. This design increases the magnetic relaxation rate of the 19F nuclei 4-fold at 1.5 T and effects a 125% increase in signal, an effect which is maintained when they are targeted to human plasma clots. By varying the surface concentration of gadolinium, the relaxation effect can be quantitatively modulated to tailor particle properties. This novel strategy dramatically improves the sensitivity and range of 19F MRI/MRS and forms the basis for designing contrast agents capable of sensing their surface chemistry. PMID:18956457

  9. Hyperintense Dentate Nuclei on T1-Weighted MRI: Relation to Repeat Gadolinium Administration

    PubMed Central

    Adin, M.E.; Kleinberg, L.; Vaidya, D.; Zan, E.; Mirbagheri, S.; Yousem, D.M.

    2016-01-01

    BACKGROUND AND PURPOSE A hyperintense appearance of the dentate nucleus on T1-weighted MR images has been related to various clinical conditions, but the etiology remains indeterminate. We aimed to investigate the possible associations between a hyperintense appearance of the dentate nucleus on T1-weighted MR images in patients exposed to radiation and factors including, but not limited to, the cumulative number of contrast-enhanced MR images, amount of gadolinium administration, dosage of ionizing radiation, and patient demographics. MATERIALS AND METHODS The medical records of 706 consecutive patients who were treated with brain irradiation at The Johns Hopkins Medical Institutions between 1995 and 2010 were blindly reviewed by 2 readers. RESULTS One hundred eighty-four subjects were included for dentate nuclei analysis. Among the 184 subjects who cumulatively underwent 2677 MR imaging studies following intravenous gadolinium administration, 103 patients had hyperintense dentate nuclei on precontrast T1-weighted MR images. The average number of gadolinium-enhanced MR imaging studies performed in the group with normal dentate nuclei was significantly lower than that of the group with hyperintense dentate nuclei. The average follow-up time was 62.5 months. No significant difference was observed between hyperintense and normal dentate nuclei groups in terms of exposed radiation dose, serum creatinine and calcium/phosphate levels, patient demographics, history of chemotherapy, and strength of the scanner. No dentate nuclei abnormalities were found on the corresponding CT scans of patients with hyperintense dentate nuclei (n = 44). No dentate nuclei abnormalities were found in 53 healthy volunteers. CONCLUSIONS Repeat performance of gadolinium-enhanced studies likely contributes to a long-standing hyperintense appearance of dentate nuclei on precontrast T1-weighted-MR images. PMID:26294649

  10. Sensitive and transportable gadolinium-core plastic scintillator sphere for neutron detection and counting

    NASA Astrophysics Data System (ADS)

    Dumazert, Jonathan; Coulon, Romain; Carrel, Frédérick; Corre, Gwenolé; Normand, Stéphane; Méchin, Laurence; Hamel, Matthieu

    2016-08-01

    Neutron detection forms a critical branch of nuclear-related issues, currently driven by the search for competitive alternative technologies to neutron counters based on the helium-3 isotope. The deployment of plastic scintillators shows a high potential for efficient detectors, safer and more reliable than liquids, more easily scalable and cost-effective than inorganic. In the meantime, natural gadolinium, through its 155 and mostly 157 isotopes, presents an exceptionally high interaction probability with thermal neutrons. This paper introduces a dual system including a metal gadolinium core inserted at the center of a high-scale plastic scintillator sphere. Incident fast neutrons are thermalized by the scintillator shell and then may be captured with a significant probability by gadolinium 155 and 157 nuclei in the core. The deposition of a sufficient fraction of the capture high-energy prompt gamma signature inside the scintillator shell will then allow discrimination from background radiations by energy threshold, and therefore neutron detection. The scaling of the system with the Monte Carlo MCNPX2.7 code was carried out according to a tradeoff between the moderation of incident fast neutrons and the probability of slow neutron capture by a moderate-cost metal gadolinium core. Based on the parameters extracted from simulation, a first laboratory prototype for the assessment of the detection method principle has been synthetized. The robustness and sensitivity of the neutron detection principle are then assessed by counting measurement experiments. Experimental results confirm the potential for a stable, highly sensitive, transportable and cost-efficient neutron detector and orientate future investigation toward promising axes.

  11. Proposed experiment to measure {gamma}-rays from the thermal neutron capture of gadolinium

    SciTech Connect

    Yano, Takatomi; Ou, I.; Izumi, T.; Yamaguchi, R.; Mori, T.; Sakuda, M.

    2012-11-12

    Gadolinium-157 ({sup 157}Gd) has the largest thermal neutron capture cross section among any stable nuclei. The thermal neutron capture yields {gamma}-ray cascade with total energy of about 8 MeV. Because of these characteristics, Gd is applied for the recent neutrino detectors. Here, we propose an experiment to measure the multiplicity and the angular correlation of {gamma}-rays from the Gd neutron capture. With these information, we expect the improved identification of the Gd neutron capture.

  12. Nickel-based gadolinium alloy for neutron adsorption application in RAM packages.

    SciTech Connect

    Mizia, Ronald A.; Dupont, John Neuman; McConnell, Paul E.; Robino, Charles Victor

    2005-02-01

    The National Spent Nuclear Fuel Program, located at the Idaho National Laboratory (INL), coordinates and integrates national efforts in management and disposal of US Department of Energy (DOE)-owned spent nuclear fuel. These management functions include development of standardised systems for long-term disposal in the proposed Yucca Mountain repository. Nuclear criticality control measures are needed in these systems to avoid restrictive fissile loading limits because of the enrichment and total quantity of fissile material in some types of the DOE spent nuclear fuel. This need is being addressed by development of corrosion-resistant, neutron-absorbing structural alloys for nuclear criticality control. This paper outlines results of a metallurgical development programme that is investigating the alloying of gadolinium into a nickel-chromium-molybdenum alloy matrix. Gadolinium has been chosen as the neutron absorption alloying element due to its high thermal neutron absorption cross section and low solubility in the expected repository environment. The nickel-chromium-molybdenum alloy family was chosen for its known corrosion performance, mechanical properties, and weldability. The workflow of this programme includes chemical composition definition, primary and secondary melting studies, ingot conversion processes, properties testing, and national consensus codes and standards work. The microstructural investigation of these alloys shows that the gadolinium addition is present in the alloy as a gadolinium-rich second phase. The mechanical strength values are similar to those expected for commercial Ni-Cr-Mo alloys. The alloys have been corrosion tested with acceptable results. The initial results of weldability tests have also been acceptable. Neutronic testing in a moderated critical array has generated favourable results. An American Society for Testing and Materials material specification has been issued for the alloy and a Code Case has been submitted to the

  13. Gadolinium uptake by brain cancer cells: Quantitative analysis with X-PEEM spectromicroscopy for cancer therapy

    NASA Astrophysics Data System (ADS)

    De Stasio, Gelsomina; Gilbert, B.; Perfetti, P.; Margaritondo, G.; Mercanti, D.; Ciotti, M. T.; Casalbore, P.; Larocca, L. M.; Rinelli, A.; Pallini, R.

    2000-05-01

    We present the first X-PEEM spectromicroscopy semi-quantitative data, acquired on Gd in glioblastoma cell cultures from human brain cancer. The cells were treated with a Gd compound for the optimization of GdNCT (Gadolinium Neutron Capture Therapy). We analyzed the kinetics of Gd uptake as a function of exposure time, and verified that a quantitative analytical technique gives the same results as our MEPHISTO X-PEEM, demonstrating the feasibility of semi-quantitative spectromicroscopy.

  14. Phospholipid micelle encapsulated gadolinium oxide nanoparticles for imaging and gene delivery

    PubMed Central

    Dixit, Suraj; Das, Mahasweta; Alwarappan, Subbiah; Goicochea, Nancy L.; Howell, Mark; Mohapatra, Subhra

    2014-01-01

    We encapsulated gadolinium oxide (Gd2O3) nanoparticles within phospholipid micelles as a novel low cytotoxic T1-weighted MRI imaging contrast agent (MGdNPs) that can also deliver small molecules such as DNA plasmids. MGdNPs show relatively good MRI relaxivity values, negligible cytotoxicity, excellent cellular uptake and expression of DNA plasmids in vivo. Biodistribution studies in mice show that intranasal and intraperitoneal administration of MGdNPs can effectively target specific organs. PMID:24724012

  15. Gadolinium neutron capture brachytherapy (GdNCB), a new treatment method for intravascular brachytherapy.

    PubMed

    Enger, Shirin A; Rezaei, Arash; Munck af Rosenschöld, Per; Lundqvist, Hans

    2006-01-01

    Restenosis is a major problem after balloon angioplasty and stent implantation. The aim of this study is to introduce gadolinium neutron capture brachytherapy (GdNCB) as a suitable modality for treatment of stenosis. The utility of GdNCB in intravascular brachytherapy (IVBT) of stent stenosis is investigated by using the GEANT4 and MCNP4B Monte Carlo radiation transport codes. To study capture rate, Kerma, absorbed dose and absorbed dose rate around a Gd-containing stent activated with neutrons, a 30 mm long, 5 mm diameter gadolinium foil is chosen. The input data is a neutron spectrum used for clinical neutron capture therapy in Studsvik, Sweden. Thermal neutron capture in gadolinium yields a spectrum of high-energy gamma photons, which due to the build-up effect gives an almost flat dose delivery pattern to the first 4 mm around the stent. The absorbed dose rate is 1.33 Gy/min, 0.25 mm from the stent surface while the dose to normal tissue is in order of 0.22 Gy/min, i.e., a factor of 6 lower. To spare normal tissue further fractionation of the dose is also possible. The capture rate is relatively high at both ends of the foil. The dose distribution from gamma and charge particle radiation at the edges and inside the stent contributes to a nonuniform dose distribution. This will lead to higher doses to the surrounding tissue and may prevent stent edge and in-stent restenosis. The position of the stent can be verified and corrected by the treatment plan prior to activation. Activation of the stent by an external neutron field can be performed days after catherization when the target cells start to proliferate and can be expected to be more radiation sensitive. Another advantage of the nonradioactive gadolinium stent is the possibility to avoid radiation hazard to personnel. PMID:16485408

  16. Gadolinium neutron capture brachytherapy (GdNCB), a new treatment method for intravascular brachytherapy

    SciTech Connect

    Enger, Shirin A.; Rezaei, Arash; Munck af Rosenschoeld, Per; Lundqvist, Hans

    2006-01-15

    Restenosis is a major problem after balloon angioplasty and stent implantation. The aim of this study is to introduce gadolinium neutron capture brachytherapy (GdNCB) as a suitable modality for treatment of stenosis. The utility of GdNCB in intravascular brachytherapy (IVBT) of stent stenosis is investigated by using the GEANT4 and MCNP4B Monte Carlo radiation transport codes. To study capture rate, Kerma, absorbed dose and absorbed dose rate around a Gd-containing stent activated with neutrons, a 30 mm long, 5 mm diameter gadolinium foil is chosen. The input data is a neutron spectrum used for clinical neutron capture therapy in Studsvik, Sweden. Thermal neutron capture in gadolinium yields a spectrum of high-energy gamma photons, which due to the build-up effect gives an almost flat dose delivery pattern to the first 4 mm around the stent. The absorbed dose rate is 1.33 Gy/min, 0.25 mm from the stent surface while the dose to normal tissue is in order of 0.22 Gy/min, i.e., a factor of 6 lower. To spare normal tissue further fractionation of the dose is also possible. The capture rate is relatively high at both ends of the foil. The dose distribution from gamma and charge particle radiation at the edges and inside the stent contributes to a nonuniform dose distribution. This will lead to higher doses to the surrounding tissue and may prevent stent edge and in-stent restenosis. The position of the stent can be verified and corrected by the treatment plan prior to activation. Activation of the stent by an external neutron field can be performed days after catherization when the target cells start to proliferate and can be expected to be more radiation sensitive. Another advantage of the nonradioactive gadolinium stent is the possibility to avoid radiation hazard to personnel.

  17. Optical absorption of Ni2+ and Ni3+ ions in gadolinium gallium garnet epitaxial films

    NASA Astrophysics Data System (ADS)

    Vasileva, N. V.; Gerus, P. A.; Sokolov, V. O.; Plotnichenko, V. G.

    2012-12-01

    Single-crystal Ni-doped gadolinium gallium garnet films were grown for the first time from supercooled Bi2O3-B2O3-based melt solutions by liquid-phase epitaxy. Optical absorption bands due to Ni2+, Ni3+ and Bi3+ ions were observed in those films. Interpretation and tabulation of all absorption bands of nickel ions occupying octahedral and tetrahedral sites in the garnet lattice are presented.

  18. Assembly of Double-Hydrophilic Block Copolymers Triggered by Gadolinium Ions: New Colloidal MRI Contrast Agents.

    PubMed

    Frangville, Camille; Li, Yichen; Billotey, Claire; Talham, Daniel R; Taleb, Jacqueline; Roux, Patrick; Marty, Jean-Daniel; Mingotaud, Christophe

    2016-07-13

    Mixing double-hydrophilic block copolymers containing a poly(acrylic acid) block with gadolinium ions in water leads to the spontaneous formation of polymeric nanoparticles. With an average diameter near 20 nm, the nanoparticles are exceptionally stable, even after dilution and over a large range of pH and ionic strength. High magnetic relaxivities were measured in vitro for these biocompatible colloids, and in vivo magnetic resonance imaging on rats demonstrates the potential utility of such polymeric assemblies. PMID:27224089

  19. Theory of morphological transformation of viral capsid shell during the maturation process in the HK97 bacteriophage and similar viruses.

    PubMed

    Konevtsova, O V; Lorman, V L; Rochal, S B

    2016-05-01

    We consider the symmetry and physical origin of collective displacement modes playing a crucial role in the morphological transformation during the maturation of the HK97 bacteriophage and similar viruses. It is shown that the experimentally observed hexamer deformation and pentamer twist in the HK97 procapsid correspond to the simplest irreducible shear strain mode of a spherical shell. We also show that the icosahedral faceting of the bacteriophage capsid shell is driven by the simplest irreducible radial displacement field. The shear field has the rotational icosahedral symmetry group I while the radial field has the full icosahedral symmetry I_{h}. This difference makes their actions independent. The radial field sign discriminates between the icosahedral and the dodecahedral shapes of the faceted capsid shell, thus making the approach relevant not only for the HK97-like viruses but also for the parvovirus family. In the frame of the Landau-Ginzburg formalism we propose a simple phenomenological model valid for the first reversible step of the HK97 maturation process. The calculated phase diagram illustrates the discontinuous character of the virus shape transformation. The characteristics of the virus shell faceting and expansion obtained in the in vitro and in vivo experiments are related to the decrease in the capsid shell thickness and to the increase of the internal capsid pressure. PMID:27300929

  20. The Capsid Protein of Turnip Crinkle Virus Overcomes two Separate Defense Barriers to Facilitate Viral Systemic Movement in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The capsid protein (CP) of Turnip crinkle virus (TCV) is a multi-functional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV...

  1. RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

  2. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

    PubMed Central

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research. PMID:24435020

  3. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to CPV40 capsid protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAb) were prepared against the 40 kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. By immunoblotting analysis, one MAb, designated MAbCPV40-1, bound to a 40 kDa protein in extracts of C. parvum oocysts, which...

  4. Theory of morphological transformation of viral capsid shell during the maturation process in the HK97 bacteriophage and similar viruses

    NASA Astrophysics Data System (ADS)

    Konevtsova, O. V.; Lorman, V. L.; Rochal, S. B.

    2016-05-01

    We consider the symmetry and physical origin of collective displacement modes playing a crucial role in the morphological transformation during the maturation of the HK97 bacteriophage and similar viruses. It is shown that the experimentally observed hexamer deformation and pentamer twist in the HK97 procapsid correspond to the simplest irreducible shear strain mode of a spherical shell. We also show that the icosahedral faceting of the bacteriophage capsid shell is driven by the simplest irreducible radial displacement field. The shear field has the rotational icosahedral symmetry group I while the radial field has the full icosahedral symmetry Ih. This difference makes their actions independent. The radial field sign discriminates between the icosahedral and the dodecahedral shapes of the faceted capsid shell, thus making the approach relevant not only for the HK97-like viruses but also for the parvovirus family. In the frame of the Landau-Ginzburg formalism we propose a simple phenomenological model valid for the first reversible step of the HK97 maturation process. The calculated phase diagram illustrates the discontinuous character of the virus shape transformation. The characteristics of the virus shell faceting and expansion obtained in the in vitro and in vivo experiments are related to the decrease in the capsid shell thickness and to the increase of the internal capsid pressure.

  5. Pathogenesis of type 2 turkey astroviruses with variant capsid genes in 2-day-old specific pathogen free poults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenicity of three different type 2 turkey astroviruses (TAstV-2) was studied in specific-pathogen-free (SPF) turkeys. These viruses differ based on sequence analysis of the capsid gene. Poults were inoculated at two days of age and examined during 14 days for clinical signs and virus sheddi...

  6. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

  7. Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

    PubMed Central

    MacBeth, K J; Patterson, J L

    1995-01-01

    Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568059

  8. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    PubMed

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  9. Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

    PubMed Central

    Dobos, P.; Kerr, Ian M.; Martin, E. M.

    1971-01-01

    The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of 35S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides. Images PMID:4331652

  10. Homology-based Identification of Capsid Determinants That Protect HIV1 from Human TRIM5α Restriction*

    PubMed Central

    Maillard, Pierre V.; Zoete, Vincent; Michielin, Olivier; Trono, Didier

    2011-01-01

    The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid. PMID:21169362

  11. Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids.

    PubMed

    Pierson, Elizabeth E; Keifer, David Z; Kukreja, Alexander A; Wang, Joseph C-Y; Zlotnick, Adam; Jarrold, Martin F

    2016-01-29

    Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices. PMID:26151485

  12. Large Scale Testing and Development of Gadolinium Trichloride for Use in Neutron Detection in Large Water

    SciTech Connect

    Mark Vagine

    2007-09-18

    Water Cherenkov detectors have been used for many years as inexpensive, effective detectors for neutrino interactions and nucleon decay searches. While many important measurements have been made with these detectors a major drawback has been their inability to detect the absorption of thermal neutrons. We believe an inexpensive, effective technique could be developed to overcome this situation via the addition to water of a solute with a large neutron cross section and energetic gamma daughters which would make neutrons detectable. Gadolinium seems an excellent candidate especially since in recent years it has become very inexpensive, now less than $8 per kilogram in the form of commercially-available gadolinium trichloride, GdCl{sub 3}. This non-toxic, non-reactive substance is highly soluble in water. Neutron capture on gadolinium yields a gamma cascade which would be easily seen in detectors like Super-Kamiokande. We have begun to investigate the use of GdCl{sub 3} as a possible upgrade for the Super-Kamiokande detector with a view toward improving its performance as a detector for atmospheric neutrinos, supernova neutrinos, wrong-sign solar neutrinos, reactor neutrinos, proton decay, and also as a target for the coming T2K long-baseline neutrino experiment. This large-scale investigation, conducted in the one kiloton water Cherenkov detector built for the K2K long-baseline experiment, follows up on highly promising benchtop-scale work previously carried out with the assistance of a 2003 Advanced Detector Research Program grant.

  13. Pineapple juice labeled with gadolinium: a convenient oral contrast for magnetic resonance cholangiopancreatography.

    PubMed

    Coppens, Emmanuel; Metens, Thierry; Winant, Catherine; Matos, Celso

    2005-10-01

    The aim of our study was to prepare in vitro a pineapple juice (PJ) solution labeled with a minimal gadolinium concentration working as a negative contrast agent in heavily T2-weighted imaging and to assess that solution in vivo as a negative oral contrast agent for magnetic resonance cholangiopancreatography (MRCP). Three PJs were compared in vitro according to their T2. Increasing concentrations of gadolinium (Gd)-DOTA in PJ were assessed in vitro for T2 reduction. Single-shot turbo spin echo T2-weighted MR cholangiopancreatograms were obtained for 35 patients with suspected biliopancreatic duct disease, before and after ingestion of the PJ/Gd-DOTA solution. Signal intensity (SI) measurements of gastroduodenal lumens, pancreatobiliary ducts, and image quality scores were obtained systematically before and after contrast ingestion. The in vitro selected Gd-DOTA concentration in the PJ was 2.76 mmol/l. Ingestion of 180 ml of PJ labeled with 1 ml of Gd-DOTA eliminated efficiently the gastroduodenal SI in MRCP, improving significantly the rates of complete visualization of the pancreatobiliary ducts (P<0.01) and the MRCP image quality scores (P<0.05). All patients easily ingested the contrast solution and found the solution palatable. PJ labeled with gadolinium constituted an efficient and convenient negative oral contrast agent for MRCP. PMID:15999215

  14. Breakthrough reactions of iodinated and gadolinium contrast media after oral steroid premedication protocol

    PubMed Central

    2014-01-01

    Background Adverse reactions to iodinated and gadolinium contrast media are an important clinical issue. Although some guidelines have proposed oral steroid premedication protocols to prevent adverse reactions, some patients may have reactions to contrast media in spite of premedication (breakthrough reaction; BTR). The purpose of this study was to assess the frequency, type and severity of BTR when following an oral steroid premedication protocol. Methods All iodinated and gadolinium contrast-enhanced radiologic examinations between August 2011 and February 2013 for which the premedication protocol was applied in our institution were assessed for BTRs. Results The protocol was applied to a total of 252 examinations (153 patients, ages 15–87 years; 63 males, 90 females). Of these, 152 were for prior acute adverse reactions to contrast media, 85 were for a history of bronchial asthma, and 15 were for other reasons. There were 198 contrast enhanced CTs and 54 contrast enhanced MRIs. There were nine BTR (4.5%) for iodinated contrast media, and only one BTR (1.9%) for gadolinium contrast media: eight were mild and one was moderate. No patient who had a mild index reaction (IR) had a severe BTR. Conclusion Incidence of BTRs when following the premedication protocol was low. This study by no means proves the efficacy of premedication, but provides some support for following a premedication protocol to improve safety of contrast-enhanced examinations when prior adverse reactions are mild, or when there is a history of asthma. PMID:25287952

  15. Thermal Excitation of Gadolinium-Based Contrast Agents Using Spin Resonance

    PubMed Central

    Fridjhon, Peter; Rubin, David M.

    2016-01-01

    Theoretical and experimental investigations into the thermal excitation of liquid paramagnetic contrast agents using the spin resonance relaxation mechanism are presented. The electronic spin-lattice relaxation time τ1e of gadolinium-based contrast agents, which is estimated at 0.1 ns, is ten orders of magnitude faster than the relaxation time of protons in water. The shorter relaxation time is found to significantly increase the rate of thermal energy deposition. To the authors’ knowledge this is the first study of gadolinium based contrast agents in a liquid state used as thermal agents. Analysis shows that when τ1e and other experimental parameters are optimally selected, a maximum theoretical heating rate of 29.4 °C.s−1 could be achieved which would suffice for clinical thermal ablation of neoplasms. The experimental results show a statistically significant thermal response for two out of the four contrast agents tested. The results are compared to the simulated estimates via analysis of a detailed model of the system. While these experimentally determined temperature rises are small and thus of no clinical utility, their presence supports the theoretical analysis and strongly suggests that the chemical structure of the selected compounds plays an important role in this mechanism of heat deposition. There exists an opportunity for the development of alternative gadolinium-based compounds with an order of magnitude longer τ1e in a diluted form to be used as an efficient hyperthermia agent for clinical use. PMID:27341338

  16. Thermal Excitation of Gadolinium-Based Contrast Agents Using Spin Resonance.

    PubMed

    Dinger, Steven C; Fridjhon, Peter; Rubin, David M

    2016-01-01

    Theoretical and experimental investigations into the thermal excitation of liquid paramagnetic contrast agents using the spin resonance relaxation mechanism are presented. The electronic spin-lattice relaxation time τ1e of gadolinium-based contrast agents, which is estimated at 0.1 ns, is ten orders of magnitude faster than the relaxation time of protons in water. The shorter relaxation time is found to significantly increase the rate of thermal energy deposition. To the authors' knowledge this is the first study of gadolinium based contrast agents in a liquid state used as thermal agents. Analysis shows that when τ1e and other experimental parameters are optimally selected, a maximum theoretical heating rate of 29.4 °C.s-1 could be achieved which would suffice for clinical thermal ablation of neoplasms. The experimental results show a statistically significant thermal response for two out of the four contrast agents tested. The results are compared to the simulated estimates via analysis of a detailed model of the system. While these experimentally determined temperature rises are small and thus of no clinical utility, their presence supports the theoretical analysis and strongly suggests that the chemical structure of the selected compounds plays an important role in this mechanism of heat deposition. There exists an opportunity for the development of alternative gadolinium-based compounds with an order of magnitude longer τ1e in a diluted form to be used as an efficient hyperthermia agent for clinical use. PMID:27341338

  17. T1 assessment of hip joint cartilage following intra-articular gadolinium injection: a pilot study.

    PubMed

    Bittersohl, Bernd; Hosalkar, Harish S; Kim, Young-Jo; Werlen, Stefan; Trattnig, Siegfried; Siebenrock, Klaus A; Mamisch, Tallal C

    2010-10-01

    This pilot study defines the feasibility of cartilage assessment in symptomatic femoroacetabular impingement patients using intra-articular delayed gadolinium-enhanced MRI of cartilage (ia-dGEMRIC). Nine patients were scanned preliminary to study the contrast infiltration process into hip joint cartilage. Twenty-seven patients with symptomatic femoroacetabular impingement were subsequently scanned with intra-articular delayed gadolinium-enhanced MRI of cartilage. These T(1) findings were correlated to morphological findings. Zonal variations were studied. This pilot study demonstrates a significant difference between the pre- and postcontrast T(1) values (P < 0.001) remaining constant for 45 min. We noted higher mean T(1) values in morphologically normal-appearing cartilage than in damaged cartilage, which was statistically significant for all zones except the anterior-superior zone. Intraobserver (0.972) and interobserver correlation coefficients (0.933) were statistically significant. This study outlines the feasibility of intra-articular delayed gadolinium-enhanced MRI of cartilage for assessment of cartilage changes in patients with femoroacetabular impingement. It can also define the topographic extent and differing severities of cartilage damage. PMID:20872764

  18. Study of the Photon Strength Functions for Gadolinium Isotopes with the DANCE Array

    SciTech Connect

    Dashdorj, D.; Mitchell, G. E.; Baramsai, B.; Chankova, R.; Chyzh, A.; Walker, C.; Agvaanluvsan, U.; Becker, J. A.; Parker, W.; Sleaford, B.; Wu, C. Y.; Bredeweg, T. A.; Couture, A.; Haight, R. C.; Jandel, M.; Rundberg, R. S.; Ullmann, J. L.; Vieira, D. J.; Wouters, J. M.; Krticka, M.

    2009-03-10

    The gadolinium isotopes are interesting for reactor applications as well as for medicine and astrophysics. The gadolinium isotopes have some of the largest neutron capture cross sections. As a consequence they are used in the control rod in reactor fuel assembly. From the basic science point of view, there are seven stable isotopes of gadolinium with varying degrees of deformation. Therefore they provide a good testing ground for the study of deformation dependent structure such as the scissors mode. Decay gamma rays following neutron capture on Gd isotopes are detected by the DANCE array, which is located at flight path 14 at the Lujan Neutron Scattering Center at Los Alamos National Laboratory. The high segmentation and close packing of the detector array enable gamma-ray multiplicity measurements. The calorimetric properties of the DANCE array coupled with the neutron time-of-flight technique enables one to gate on a specific resonance of a specific isotope in the time-of-flight spectrum and obtain the summed energy spectrum for that isotope. The singles gamma-ray spectrum for each multiplicity can be separated by their DANCE cluster multiplicity. Various photon strength function models are used for comparison with experimentally measured DANCE data and provide insight for understanding the statistical decay properties of deformed nuclei.

  19. Selective Filtration of Gadolinium Trichloride for Use in Neutron Detection in Large Water Cherenkov Detectors

    SciTech Connect

    Vagins, Mark R.

    2013-04-10

    Water Cherenkov detectors have been used for many years as inexpensive, effective detectors for neutrino interactions and nucleon decay searches. While many important measurements have been made with these detectors a major drawback has been their inability to detect the absorption of thermal neutrons. We believe an inexpensive, effective technique could be developed to overcome this situation via the addition to water of a solute with a large neutron cross section and energetic gamma daughters which would make neutrons detectable. Gadolinium seems an excellent candidate especially since in recent years it has become very inexpensive, now less than $8 per kilogram in the form of commercially-available gadolinium trichloride, GdCl{sub 3}. This non-toxic, non-reactive substance is highly soluble in water. Neutron capture on gadolinium yields a gamma cascade which would be easily seen in detectors like Super-Kamiokande. We have been investigating the use of GdCl{sub 3} as a possible upgrade for the Super-Kamiokande detector with a view toward improving its performance as a detector for atmospheric neutrinos, supernova neutrinos, wrong-sign solar neutrinos, reactor neutrinos, proton decay, and also as a target for the coming T2K long-baseline neutrino experiment. This focused study of selective water filtration and GdCl{sub 3} extraction techniques, conducted at UC Irvine, followed up on highly promising benchtop-scale and kiloton-scale work previously carried out with the assistance of 2003 and 2005 Advanced Detector Research Program grants.

  20. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    PubMed Central

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J.L.; Roos, Wouter H.; Barsegov, Valeri

    2013-01-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔHind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔSind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function. PMID:24138865

  1. Deletion of Murid Herpesvirus 4 ORF63 Affects the Trafficking of Incoming Capsids toward the Nucleus

    PubMed Central

    Latif, Muhammad Bilal; Machiels, Bénédicte; Xiao, Xue; Mast, Jan; Vanderplasschen, Alain

    2015-01-01

    ABSTRACT Gammaherpesviruses are important human and animal pathogens. Despite the fact that they display the classical architecture of herpesviruses, the function of most of their structural proteins is still poorly defined. This is especially true for tegument proteins. Interestingly, a potential role in immune evasion has recently been proposed for the tegument protein encoded by Kaposi's sarcoma-associated herpesvirus open reading frame 63 (ORF63). To gain insight about the roles of ORF63 in the life cycle of a gammaherpesvirus, we generated null mutations in the ORF63 gene of murid herpesvirus 4 (MuHV-4). We showed that disruption of ORF63 was associated with a severe MuHV-4 growth deficit both in vitro and in vivo. The latter deficit was mainly associated with a defect of replication in the lung but did not affect the establishment of latency in the spleen. From a functional point of view, inhibition of caspase-1 or the inflammasome did not restore the growth of the ORF63-deficient mutant, suggesting that the observed deficit was not associated with the immune evasion mechanism identified previously. Moreover, this growth deficit was also not associated with a defect in virion egress from the infected cells. In contrast, it appeared that MuHV-4 ORF63-deficient mutants failed to address most of their capsids to the nucleus during entry into the host cell, suggesting that ORF63 plays a role in capsid movement. In the future, ORF63 could therefore be considered a target to block gammaherpesvirus infection at a very early stage of the infection. IMPORTANCE The important diseases caused by gammaherpesviruses in human and animal populations justify a better understanding of their life cycle. In particular, the role of most of their tegument proteins is still largely unknown. In this study, we used murid herpesvirus 4, a gammaherpesvirus infecting mice, to decipher the role of the protein encoded by the viral ORF63 gene. We showed that the absence of this protein is

  2. A trimer of dimers is the basic building block for human immunodeficiency virus-1 capsid assembly.

    PubMed

    Tsiang, Manuel; Niedziela-Majka, Anita; Hung, Magdeleine; Jin, Debi; Hu, Eric; Yant, Stephen; Samuel, Dharmaraj; Liu, Xiaohong; Sakowicz, Roman

    2012-06-01

    Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 μM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small

  3. Chasing the Origin of Viruses: Capsid-Forming Genes as a Life-Saving Preadaptation within a Community of Early Replicators

    PubMed Central

    Jalasvuori, Matti; Mattila, Sari; Hoikkala, Ville

    2015-01-01

    Virus capsids mediate the transfer of viral genetic information from one cell to another, thus the origin of the first viruses arguably coincides with the origin of the viral capsid. Capsid genes are evolutionarily ancient and their emergence potentially predated even the origin of first free-living cells. But does the origin of the capsid coincide with the origin of viruses, or is it possible that capsid-like functionalities emerged before the appearance of true viral entities? We set to investigate this question by using a computational simulator comprising primitive replicators and replication parasites within a compartment matrix. We observe that systems with no horizontal gene transfer between compartments collapse due to the rapidly emerging replication parasites. However, introduction of capsid-like genes that induce the movement of randomly selected genes from one compartment to another rescues life by providing the non-parasitic replicators a mean to escape their current compartments before the emergence of replication parasites. Capsid-forming genes can mediate the establishment of a stable meta-population where parasites cause only local tragedies but cannot overtake the whole community. The long-term survival of replicators is dependent on the frequency of horizontal transfer events, as systems with either too much or too little genetic exchange are doomed to succumb to replication-parasites. This study provides a possible scenario for explaining the origin of viral capsids before the emergence of genuine viruses: in the absence of other means of horizontal gene transfer between compartments, evolution of capsid-like functionalities may have been necessary for early life to prevail. PMID:25955384

  4. Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane.

    PubMed Central

    Rhee, S S; Hui, H X; Hunter, E

    1990-01-01

    The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane. Images PMID:2370682

  5. The Last C-Terminal Residue of VP3, Glutamic Acid 257, Controls Capsid Assembly of Infectious Bursal Disease Virus

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Da Costa, Bruno; Delmas, Bernard

    2004-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH2-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process. PMID:15016850

  6. Antivirals interacting with hepatitis B virus core protein and core mutations may misdirect capsid assembly in a similar fashion.

    PubMed

    Hacker, Hans Jörg; Deres, Karl; Mildenberger, Maria; Schröder, Claus H

    2003-12-15

    Recently, heteroarylpyrimidines (HAP) have been identified as potent inhibitors of capsid maturation. Here we discuss the HAP mode of action comparing the aggregation phenotype of wild-type and mutant core proteins with the respective phenotype imposed by HAP or other agents interacting with core protein. Pertinent tests include core fusion protein-mediated transactivation in a two-hybrid system and capsid formation. The finding that transactivation appeared to be unaffected by HAP, or by mutations preventing assembly, is surprising and raises the question for the structure of the interacting hybrid core proteins: Are they monomers, dimers or even oligomers? A direct activity of core fusion monomers is not excluded but considered to be highly unlikely due to rapid homodimerisation. A role of core fusion dimers in transactivation would indicate distinct interactions with a differential sensitivity to HAP. Regarding significance of data gained in two-hybrid systems, caution is necessary, since the site of transactivation is the nucleus, whereas the real site of the core protein interactions during replication is the cytoplasm. Apparently, HAP leave the monomer-monomer interface of HBV core protein unaffected but prevent capsid maturation by interacting with a region known to be crucial for dimer multimerisation and formation of stable capsids. It is suggested to use antivirals as tools for the elucidation of early steps in genome replication and capsid assembly. A frame for this could be the hypothesis that the virus uses soluble core protein, namely intracellular maturation intermediates of HbeAg for a core targeted self-restriction of replication. PMID:14637185

  7. Gadolinium-148 and other spallation production cross section measurements for accelerator target facilities

    NASA Astrophysics Data System (ADS)

    Kelley, Karen Corzine

    At the Los Alamos Neutron Science Center accelerator complex, protons are accelerated to 800 MeV and directed to two tungsten targets, Target 4 at the Weapons Neutron Research facility and the 1L target at the Lujan Center. The Department of Energy requires hazard classification analyses to be performed on these targets and places limits on certain radionuclide inventories in the targets to avoid characterizing the facilities as "nuclear facilities." Gadolinium-148 is a radionuclide created from the spallation of tungsten. Allowed isotopic inventories are particularly low for this isotope because it is an alpha-particle emitter with a 75-year half-life. The activity level of Gadolinium-148 is low, but it encompasses almost two-thirds of the total dose burden for the two tungsten targets based on present yield estimates. From a hazard classification standpoint, this severely limits the lifetime of these tungsten targets. The cross section is not well-established experimentally and this is the motivation for measuring the Gadolinium-148 production cross section from tungsten. In a series of experiments at the Weapons Neutron Research facility, Gadolinium-148 production was measured for 600- and 800-MeV protons on tungsten, tantalum, and gold. These experiments used 3 mum thin tungsten, tantalum, and gold foils and 10 mum thin aluminum activation foils. In addition, spallation yields were determined for many short-lived and long-lived spallation products with these foils using gamma and alpha spectroscopy and compared with predictions of the Los Alamos National Laboratory codes CEM2k+GEM2 and MCNPX. The cumulative Gadolinium-148 production cross section measured from tantalum, tungsten, and gold for incident 600-MeV protons were 15.2 +/- 4.0, 8.31 +/- 0.92, and 0.591 +/- 0.155, respectively. The average production cross sections measured at 800 MeV were 28.6 +/- 3.5, 19.4 +/- 1.8, and 3.69 +/- 0.50 for tantalum, tungsten, and gold, respectively. These cumulative

  8. Simple method for quantification of gadolinium magnetic resonance imaging contrast agents using ESR spectroscopy.

    PubMed

    Takeshita, Keizo; Kinoshita, Shota; Okazaki, Shoko

    2012-01-01

    To develop an estimation method of gadolinium magnetic resonance imaging (MRI) contrast agents, the effect of concentration of Gd compounds on the ESR spectrum of nitroxyl radical was examined. A solution of either 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) was mixed with a solution of Gd compound and the ESR spectrum was recorded. Increased concentration of gadolinium-diethylenetriamine pentaacetic acid chelate (Gd-DTPA), an MRI contrast agent, increased the peak-to-peak line widths of ESR spectra of the nitroxyl radicals, in accordance with a decrease of their signal heights. A linear relationship was observed between concentration of Gd-DTPA and line width of ESR signal, up to approximately 50 mmol/L Gd-DTPA, with a high correlation coefficient. Response of TEMPONE was 1.4-times higher than that of TEMPOL as evaluated from the slopes of the lines. The response was slightly different among Gd compounds; the slopes of calibration curves for acua[N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]glycinato(3-)]gadolinium hydrate (Gd-DTPA-BMA) (6.22 μT·L/mmol) and gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate (Gd-DOTA) (6.62 μT·L/mmol) were steeper than the slope for Gd-DTPA (5.45 μT·L/mmol), whereas the slope for gadolinium chloride (4.94 μT·L/mmol) was less steep than that for Gd-DTPA. This method is simple to apply. The results indicate that this method is useful for rough estimation of the concentration of Gd contrast agents if calibration is carried out with each standard compound. It was also found that the plot of the reciprocal square root of signal height against concentrations of contrast agents could be useful for the estimation if a constant volume of sample solution is taken and measured at the same position in the ESR cavity every time. PMID:22223372

  9. Incorporation of Integral Fuel Burnable Absorbers Boron and Gadolinium into Zirconium-Alloy Fuel Clad Material

    SciTech Connect

    Sridharan, K.; Renk, T.J.; Lahoda, E.J.; Corradini, M.L

    2004-12-14

    Long-lived fuels require the use of higher enrichments of 235U or other fissile materials. Such high levels of fissile material lead to excessive fuel activity at the beginning of life. To counteract this excessive activity, integral fuel burnable absorbers (IFBA) are added to some rods in the fuel assembly. The two commonly used IFBA elements are gadolinium, which is added as gadolinium-oxide to the UO2 powder, and boron, which is applied as a zirconium-diboride coating on the UO2 pellets using plasma spraying or chemical vapor deposition techniques. The incorporation of IFBA into the fuel has to be performed in a nuclear-regulated facility that is physically separated from the main plant. These operations tend to be very costly because of their small volume and can add from 20 to 30% to the manufacturing cost of the fuel. Other manufacturing issues that impact cost and performance are maintaining the correct levels of dosing, the reduction in fuel melting point due to gadolinium-oxide additions, and parasitic neutron absorption at fuel's end-of-life. The goal of the proposed research is to develop an alternative approach that involves incorporation of boron or gadolinium into the outer surface of the fuel cladding material rather than as an additive to the fuel pellets. This paradigm shift will allow for the introduction of the IFBA in a non-nuclear regulated environment and will obviate the necessity of additional handling and processing of the fuel pellets. This could represent significant cost savings and potentially lead to greater reproducibility and control of the burnable fuel in the early stages of the reactor operation. The surface alloying is being performed using the IBEST (Ion Beam Surface Treatment) process developed at Sandia National Laboratories. IBEST involves the delivery of energetic ion beam pulses onto the surface of a material, near-surface melting, and rapid solidification. The non-equilibrium nature of such processing allows for surface

  10. Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion

    SciTech Connect

    Kuznetsov, Yu. G.; McPherson, Alexander . E-mail: amcphers@uci.edu

    2006-09-01

    Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70-75 deg. C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.

  11. Immunogenicity of bacteriophage lambda particles displaying porcine Circovirus 2 (PCV2) capsid protein epitopes.

    PubMed

    Gamage, Lakshman N A; Ellis, John; Hayes, Sidney

    2009-11-01

    Phage lambda particles displaying four immunodominant regions of porcine Circovirus 2 (PCV2) capsid protein (LDP-D-CAP) was shown to be immunogenic in pigs. The immunodominant regions were fused to the carboxyl-terminal of lambda head protein D. Expression of D-CAP on lambda display particles was demonstrated by ELISA and Western blots. Pigs receiving LDP-D-CAP, without incorporating adjuvant, showed significant anti-PCV2 immune response following the primary vaccination. The LDP-D-CAP preparation induced PCV2 neutralizing antibodies. Delayed type hypersensitivity (DTH) reaction scores revealed that the immunized pigs were hypersensitive to both lambda phage and PCV2 antigens. The LDP-D-CAP elicited both cellular and humoral immune responses. Neither LDP-D-CAP nor the lambda control elicited any untoward local or systemic reactions following immunization. These studies produced the first potential phage vaccine to porcine Circovirus 2. PMID:19712770

  12. Defensiveness, trait anxiety, and Epstein-Barr viral capsid antigen antibody titers in healthy college students.

    PubMed

    Esterling, B A; Antoni, M H; Kumar, M; Schneiderman, N

    1993-03-01

    The relationship of individual differences in repressive coping styles with differences in antibody titer to Epstein-Barr viral capsid antigen (EBV-VCA) were investigated in a normal, healthy college population made up of people previously exposed to EBV. Each of 54 1st-year undergraduates completed a battery of physical-status questions and items pertaining to potential behavioral immunomodulatory confounds, along with the Taylor Manifest Anxiety Scale (T-MAS) and the Marlowe-Crowne Social Desirability Scale (MC-SDS). Ss reporting high and middle levels of anxiety had higher antibody titers to EBV, suggesting poorer immune control over the latent virus, as compared with the low-anxious group. Similarly, high-defensive Ss had higher antibody titers than their low-defensive counterparts, and neither group differed from the middle group. PMID:8500440

  13. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    SciTech Connect

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  14. Defining criteria for oligomannose immunogens for HIV using icosahedral virus capsid scaffolds.

    PubMed

    Astronomo, Rena D; Kaltgrad, Eiton; Udit, Andrew K; Wang, Sheng-Kai; Doores, Katie J; Huang, Cheng-Yuan; Pantophlet, Ralph; Paulson, James C; Wong, Chi-Huey; Finn, M G; Burton, Dennis R

    2010-04-23

    The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV. PMID:20416507

  15. Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids.

    PubMed

    Hugel, Thorsten; Michaelis, Jens; Hetherington, Craig L; Jardine, Paul J; Grimes, Shelley; Walter, Jessica M; Falk, Wayne; Anderson, Dwight L; Bustamante, Carlos

    2007-03-01

    The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question. PMID:17311473

  16. Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

    PubMed

    Frutos, Marta de; Leforestier, Amélie; Degrouard, Jéril; Zambrano, Nebraska; Wien, Frank; Boulanger, Pascale; Brasilès, Sandrine; Renouard, Madalena; Durand, Dominique; Livolant, Françoise

    2016-07-01

    We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine. PMID:27152667

  17. Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

    SciTech Connect

    Bhuvanakantham, Raghavan; Chong, Mun-Keat; Ng, Mah-Lee

    2009-11-06

    West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.

  18. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.

    PubMed

    Schur, Florian K M; Obr, Martin; Hagen, Wim J H; Wan, William; Jakobi, Arjen J; Kirkpatrick, Joanna M; Sachse, Carsten; Kräusslich, Hans-Georg; Briggs, John A G

    2016-07-29

    Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1. PMID:27417497

  19. Multiple roles of the capsid protein in the early steps of HIV-1 infection.

    PubMed

    Fassati, Ariberto

    2012-12-01

    The early steps of HIV-1 infection starting after virus entry into cells up to integration of its genome into host chromosomes are poorly understood. From seminal work showing that HIV-1 and oncoretroviruses follow different steps in the early stages post-entry, significant advances have been made in recent years and an important role for the HIV-1 capsid (CA) protein, the constituent of the viral core, has emerged. CA appears to orchestrate several events, such as virus uncoating, recognition by restriction factors and the innate immune system. It also plays a role in nuclear import and integration of HIV-1 and has become a novel target for antiretroviral drugs. Here we describe the different functions of CA and how they may be integrated into one or more coherent models that illuminate the early events in HIV-1 infection and their relations with the host cell. PMID:23041358

  20. Roles of HIV-1 capsid in viral replication and immune evasion.

    PubMed

    Le Sage, Valerie; Mouland, Andrew J; Valiente-Echeverría, Fernando

    2014-11-26

    The primary roles of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein are to encapsidate and protect the viral RNA genome. It is becoming increasing apparent that HIV-1 CA is a multifunctional protein that acts early during infection to coordinate uncoating, reverse transcription, nuclear import of the pre-integration complex and integration of double stranded viral DNA into the host genome. Additionally, numerous recent studies indicate that CA is playing a crucial function in HIV-1 immune evasion. Here we summarize the current knowledge on HIV-1 CA and its interactions with the host cell to promote infection. The fact that CA engages in a number of different protein-protein interactions with the host makes it an interesting target for the development of new potent antiviral agents. PMID:25036886