scholarly journals Abstract OR-7: Genome Release Mechanism of Picorna-Like Viruses

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S9-S10
Author(s):  
Pavel Plevka ◽  
Karel Škubník ◽  
Lukáš Sukeník ◽  
David Buchta ◽  
Tibor Füzik ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Virus Genome ◽  
Release Mechanism ◽  
Icosahedral Symmetry ◽  
Acidic Ph ◽  
Virus Particles ◽  
Animal Pathogens ◽  
Virus Capsids ◽  
Virus Genomes

Protein capsids protect the genomes of viruses from degradation in the extracellular environment. However, virus capsids must release genomes into a host cell to initiate infection. We used cryo-electron microscopy to characterize the genome release of viruses from the order Picornavirales: picornaviruses, dicistroviruses, and iflaviruses. These virus families include numerous human and animal pathogens. The viruses have non-enveloped virions and capsids organized with icosahedral symmetry. Their genome release can be induced in vitro by exposure to acidic pH, mimicking conditions in endosomes. We show that conformational changes of capsids and expansion of viral RNA genomes, which are induced by acidic pH, trigger the opening of picorna-like virus particles. The capsids of the studied viruses crack into pieces or open like flowers to release their genomes. The large openings of capsids enable the virus genomes to exit within microseconds, which limits the probability of their degradation by the RNases. Characterization of the virus genome release is the first step towards developing inhibitors of the process.

scholarly journals Capsid opening enables genome release of iflaviruses

2021 ◽  
Vol 7 (1) ◽  
pp. eabd7130
Author(s):  
Karel Škubník ◽  
Lukáš Sukeník ◽  
David Buchta ◽  
Tibor Füzik ◽  
Michaela Procházková ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Icosahedral Symmetry ◽  
Acidic Ph ◽  
Deformed Wing Virus ◽  
Sacbrood Virus ◽  
The Family ◽  
Paralysis Virus ◽  
Genome Degradation

The family Iflaviridae includes economically important viruses of the western honeybee such as deformed wing virus, slow bee paralysis virus, and sacbrood virus. Iflaviruses have nonenveloped virions and capsids organized with icosahedral symmetry. The genome release of iflaviruses can be induced in vitro by exposure to acidic pH, implying that they enter cells by endocytosis. Genome release intermediates of iflaviruses have not been structurally characterized. Here, we show that conformational changes and expansion of iflavirus RNA genomes, which are induced by acidic pH, trigger the opening of iflavirus particles. Capsids of slow bee paralysis virus and sacbrood virus crack into pieces. In contrast, capsids of deformed wing virus are more flexible and open like flowers to release their genomes. The large openings in iflavirus particles enable the fast exit of genomes from capsids, which decreases the probability of genome degradation by the RNases present in endosomes.

scholarly journals B23/nucleophosmin is involved in regulation of adenovirus chromatin structure at late infection stages, but not in virus replication and transcription

2012 ◽  
Vol 93 (6) ◽  
pp. 1328-1338 ◽  
Author(s):  
Mohammad Abdus Samad ◽  
Tetsuro Komatsu ◽  
Mitsuru Okuwaki ◽  
Kyosuke Nagata
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Genome ◽  
Virus Particles ◽  
Core Proteins ◽  
Stimulatory Factor ◽  
Interfering Rna

B23/nucleophosmin has been identified in vitro as a stimulatory factor for replication of adenovirus DNA complexed with viral basic core proteins. In the present study, the in vivo function of B23 in the adenovirus life cycle was studied. It was found that both the expression of a decoy mutant derived from adenovirus core protein V that tightly associates with B23 and small interfering RNA-mediated depletion of B23 impeded the production of progeny virions. However, B23 depletion did not significantly affect the replication and transcription of the virus genome. Chromatin immunoprecipitation analyses revealed that B23 depletion significantly increased the association of viral DNA with viral core proteins and cellular histones. These results suggest that B23 is involved in the regulation of association and/or dissociation of core proteins and cellular histones with the virus genome. In addition, these results suggest that proper viral chromatin assembly, regulated in part by B23, is crucial for the maturation of infectious virus particles.

scholarly journals Virion structure and genome delivery mechanism of sacbrood honeybee virus

2018 ◽  
Vol 115 (30) ◽  
pp. 7759-7764 ◽  
Author(s):  
Michaela Procházková ◽  
Tibor Füzik ◽  
Karel Škubník ◽  
Jana Moravcová ◽  
Zorica Ubiparip ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Honey Bee ◽  
Virus Genome ◽  
Icosahedral Symmetry ◽  
The Family ◽  
Virion Structure ◽  
Negative Effect ◽  
A Minor ◽  
Bee Colonies

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.

scholarly journals In Vitro-Assembled Alphavirus Core-Like Particles Maintain a Structure Similar to That of Nucleocapsid Cores in Mature Virus

2002 ◽  
Vol 76 (21) ◽  
pp. 11128-11132 ◽  
Author(s):  
Suchetana Mukhopadhyay ◽  
Paul R. Chipman ◽  
Eunmee M. Hong ◽  
Richard J. Kuhn ◽  
Michael G. Rossmann
Keyword(s):  
Nucleic Acid ◽  
Capsid Protein ◽  
Structural Information ◽  
Poor Quality ◽  
Icosahedral Symmetry ◽  
Virus Particles ◽  
Surface Lattice ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 Å with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.

scholarly journals The ND10 Complex Represses Lytic Human Herpesvirus 6A Replication and Promotes Silencing of the Viral Genome

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 401 ◽  
Author(s):  
Anirban Sanyal ◽  
Nina Wallaschek ◽  
Mandy Glass ◽  
Louis Flamand ◽  
Darren Wight ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Virus Genome ◽  
Lytic Replication ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Infected Cells ◽  
T Cell Lines ◽  
Virus Genomes

Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells.

scholarly journals Sindbis Virus Conformational Changes Induced by a Neutralizing Anti-E1 Monoclonal Antibody

2008 ◽  
Vol 82 (12) ◽  
pp. 5750-5760 ◽  
Author(s):  
Raquel Hernandez ◽  
Angel Paredes ◽  
Dennis T. Brown
Keyword(s):  
Monoclonal Antibody ◽  
Virus Particle ◽  
Conformational Changes ◽  
Sindbis Virus ◽  
Low Ph ◽  
General Mechanism ◽  
Virus Infectivity ◽  
Virus Particles ◽  
Infectious State

ABSTRACT A rare Sindbis virus anti-E1 neutralizing monoclonal antibody, Sin-33, was investigated to determine the mechanism of in vitro neutralization. A cryoelectron microscopic reconstruction of Sindbis virus (SVHR) neutralized with FAb from Sin-33 (FAb-33) revealed conformational changes on the surface of the virion at a resolution of 24 Å. FAb-33 was found to bind E1 in less than 1:1 molar ratios, as shown by the absence of FAb density in the reconstruction and stoichiometric measurements using radiolabeled FAb-33, which determined that about 60 molecules of FAb-33 bound to the 240 possible sites in a single virus particle. FAb-33-neutralized virus particles became sensitive to digestion by endoproteinase Glu-C, providing further evidence of antibody-induced structural changes within the virus particle. The treatment of FAb-33-neutralized or Sin-33-neutralized SVHR with low pH did not induce the conformational rearrangements required for virus membrane-cell membrane fusion. Exposure to low pH, however, increased the amount of Sin-33 or FAb-33 that bound to the virus particles, indicating the exposure of additional epitopes. The neutralization of SVHR infection by FAb-33 or Sin-33 did not prevent the association of virus with host cells. These data are in agreement with the results of previous studies that demonstrated that specific antibodies can inactivate the infectious state of a metastable virus in vitro by the induction of conformational changes to produce an inactive structure. A model is proposed which postulates that the induction of conformational changes in the infectious state of a metastable enveloped virus may be a general mechanism of antibody inactivation of virus infectivity.

scholarly journals Activities of Ceftobiprole and Other Cephalosporins against Extracellular and Intracellular (THP-1 Macrophages and Keratinocytes) Forms of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus

2009 ◽  
Vol 53 (6) ◽  
pp. 2289-2297 ◽  
Author(s):  
Sandrine Lemaire ◽  
Youri Glupczynski ◽  
Valérie Duval ◽  
Bernard Joris ◽  
Paul M. Tulkens ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Conformational Changes ◽  
Acidic Ph ◽  
Binding Assays ◽  
Methicillin Resistant ◽  
Partial Restoration ◽  
Drug Concentrations ◽  
Mrsa Strains ◽  
Hospital Acquired

ABSTRACT Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, β-lactams show activity against intracellular methicillin (meticillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these β-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (E max), relative potency (the concentration causing a reduction of the inoculum halfway between E 0 and E max [EC50]), and static concentration (Cs )} were measured. All strains showed sigmoidal dose-responses, with E max being about a 1 log10 CFU decrease from the postphagocytosis inoculum, and EC50 and Cs being 0.2 to 0.3× and 0.6 to 0.9× the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.

scholarly journals The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs

2016 ◽  
Vol 2 (8) ◽  
pp. e1501929 ◽  
Author(s):  
Hyunwook Lee ◽  
Kristin L. Shingler ◽  
Lindsey J. Organtini ◽  
Robert E. Ashley ◽  
Alexander M. Makhov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Viral Proteins ◽  
Receptor Interaction ◽  
Icosahedral Symmetry ◽  
The Novel ◽  
Structural Studies ◽  
Cryo Electron Microscopy ◽  
Unique Site

Many nonenveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on the mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules to trigger the entry intermediate or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. These first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 Å, respectively) revealed a novel A-particle that is markedly different from the classical A-particles. The asymmetric receptor binding triggers minimal global capsid expansion but marked local conformational changes at the site of receptor interaction. In addition, viral proteins extrude from the capsid only at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of the receptor triggers formation of a unique site in preparation for genome release.

scholarly journals Virion structure and in vitro genome release mechanism of dicistrovirus Kashmir bee virus

2021 ◽  
Author(s):  
Liya Mukhamedova ◽  
Tibor Füzik ◽  
Jiří Nováček ◽  
Dominik Hrebík ◽  
Antonín Přidal ◽  
...  
Keyword(s):  
Ecological Impact ◽  
Infection Process ◽  
Capsid Proteins ◽  
Release Mechanism ◽  
Acidic Ph ◽  
Colony Collapse Disorder ◽  
Colony Collapse ◽  
Kashmir Bee Virus ◽  
Bee Virus

Infections of Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here we present the structure of the virion of KBV determined to a resolution of 2.8 Å. We show that the exposure of KBV to acidic pH induces a reduction in inter-pentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae. Importance The western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the US, and no cure of the disease is currently available. Here we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

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