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 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 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 Virus Status, Varroa Levels, and Survival of 20 Managed Honey Bee Colonies Monitored in Luxembourg Between the Summer of 2011 and the Spring of 2013

2015 ◽  
Vol 59 (1) ◽  
pp. 59-73 ◽  
Author(s):  
Antoine Clermont ◽  
Matias Pasquali ◽  
Michael Eickermann ◽  
François Kraus ◽  
Lucien Hoffmann ◽  
...  
Keyword(s):  
Honey Bee ◽  
Varroa Destructor ◽  
Principal Component ◽  
Deformed Wing Virus ◽  
Sacbrood Virus ◽  
Queen Cell ◽  
Varroa Mites ◽  
Kashmir Bee Virus ◽  
Bee Colonies ◽  
Paralysis Virus

Abstract Twenty managed honey bee colonies, split between 5 apiaries with 4 hives each, were monitored between the summer of 2011 and spring of 2013. Living bees were sampled in July 2011, July 2012, and August 2012. Twenty-five, medium-aged bees, free of varroa mites, were pooled per colony and date, to form one sample. Unlike in France and Belgium, Chronic Bee Paralysis Virus (CBPV) has not been found in Luxembourg. Slow Bee Paralysis Virus (SBPV) and Israeli Acute Paralysis Virus (IAPV) levels were below detection limits. Traces of Kashmir Bee Virus (KBV) were amplified. Black Queen Cell Virus (BQCV), Varroa destructor Virus-1 (VDV-1), and SacBrood Virus (SBV) were detected in all samples and are reported from Luxembourg for the first time. Varroa destructor Macula- Like Virus (VdMLV), Deformed Wing Virus (DWV), and Acute Bee Paralysis Virus (ABPV) were detected at all locations, and in most but not all samples. There was a significant increase in VDV-1 and DWV levels within the observation period. A principal component analysis was unable to separate the bees of colonies that survived the following winter from bees that died, based on their virus contents in summer. The number of dead varroa mites found below colonies was elevated in colonies that died in the following winter. Significant positive relationships were found between the log-transformed virus levels of the bees and the log-transformed number of mites found below the colonies per week, for VDV-1 and DWV. Sacbrood virus levels were independent of varroa levels, suggesting a neutral or competitive relationship between this virus and varroa.

Occurrence, detection, and quantification of economically important viruses in healthy and unhealthy honey bee (Hymenoptera: Apidae) colonies in Canada

2015 ◽  
Vol 148 (1) ◽  
pp. 22-35 ◽  
Author(s):  
Suresh D. Desai ◽  
Santosh Kumar ◽  
Robert W. Currie
Keyword(s):  
Polymerase Chain Reaction ◽  
Honey Bee ◽  
Multiple Infections ◽  
Black Queen Cell Virus ◽  
Chain Reaction ◽  
Deformed Wing Virus ◽  
Sacbrood Virus ◽  
Queen Cell ◽  
Polymerase Chain ◽  
Paralysis Virus

AbstractThe occurrence, quantification, and distribution patterns of deformed wing virus (DWV) and sacbrood virus (SBV), (family Iflaviridae); black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and acute bee paralysis virus (ABPV) (family Dicistroviridae), and chronic bee paralysis virus (CBPV) (unclassified), were characterised in 80 “healthy” honey bee (Apis mellifera Linnaeus; Hymenoptera: Apidae) colonies and 23 “unhealthy” colonies by employing reverse transcription polymerase chain reaction (RT-PCR) for virus identification and quantitative real-time polymerase chain reaction (qPCR) for quantification. All seven viruses were common but the most prevalent viruses were DWV, followed by BQCV and IAPV. For most viruses, prevalence in surviving but unhealthy colonies in spring did not differ from that of healthy baseline colony levels in fall suggesting spring prevalence level would not be a useful metric for diagnosis of factors contributing to colony loss. Sacbrood virus was the only virus that was more prevalent in unhealthy colonies from Manitoba, Canada than in healthy from colonies across Canada but did not differ from healthy colonies within Manitoba. Multiple infections were ubiquitous with a few colonies having simultaneous infection with as many as five viruses. Among the three viruses quantified by qPCR, DWV had the highest relative concentrations in pooled samples of worker bees. Deformed wing virus was the only virus within healthy colonies that differed in fall concentration among provinces and was at high levels in unhealthy colonies. Black queen cell virus was positively correlated with IAPV across all samples. Our study provides the first major baseline study of viruses in Canadian honey bees.

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.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

QUALITATIVE AND QUANTITATIVE PHYTOCHEMICAL SCREENING AND IN VITRO ANTIOXIDANT EVALUATION OF PERGULARIA DAEMIA

2018 ◽  
Vol 24 (2) ◽  
Author(s):  
GITA MISHRA ◽  
HEMESHWER KUMAR CHANDRA ◽  
NISHA SAHU ◽  
SATENDRA KUMAR NIRALA ◽  
MONIKA BHADAURIA
Keyword(s):  
Reducing Sugars ◽  
Antioxidant Properties ◽  
Phytochemical Analysis ◽  
Phytochemical Screening ◽  
Qualitative And Quantitative ◽  
The Family ◽  
Ethanolic Extracts ◽  
Antioxidant Evaluation ◽  
In Vitro Antioxidant Activity

Pergularia daemia belongs to the family Asclepiadaceae, known to have anticancer, anti-inflammatory activity. Aim of the present study was to evaluate qualitative and quantitative phytochemical and antioxidant properties of ethanolic extracts of leaf, stem and root parts of P. daemia . Preliminary phytochemical analysis and in vitro antioxidant properties were evaluated by standard methods. The qualitative phytochemical analysis of P. daemia showed presence of flavonoids, tannins, alkaloid, phytosterol, carbohydrate, phenol, saponin, glycosides, terpenoids, steroids proteins and reducing sugars. Quantitative analysis showed polyphenol, flavonoid, flavonone, flavone and flavonol in P. daemia leaves, stem and root in considerable quantity. The in vitro antioxidant activity of P. daemia clearly demonstrated that leaf, stem and root parts have prominent antioxidant properties and was effective in scavenging free radicals.

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