Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.
- MeSH
- akutní hemoragická konjunktivitida virologie MeSH
- antivirové látky * farmakologie MeSH
- elektronová kryomikroskopie MeSH
- kapsida * ultrastruktura MeSH
- lidé MeSH
- lidský enterovirus D * účinky léků ultrastruktura MeSH
- oxadiazoly farmakologie MeSH
- oxazoly farmakologie MeSH
- virion účinky léků ultrastruktura MeSH
- virové plášťové proteiny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.
- MeSH
- genetické vektory * MeSH
- HEK293 buňky MeSH
- kapsida metabolismus ultrastruktura MeSH
- lidé MeSH
- myši MeSH
- oligopeptidy chemie metabolismus MeSH
- penetrační peptidy chemie metabolismus MeSH
- Polyomavirus genetika metabolismus ultrastruktura MeSH
- transdukce genetická * MeSH
- virion genetika metabolismus ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Bacteriophages of the significant veterinary pathogen Staphylococcus pseudintermedius are rarely described morphologically and genomically in detail, and mostly include phages of the Siphoviridae family. There is currently no taxonomical classification for phages of this bacterial species. Here we describe a new phage designated vB_SpsS_QT1, which is related to phage 2638A originally described as a Staphylococcus aureus phage. Propagating strain S. aureus 2854 of the latter was reclassified by rpoB gene sequencing as S. pseudintermedius 2854 in this work. Both phages have a narrow but different host range determined on 54 strains. Morphologically, both of them belong to the family Siphoviridae, share the B1 morphotype, and differ from other staphylococcal phage genera by a single long fibre at the terminus of the tail. The complete genome of phage vB_SpsS_QT1 was sequenced with the IonTorrent platform and expertly annotated. Its linear genome with cohesive ends is 43,029 bp long and encodes 60 predicted genes with the typical modular structure of staphylococcal siphophages. A global alignment found the genomes of vB_SpsS_QT1 and 2638A to share 84% nucleotide identity, but they have no significant similarity of nucleotide sequences with other phage genomes available in public databases. Based on the morphological, phylogenetic, and genomic analyses, a novel genus Fibralongavirus in the family Siphoviridae is described with phage species vB_SpsS_QT1 and 2638A.
Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.
- MeSH
- Cercopithecus aethiops MeSH
- dvouvláknová RNA chemie genetika MeSH
- elektronová kryomikroskopie MeSH
- enterovirus B lidský genetika ultrastruktura MeSH
- epitelové buňky ultrastruktura virologie MeSH
- genom virový * MeSH
- kapsida chemie ultrastruktura MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- RNA virová chemie genetika MeSH
- simulace molekulární dynamiky MeSH
- svlékání virového obalu genetika MeSH
- virion genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
- MeSH
- elektronová kryomikroskopie MeSH
- genové produkty gag chemie genetika ultrastruktura MeSH
- HEK293 buňky MeSH
- HIV-1 chemie genetika ultrastruktura MeSH
- kapsida chemie ultrastruktura MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- myši MeSH
- proteinové domény MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- tomografie elektronová MeSH
- virion chemie genetika ultrastruktura MeSH
- virové plášťové proteiny chemie genetika ultrastruktura MeSH
- virus myší leukemie chemie genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- srovnávací studie MeSH
Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.
- MeSH
- DNA viry genetika fyziologie ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- genom virový MeSH
- kapsida metabolismus ultrastruktura MeSH
- sestavení viru MeSH
- virion genetika fyziologie ultrastruktura MeSH
- virové proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, we present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.
- MeSH
- elektronová kryomikroskopie MeSH
- exprese genu MeSH
- fúze membrán genetika MeSH
- imunoglobuliny - Fab fragmenty biosyntéza chemie MeSH
- internalizace viru MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- nádorové buněčné linie MeSH
- neurony patologie virologie MeSH
- neutralizující protilátky biosyntéza chemie MeSH
- proteinové domény MeSH
- protilátky virové biosyntéza chemie MeSH
- virion genetika metabolismus ultrastruktura MeSH
- virové proteiny chemie genetika metabolismus MeSH
- viry klíšťové encefalitidy genetika metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.
The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.
- MeSH
- elektronová kryomikroskopie MeSH
- kapsida ultrastruktura MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- počítačové zpracování obrazu MeSH
- proteinové domény MeSH
- RNA-viry ultrastruktura MeSH
- sekvence aminokyselin MeSH
- včely virologie MeSH
- virion ultrastruktura MeSH
- virové plášťové proteiny chemie ultrastruktura MeSH
- viry hmyzu ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.
- MeSH
- Dicistroviridae ultrastruktura MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- včely virologie MeSH
- virion ultrastruktura MeSH
- virové plášťové proteiny chemie MeSH
- virové struktury MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
