Membrane penetration by non-enveloped viruses is diverse and generally not well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. Enteroviruses enter cells by receptor-mediated endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing enteroviruses deform, rupture, and release the virus particles into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of particles that released their genomes, but did not prevent them from reaching the cytoplasm. Inhibiting post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. The rupture of endosomes also occurs in control cells and after the endocytosis of very low-density lipoprotein. In summary, our results show that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus particles into the cytoplasm to initiate infection. Since the studied enteroviruses employ different receptors for cell entry but are delivered into the cytoplasm by cell-mediated endosome disruption, it is likely that most if not all enteroviruses, and probably numerous other viruses from the family Picornaviridae, can utilize endosome rupture to infect cells.

• MeSH
• buněčná membrána ultrastruktura   virologie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• cytoplazma virologie   MeSH
• elektronová kryomikroskopie MeSH
• endocytóza * MeSH
• endozomy * patologie   virologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• makrolidy farmakologie   MeSH
• pikornavirové infekce * virologie   MeSH
• Rhinovirus * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bafilomycin A1 MeSH Prohlížeč
• makrolidy MeSH

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

• Klíčová slova
• cryo-electron microscopy, genome release, receptor, structure, virus,
• MeSH
• aktivace viru fyziologie   MeSH
• elektronová kryomikroskopie MeSH
• enterovirové infekce metabolismus   virologie   MeSH
• genom virový genetika   MeSH
• HeLa buňky MeSH
• kapsida metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mezibuněčná adhezivní molekula-1 chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• Rhinovirus genetika   metabolismus   fyziologie   MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• svlékání virového obalu fyziologie   MeSH
• vazba proteinů MeSH
• virion genetika   metabolismus   ultrastruktura   MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ICAM1 protein, human MeSH Prohlížeč
• mezibuněčná adhezivní molekula-1 MeSH
• RNA virová MeSH
• virové plášťové proteiny MeSH

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 ImportanceThe 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.

• Publikační typ
• časopisecké články MeSH

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.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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
• Názvy látek
• dvouvláknová RNA MeSH
• RNA virová MeSH

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.

• Klíčová slova
• genome, honeybee, release, structure, virus,
• MeSH
• endozomy chemie   metabolismus   virologie   MeSH
• genom virový * MeSH
• krystalografie rentgenová MeSH
• RNA-viry * chemie   metabolismus   MeSH
• včely virologie   MeSH
• virion * chemie   metabolismus   MeSH
• virové plášťové proteiny * chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny * 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.

• Klíčová slova
• Apis mellifera, Cripavirus, Dicistroviridae, Picornavirales, Triatovirus, X ray, X-ray crystallography, capsid, crystallography, honey bee, honeybee, insect disease, structure, virion, virus,
• 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
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

UNLABELLED: Viruses of the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-electron microscopy analysis of IAPV virions induced to release their genomes in vitro We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 Å and of empty capsids to a resolution of 3.9 Å. We show that IAPV does not form expanded A particles before genome release as in the case of related enteroviruses of the family Picornaviridae The structural changes observed in the empty IAPV particles include detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike the case for many picornaviruses, the empty particles of IAPV are not expanded relative to the native virions and do not contain pores in their capsids that might serve as channels for genome release. Therefore, rearrangement of a unique region of the capsid is probably required for IAPV genome release. IMPORTANCE: Honeybee populations in Europe and North America are declining due to pressure from pathogens, including viruses. Israeli acute bee paralysis virus (IAPV), a member of the family Dicistroviridae, causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present a structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that genome release is not preceded by an expansion of IAPV virions as in the case of related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon genome release. The genome leaves behind empty particles that have compact protein shells.

• Klíčová slova
• Aparavirus, Apis mellifera, CCD, Dicistroviridae, Picornavirales, capsid, colony collapse disorder, cryo, electron microscopy, empty, genome, honey bee, honeybee, release, structure, uncoating, virion, virus,
• MeSH
• Dicistroviridae fyziologie   ultrastruktura   MeSH
• elektronová kryomikroskopie * MeSH
• genom virový * MeSH
• kapsida metabolismus   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• sestavení viru MeSH
• svlékání virového obalu * MeSH
• včely virologie   MeSH
• virion fyziologie   ultrastruktura   MeSH
• virové plášťové proteiny chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Viruses from the family Iflaviridae are insect pathogens. Many of them, including slow bee paralysis virus (SBPV), cause lethal diseases in honeybees and bumblebees, resulting in agricultural losses. Iflaviruses have nonenveloped icosahedral virions containing single-stranded RNA genomes. However, their genome release mechanism is unknown. Here, we show that low pH promotes SBPV genome release, indicating that the virus may use endosomes to enter host cells. We used cryo-EM to study a heterogeneous population of SBPV virions at pH 5.5. We determined the structures of SBPV particles before and after genome release to resolutions of 3.3 and 3.4 Å, respectively. The capsids of SBPV virions in low pH are not expanded. Thus, SBPV does not appear to form "altered" particles with pores in their capsids before genome release, as is the case in many related picornaviruses. The egress of the genome from SBPV virions is associated with a loss of interpentamer contacts mediated by N-terminal arms of VP2 capsid proteins, which result in the expansion of the capsid. Pores that are 7 Å in diameter form around icosahedral threefold symmetry axes. We speculate that they serve as channels for the genome release. Our findings provide an atomic-level characterization of the genome release mechanism of iflaviruses.

• Klíčová slova
• electron microscopy, honeybee, structure, uncoating, virus,
• MeSH
• Dicistroviridae genetika   fyziologie   ultrastruktura   MeSH
• elektronová kryomikroskopie MeSH
• genom virový MeSH
• kapsida ultrastruktura   MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• Picornaviridae genetika   fyziologie   ultrastruktura   MeSH
• statická elektřina MeSH
• svlékání virového obalu fyziologie   MeSH
• včely virologie   MeSH
• virové plášťové proteiny chemie   ultrastruktura   MeSH
• viry hmyzu genetika   fyziologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Aichi virus 1 (AiV-1) is a human pathogen from the Kobuvirus genus of the Picornaviridae family. Worldwide, 80 to 95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. We present here the structure of the AiV-1 virion determined to a resolution of 2.1 Å using X-ray crystallography. The surface loop puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue BC loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 Å using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at least in vitro, and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release. IMPORTANCE Aichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80 to 95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from the Picornaviridae family. We also determined that AiV-1 capsids form compact shells even after genome release. Therefore, AiV-1 genome release requires large localized and probably reversible reorganization of the capsid.

• Publikační typ
• časopisecké články MeSH