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1.
Nature ; 550(7674): 114-118, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28953874

RESUMO

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.


Assuntos
Bancos de Espécimes Biológicos , Genômica/métodos , Haploidia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Animais , Vasos Sanguíneos/citologia , Linhagem da Célula/genética , Resfriado Comum/genética , Resfriado Comum/virologia , Genes Essenciais/genética , Testes Genéticos , Células HEK293 , Homozigoto , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Neovascularização Fisiológica/genética , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Independentes de Cálcio/metabolismo , Rhinovirus/patogenicidade
2.
Proc Natl Acad Sci U S A ; 116(38): 19109-19115, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31462495

RESUMO

Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.


Assuntos
Antivirais/metabolismo , Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Farmacorresistência Viral , Oxidiazóis/farmacologia , Rhinovirus/metabolismo , Proteínas Virais/química , Antivirais/farmacologia , Sítios de Ligação , Capsídeo/efeitos dos fármacos , Capsídeo/ultraestrutura , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/virologia , Ligação Proteica , Conformação Proteica , Rhinovirus/efeitos dos fármacos , Rhinovirus/ultraestrutura , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Proc Natl Acad Sci U S A ; 115(30): E7158-E7165, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987044

RESUMO

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.


Assuntos
Infecções por Picornaviridae/metabolismo , Rhinovirus/fisiologia , Replicação Viral/fisiologia , Animais , Desoxiglucose/farmacologia , Feminino , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Nucleotídeos/biossíntese , Nucleotídeos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/patologia , Replicação Viral/efeitos dos fármacos
4.
PLoS Pathog ; 14(8): e1007203, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30080883

RESUMO

In nearly all picornaviruses the precursor of the smallest capsid protein VP4 undergoes co-translational N-terminal myristoylation by host cell N-myristoyltransferases (NMTs). Curtailing this modification by mutation of the myristoylation signal in poliovirus has been shown to result in severe assembly defects and very little, if any, progeny virus production. Avoiding possible pleiotropic effects of such mutations, we here used pharmacological abrogation of myristoylation with the NMT inhibitor DDD85646, a pyrazole sulfonamide originally developed against trypanosomal NMT. Infection of HeLa cells with coxsackievirus B3 in the presence of this drug decreased VP0 acylation at least 100-fold, resulting in a defect both early and late in virus morphogenesis, which diminishes the yield of viral progeny by about 90%. Virus particles still produced consisted mainly of provirions containing RNA and uncleaved VP0 and, to a substantially lesser extent, of mature virions with cleaved VP0. This indicates an important role of myristoylation in the viral maturation cleavage. By electron microscopy, these RNA-filled particles were indistinguishable from virus produced under control conditions. Nevertheless, their specific infectivity decreased by about five hundred fold. Since host cell-attachment was not markedly impaired, their defect must lie in the inability to transfer their genomic RNA into the cytosol, likely at the level of endosomal pore formation. Strikingly, neither parechoviruses nor kobuviruses are affected by DDD85646, which appears to correlate with their native capsid containing only unprocessed VP0. Individual knockout of the genes encoding the two human NMT isozymes in haploid HAP1 cells further demonstrated the pivotal role for HsNMT1, with little contribution by HsNMT2, in the virus replication cycle. Our results also indicate that inhibition of NMT can possibly be exploited for controlling the infection by a wide spectrum of picornaviruses.


Assuntos
Aciltransferases/metabolismo , Aminopiridinas/farmacologia , Enterovirus/efeitos dos fármacos , Enterovirus/fisiologia , Sulfonamidas/farmacologia , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/metabolismo , Infecções por Coxsackievirus/metabolismo , Células HeLa , Humanos , Vírion/efeitos dos fármacos , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos
5.
PLoS Pathog ; 13(9): e1006643, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28915259

RESUMO

Antibody-dependent enhancement of viral infection is a well-described phenomenon that is based on the cellular uptake of infectious virus-antibody complexes following their interaction with Fcγ receptors expressed on myeloid cells. Here we describe a novel mechanism of antibody-mediated enhancement of infection by a flavivirus (tick-borne encephalitis virus) in transformed and primary human cells, which is independent of the presence of Fcγ receptors. Using chemical cross-linking and immunoassays, we demonstrate that the monoclonal antibody (mab) A5, recognizing an epitope at the interface of the dimeric envelope protein E, causes dimer dissociation and leads to the exposure of the fusion loop (FL). Under normal conditions of infection, this process is triggered only after virus uptake by the acidic pH in endosomes, resulting in the initiation of membrane fusion through the interaction of the FL with the endosomal membrane. Analysis of virus binding and cellular infection, together with inhibition by the FL-specific mab 4G2, indicated that the FL, exposed after mab A5- induced dimer-dissociation, mediated attachment of the virus to the plasma membrane also at neutral pH, thereby increasing viral infectivity. Since antibody-induced enhancement of binding was not only observed with cells but also with liposomes, it is likely that increased infection was due to FL-lipid interactions and not to interactions with cellular plasma membrane proteins. The novel mechanism of antibody-induced infection enhancement adds a new facet to the complexity of antibody interactions with flaviviruses and may have implications for yet unresolved effects of polyclonal antibody responses on biological properties of these viruses.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Infecções por Flavivirus/virologia , Flavivirus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Facilitadores/imunologia , Flavivirus/isolamento & purificação , Humanos , Lipossomos/imunologia , Proteínas do Envelope Viral/metabolismo
6.
J Virol ; 90(17): 7934-42, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27334586

RESUMO

UNLABELLED: Human rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating. IMPORTANCE: Based on similarity of their RNA genomic sequences, the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A viruses and all HRV-B viruses use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B, but entering the cell via the same receptors, direct the endocytosis machinery to ferry them along distinct pathways toward different endocytic compartments for uncoating.


Assuntos
Endossomos/virologia , Molécula 1 de Adesão Intercelular/metabolismo , Rhinovirus/fisiologia , Ligação Viral , Desenvelopamento do Vírus , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas rab de Ligação ao GTP/metabolismo
7.
J Virol ; 89(22): 11723-6, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26339060

RESUMO

Production of a vesicular stomatitis virus spike protein G (VSVG)-pseudotyped lentiviral expression vector in HEK293 cells decreased on overexpression of low-density lipoprotein receptor (LDLR) but not that of ICAM1 or TfR1. Reverse transcription-quantitative PCR (RT-qPCR) revealed a reduction in vector RNA as a function of LDLR expression. Decreased syncytium formation suggested diminished surface expression of VSVG. Intracellular VSVG granules colocalized with LDLR, ER-Golgi intermediate compartment protein 53 (ERGIC53), LAMP2, and vimentin but not with GM130 or calnexin, suggesting that VSVG interacts with LDLR within the ERGIC, resulting in rerouting into the aggresome/autophagosome pathway.


Assuntos
Glicoproteínas de Membrana/biossíntese , Receptores de LDL/biossíntese , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/biossíntese , Liberação de Vírus/fisiologia , Antígenos CD/biossíntese , Autoantígenos/metabolismo , Calnexina/metabolismo , Linhagem Celular , Células Gigantes/citologia , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Lentivirus/genética , Lentivirus/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores da Transferrina/biossíntese , Vimentina/metabolismo , Proteínas do Envelope Viral/genética
8.
Anal Bioanal Chem ; 408(16): 4209-17, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27020928

RESUMO

Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.


Assuntos
Eletroforese Capilar/métodos , Infecções por Picornaviridae/virologia , RNA Viral/química , Rhinovirus/isolamento & purificação , Cromanos/química , Eletroforese Capilar/instrumentação , Fluorescência , Corantes Fluorescentes/química , Humanos , RNA Viral/genética , Rhinovirus/genética
9.
Proc Natl Acad Sci U S A ; 110(50): 20063-8, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24277846

RESUMO

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA-protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp(38) of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.


Assuntos
Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/metabolismo , Rhinovirus/fisiologia , Vírion/química , Desenvelopamento do Vírus/fisiologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Processamento de Imagem Assistida por Computador , RNA Viral/química , Rhinovirus/genética
10.
Wien Med Wochenschr ; 166(7-8): 211-26, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27174165

RESUMO

For infection, viruses deliver their genomes into the host cell. These nucleic acids are usually tightly packed within the viral capsid, which, in turn, is often further enveloped within a lipid membrane. Both protect them against the hostile environment. Proteins and/or lipids on the viral particle promote attachment to the cell surface and internalization. They are likewise often involved in release of the genome inside the cell for its use as a blueprint for production of new viruses. In the following, I shall cursorily discuss the early more general steps of viral infection that include receptor recognition, uptake into the cell, and uncoating of the viral genome. The later sections will concentrate on human rhinoviruses, the main cause of the common cold, with respect to the above processes. Much of what is known on the underlying mechanisms has been worked out by Renate Fuchs at the Medical University of Vienna.


Assuntos
Resfriado Comum/fisiopatologia , Resfriado Comum/virologia , Rhinovirus/fisiologia , Genoma Viral/genética , Humanos , Lisossomos/fisiologia , Rhinovirus/genética , Ligação Viral , Internalização do Vírus , Replicação Viral/genética , Replicação Viral/fisiologia , Desenvelopamento do Vírus/fisiologia
11.
Anal Chem ; 87(17): 8709-17, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26221912

RESUMO

Gas-phase electrophoretic mobility molecular analysis (GEMMA) separates nanometer-sized, single-charged particles according to their electrophoretic mobility (EM) diameter after transition to the gas-phase via a nano electrospray process. Electrospraying as a soft desorption/ionization technique preserves noncovalent biospecific interactions. GEMMA is therefore well suited for the analysis of intact viruses and subviral particles targeting questions related to particle size, bioaffinity, and purity of preparations. By correlating the EM diameter to the molecular mass (Mr) of standards, the Mr of analytes can be determined. Here, we demonstrate (i) the use of GEMMA in purity assessment of a preparation of a common cold virus (human rhinovirus serotype 2, HRV-A2) and (ii) the analysis of subviral HRV-A2 particles derived from such a preparation. (iii) Likewise, native mass spectrometry was employed to obtain spectra of intact HRV-A2 virions and empty viral capsids (B-particles). Charge state resolution for the latter allowed its Mr determination. (iv) Cumulatively, the data measured and published earlier were used to establish a correlation between the Mr and EM diameter for a range of globular proteins and the intact virions. Although a good correlation resulted from this analysis, we noticed a discrepancy especially for the empty and subviral particles. This demonstrates the influence of genome encapsulation (preventing analytes from shrinking upon transition into the gas-phase) on the measured analyte EM diameter. To conclude, GEMMA is useful for the determination of the Mr of intact viruses but needs to be employed with caution when subviral particles or even empty viral capsids are targeted. The latter could be analyzed by native MS.


Assuntos
Ensaio de Desvio de Mobilidade Eletroforética , Rhinovirus/química , Espectrometria de Massas por Ionização por Electrospray , Vírion/química , Virologia/métodos , Humanos
12.
J Virol ; 88(11): 6307-17, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24672023

RESUMO

UNLABELLED: Enteroviruses, which represent a large genus within the family Picornaviridae, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that in vitro acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5'-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment in vivo. This finding suggests that the exposed 3'-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded. IMPORTANCE: For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification in vitro results in RNA exit starting from the 3'-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.


Assuntos
Modelos Biológicos , Sinais de Poliadenilação na Ponta 3' do RNA/fisiologia , RNA Viral/genética , Rhinovirus/genética , Vírion/genética , Liberação de Vírus/fisiologia , Western Blotting , Fracionamento Celular , Primers do DNA/genética , Eletroforese Capilar , Células HeLa , Humanos , Imunoprecipitação , Bicamadas Lipídicas/metabolismo , Sinais de Poliadenilação na Ponta 3' do RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Fluorescência , Liberação de Vírus/genética
13.
PLoS Pathog ; 9(4): e1003270, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23592991

RESUMO

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.


Assuntos
RNA Viral/genética , RNA Viral/metabolismo , Rhinovirus/fisiologia , Internalização do Vírus , Desenvelopamento do Vírus , Sequência de Bases , Capsídeo/química , Capsídeo/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Ficusina/farmacologia , Genoma Viral , Humanos , Conformação de Ácido Nucleico , Poli A/metabolismo , Conformação Proteica , Rhinovirus/genética , Análise de Sequência de RNA , Raios Ultravioleta , Montagem de Vírus , Desenvelopamento do Vírus/efeitos dos fármacos , Desenvelopamento do Vírus/genética
14.
J Virol ; 87(20): 11309-12, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23946453

RESUMO

Minor group human rhinoviruses bind low-density lipoprotein (LDL) receptors for endocytosis. Once they are inside endosomes, the acidic pH triggers their dissociation from the receptors and conversion into hydrophobic subviral A particles; these attach to the membrane and transfer their single-strand, positive-sense RNA genome into the cytosol. Here, we allowed human rhinovirus 2 (HRV2) A particles, produced in vitro by incubation at pH 5.4, to attach to liposomes; cryo-electron microscopy 3-dimensional single-particle image reconstruction revealed that they bind to the membrane around a 2-fold icosahedral symmetry axis.


Assuntos
Membrana Celular/virologia , Substâncias Macromoleculares/metabolismo , Lipídeos de Membrana/metabolismo , Rhinovirus/fisiologia , Ligação Viral , Microscopia Crioeletrônica , Humanos , Imageamento Tridimensional , Lipossomos/metabolismo , Substâncias Macromoleculares/ultraestrutura , Rhinovirus/ultraestrutura
15.
PLoS Pathog ; 8(1): e1002473, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22241997

RESUMO

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , RNA Viral/química , Rhinovirus/química , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Rhinovirus/metabolismo , Internalização do Vírus
16.
Arch Virol ; 159(1): 125-40, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23913188

RESUMO

HRV89, a major-group rhinovirus, uses intercellular adhesion molecule 1 (ICAM-1) for cell entry, while minor-group HRV2 uses the LDL receptor for clathrin-mediated endocytosis. Entry of HRV89 into HeLa epithelial cells was found to be inefficient, and infectious virus was still detected on the plasma membrane after 3 h of incubation with the cells. Endocytosis, and consequently infection, of HRV89 but not of HRV2, was almost completely blocked by the actin-polymerization inhibitor cytochalasin D, while the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on infection with either virus. Cytochalasin D also inhibited major-group HRV infection of rhabdomyosarcoma cells expressing ICAM-1 when the time available for uncoating was limited to 30 min. Although cholesterol depletion strongly inhibited HRV89 infection of HeLa cells, it only slightly affected HRV89 endocytosis, indicating that a lipid raft environment was not essential for virus uptake. The sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly reduced cell entry and infection by HRV89 only at a concentration that also inhibited HRV2 infection and Alexa 488-transferrin entry. These data rule out classical macropinocytosis as an infectious entry pathway of HRV89 in HeLa cells. Notably, the proton ATPase inhibitor bafilomycin strongly affected cell entry of both viruses, suggesting a role for submembraneous pH in rhinovirus endocytosis.


Assuntos
Actinas/metabolismo , Células Epiteliais/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Macrolídeos/farmacologia , Infecções por Picornaviridae/virologia , Rhinovirus/fisiologia , Internalização do Vírus , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Endocitose/efeitos dos fármacos , Células Epiteliais/virologia , Células HeLa , Humanos , Molécula 1 de Adesão Intercelular/genética , Infecções por Picornaviridae/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Rhinovirus/efeitos dos fármacos
17.
Virus Res ; 342: 199338, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38373599

RESUMO

The role of aichivirus A1 (AiV-A1) in acute gastroenteritis remains controversial and in vitro data illustrating its pathogenesis in suitable human models are scarce. Here, we demonstrate that AiV-A1 isolate A846/88 replicates in ApoA1- (absorptive) and Ki-67-positive (proliferative) enterocytes in stem cell-derived human small intestinal epithelium (HIE) as well as in patient biopsy samples, but not in any of the tested human cell lines. The infection did not result in tissue damage and did not trigger type I and type III interferon (IFN) signalling, whereas the control, human coxsackievirus B3 (strain Nancy), triggered both IFNs. To investigate the tissue tropism, we infected a human tracheal/bronchial epithelium model (HTBE) with AiV-A1 isolates A846/88 and kvgh99012632/2010 and, as a control, with rhinovirus A2 (RV-A2). AiV-A1 isolate kvgh99012632/2010, but not isolate A846/88, replicated in HTBE and induced type III IFN and ISGs signalling. By using various pharmacological inhibitors, we elaborated that cellular entry of AiV-A1 depends on clathrin, dynamin, and lipid rafts and is strongly reliant on endosome acidification. Viral particles co-localised with Rab5a-positive endosomes and promoted leakage of endosomal content. Our data shed light on the early events of AiV-A1 infection and reveal that different isolates exhibit distinct tissue tropism. This supports its clinical importance as a human pathogen with the potential to evolve toward broader tissue specificity.


Assuntos
Brônquios , Mucosa Intestinal , Humanos , Enterócitos , Linhagem Celular , Clatrina
18.
Antiviral Res ; 222: 105810, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38244889

RESUMO

Rhinoviruses (RVs) cause the common cold. Attempts at discovering small molecule inhibitors have mainly concentrated on compounds supplanting the medium chain fatty acids residing in the sixty icosahedral symmetry-related hydrophobic pockets of the viral capsid of the Rhinovirus-A and -B species. High-affinity binding to these pockets stabilizes the capsid against structural changes necessary for the release of the ss(+) RNA genome into the cytosol of the host cell. However, single-point mutations may abolish this binding. RV-B5 is one of several RVs that are naturally resistant against the well-established antiviral agent pleconaril. However, RV-B5 is strongly inhibited by the pyrazolopyrimidine OBR-5-340. Here, we report on isolation and characterization of RV-B5 mutants escaping OBR-5-340 inhibition and show that substitution of amino acid residues not only within the binding pocket but also remote from the binding pocket hamper inhibition. Molecular dynamics network analysis revealed that strong inhibition occurs when an ensemble of several sequence stretches of the capsid proteins enveloping OBR-5-340 move together with OBR-5-340. Mutations abrogating this dynamic, regardless of whether being localized within the binding pocket or distant from it result in escape from inhibition. Pyrazolo [3,4-d]pyrimidine derivatives overcoming OBR-5-340 escape of various RV-B5 mutants were identified. Our work contributes to the understanding of the properties of capsid-binding inhibitors necessary for potent and broad-spectrum inhibition of RVs.


Assuntos
Proteínas do Capsídeo , Infecções por Enterovirus , Humanos , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Rhinovirus/genética , Sítios de Ligação , Infecções por Enterovirus/metabolismo , Simulação de Dinâmica Molecular , Mutação , Antivirais/química
20.
Proc Natl Acad Sci U S A ; 107(14): 6146-51, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20200313

RESUMO

Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.


Assuntos
Luz , Proteínas Periplásmicas de Ligação/química , Receptores de LDL/química , Receptores Virais/química , Quelantes/química , Cromatografia em Gel , Histidina/química , Proteínas Ligantes de Maltose , Estrutura Molecular , Ácido Nitrilotriacético/química , Oligopeptídeos/química , Proteínas Periplásmicas de Ligação/isolamento & purificação , Ligação Proteica , Conformação Proteica , Receptores de LDL/metabolismo , Receptores Virais/metabolismo , Rhinovirus/química , Rhinovirus/metabolismo , Rhinovirus/efeitos da radiação , Vírion/metabolismo , Vírion/efeitos da radiação
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