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1.
Med Microbiol Immunol ; 209(3): 309-323, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31865406

RESUMO

Viruses have evolved many mechanisms to invade host cells and establish successful infections. The interaction between viral attachment proteins and host cell receptors is the first and decisive step in establishing such infections, initiating virus entry into the host cells. Therefore, the identification of host receptors is fundamental in understanding pathogenesis and tissue tropism. Furthermore, receptor identification can inform the development of antivirals, vaccines, and diagnostic technologies, which have a substantial impact on human health. Nevertheless, due to the complex nature of virus entry, the redundancy in receptor usage, and the limitations in current identification methods, many host receptors remain elusive. Recent advances in targeted gene perturbation, high-throughput screening, and mass spectrometry have facilitated the discovery of virus receptors in recent years. In this review, we compare the current methods used within the field to identify virus receptors, focussing on genomic- and interactome-based approaches.

2.
Biochem J ; 476(13): 1975-1994, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31235482

RESUMO

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid ß-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.


Assuntos
Enoil-CoA Hidratase/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Aerobiose , Anaerobiose , Catálise , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Oxirredução , Estrutura Quaternária de Proteína , Especificidade por Substrato
3.
J Virol ; 93(17)2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189702

RESUMO

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.

4.
PLoS Biol ; 17(6): e3000281, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31185007

RESUMO

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.


Assuntos
Proteínas do Capsídeo/ultraestrutura , Capsídeo/efeitos dos fármacos , Capsídeo/ultraestrutura , Sequência de Aminoácidos/genética , Aminoácidos/genética , Antígenos Virais , Antivirais , Sítios de Ligação , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Desenvolvimento de Medicamentos/métodos , Enterovirus/efeitos dos fármacos , Enterovirus/ultraestrutura , Humanos , Modelos Moleculares , Conformação Molecular , Rhinovirus/efeitos dos fármacos , Rhinovirus/ultraestrutura , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Vírion/genética
5.
Open Biol ; 9(2): 190012, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30958097

RESUMO

Viruses are obligatory parasites that take advantage of intracellular niches to replicate. During infection, their genomes are carried in capsids across the membranes of host cells to sites of virion production by exploiting cellular behaviour and resources to guide and achieve all aspects of delivery and the downstream virus manufacturing process. Successful entry hinges on execution of a precisely tuned viral uncoating program where incoming capsids disassemble in consecutive steps to ensure that genomes are released at the right time, and in the right place for replication to occur. Each step of disassembly is cell-assisted, involving individual pathways that transmit signals to regulate discrete functions, but at the same time, these signalling pathways are organized into larger networks, which communicate back and forth in complex ways in response to the presence of virus. In this review, we consider the elegant strategy by which adenoviruses (AdVs) target and navigate cellular networks to initiate the production of progeny virions. There are many remarkable aspects about the AdV entry program; for example, the virus gains targeted control of a large well-defined local network neighbourhood by coupling several interacting processes (including endocytosis, autophagy and microtubule trafficking) around a collective reference state centred on the interactional topology and multifunctional nature of protein VI. Understanding the network targeting activity of protein VI, as well as other built-in mechanisms that allow AdV particles to be efficient at navigating the subsystems of the cell, can be used to improve viral vectors, but also has potential to be incorporated for use in entirely novel delivery systems.


Assuntos
Adenoviridae/fisiologia , Capsídeo/fisiologia , Citoplasma/virologia , Vírion/fisiologia , Replicação Viral/fisiologia , Adenoviridae/metabolismo , Capsídeo/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Humanos , Microtúbulos/metabolismo , Microtúbulos/virologia , Modelos Biológicos , Vírion/metabolismo
6.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463974

RESUMO

Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.


Assuntos
Anticorpos Neutralizantes/imunologia , Parechovirus/imunologia , Parechovirus/ultraestrutura , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral , Microscopia Crioeletrônica/métodos , Epitopos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura
7.
Antiviral Res ; 161: 100-107, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472162

RESUMO

Several research groups in Europe are active on different aspects of human picornavirus research. The AIROPico (Academia-Industry R&D Opportunities for Picornaviruses) consortium combined the disciplines of pathogenesis, diagnostics and therapy development in order to fill the gaps in our understanding of how picornaviruses cause human disease and how to combat them. AIROPico was the first EU consortium dedicated to human picornavirus research and development, and has largely accelerated and improved R&D on picornavirus biology, diagnostics and therapy. In this article, we present the progress on pathogenesis, diagnostics and treatment strategy developments for human picornaviruses resulting from the structured, translational research approach of the AIROPico consortium. We here summarize new insights in protection against infection by maternal or cross-protective antibodies, the visualisation of interactions between virus and neutralizing antibodies by cryoEM structural imaging, and the outcomes from a picornavirus-infected human 3D organoid. Progress in molecular detection and a fast typing assay for rhinovirus species are presented, as well as the identification of new compounds potentially interesting as therapeutic compounds.


Assuntos
Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/tratamento farmacológico , Picornaviridae/patogenicidade , Pesquisa/organização & administração , Antivirais/uso terapêutico , Congressos como Assunto , Europa (Continente) , Humanos , Colaboração Intersetorial , Picornaviridae/genética , Pesquisa/economia
8.
Viruses ; 10(7)2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29958443

RESUMO

Tick-borne encephalitis virus (TBEV) is a growing health concern. It causes a severe disease that can lead to permanent neurological complications or death and the incidence of TBEV infections is constantly rising. Our understanding of TBEV’s structure lags behind that of other flaviviruses, but has advanced recently with the publication of a high-resolution structure of the TBEV virion. The gaps in our knowledge include: aspects of receptor binding, replication and virus assembly. Furthermore, TBEV has mostly been studied in mammalian systems, even though the virus’ interaction with its tick hosts is a central part of its life cycle. Elucidating these aspects of TBEV biology are crucial for the development of TBEV antivirals, as well as the improvement of diagnostics. In this review, we summarise the current structural knowledge on TBEV, bringing attention to the current gaps in our understanding, and propose further research that is needed to truly understand the structural-functional relationship of the virus and its hosts.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/virologia , Animais , Vírus da Encefalite Transmitidos por Carrapatos/ultraestrutura , Genoma Viral , Genômica/métodos , Humanos , Estágios do Ciclo de Vida , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral
9.
Sci Rep ; 8(1): 5820, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29643409

RESUMO

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.


Assuntos
Proteínas do Capsídeo/metabolismo , Parechovirus/fisiologia , RNA Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/química , Reagentes para Ligações Cruzadas/química , Células HT29 , Humanos , Modelos Moleculares , RNA Viral/química
10.
Sci Rep ; 7(1): 12075, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28935894

RESUMO

Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.


Assuntos
Anticorpos Neutralizantes/imunologia , Variação Antigênica/imunologia , Proteínas do Capsídeo/imunologia , Parechovirus/imunologia , Infecções por Picornaviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Variação Antigênica/genética , Proteínas do Capsídeo/genética , Humanos , Soros Imunes/imunologia , Japão , Mutação , Países Baixos , Testes de Neutralização , Parechovirus/classificação , Parechovirus/fisiologia , Infecções por Picornaviridae/virologia , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
11.
Nat Commun ; 8(1): 83, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28710463

RESUMO

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

12.
Elife ; 62017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28682240

RESUMO

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.


Assuntos
Biologia Celular , Biologia Computacional/métodos , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Curadoria de Dados
13.
Nat Commun ; 8(1): 5, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28232749

RESUMO

Assembly of the major viral pathogens of the Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.The mechanism underlying packaging of genomic RNA into viral particles is not well understood for human parechoviruses. Here the authors identify short RNA motifs in the parechovirus genome that bind capsid proteins, providing approximately 60 specific interactions for virion assembly.


Assuntos
Proteínas do Capsídeo/genética , Genoma Viral , Parechovirus/genética , RNA Viral/genética , Vírion/genética , Montagem de Vírus , Motivos de Aminoácidos , Pareamento de Bases , Sítios de Ligação , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Biologia Computacional , Sequência Conservada , Expressão Gênica , Humanos , Modelos Moleculares , Parechovirus/metabolismo , Parechovirus/ultraestrutura , Ligação Proteica , Dobramento de RNA , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Genética Reversa , Técnica de Seleção de Aptâmeros , Vírion/metabolismo , Vírion/ultraestrutura
14.
Biochim Biophys Acta ; 1859(11): 1440-1448, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27664935

RESUMO

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.


Assuntos
DNA/metabolismo , Transcrição Genética/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Ligação Proteica , Proteínas não Estruturais Virais/fisiologia , Replicação Viral
15.
Nat Commun ; 7: 11387, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27435188

RESUMO

The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.


Assuntos
Capsídeo/metabolismo , Sepse Neonatal/virologia , Parechovirus/fisiologia , Infecções por Picornaviridae/virologia , Sequência de Aminoácidos , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Parechovirus/química , Parechovirus/genética , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Montagem de Vírus
16.
J Virol ; 89(18): 9571-80, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26157123

RESUMO

UNLABELLED: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteínas do Capsídeo/química , Modelos Moleculares , Parechovirus/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral , Reações Cruzadas , Humanos , Parechovirus/imunologia , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
17.
J Virol ; 90(6): 2849-57, 2015 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-26719278

RESUMO

UNLABELLED: The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N(0)) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N(0) assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N(0)1₋408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N(0)-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N(0) binding peptide, P1₋48, to the C terminus of an N(0)21₋408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N(0)-P complex. We solved the X-ray structure of the resulting N(0)-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N(0)-P interface and explains how P chaperones N(0), preventing both self-assembly of N(0) and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization. IMPORTANCE: Measles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.


Assuntos
Vírus do Sarampo/química , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Centrifugação , Cristalografia por Raios X , Análise Mutacional de DNA , Vírus do Sarampo/genética , Modelos Moleculares , Nucleoproteínas/genética , Fosfoproteínas/genética , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Proteínas Virais/genética
18.
Cell Biosci ; 4: 37, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25105011

RESUMO

Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages.

19.
PLoS One ; 9(7): e103101, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25062251

RESUMO

In peroxisomes, peroxins (PEXs) 3 and 19 are the principal protein components of the machinery required for early peroxisomal biogenesis. For further insight into the interaction of PEX3 and PEX19, we used hydrogen exchange mass spectrometry to monitor conformational changes during complex formation between PEX3 and PEX19 in vitro. Our data showed that PEX19 remained highly flexible during interaction with PEX3. However, we could detect three changes, one each in the N-and C-terminus along with a small stretch in the middle of PEX19 (F64-L74) which became shielded from hydrogen exchange when interacting with PEX3. PEX3 became more protected from hydrogen exchange in the binding groove for PEX19 with only small changes elsewhere. Most likely the N-terminus of PEX19 initiates the binding to PEX3, and then subtle conformational changes in PEX3 affect the surface of the PEX3 molecule. PEX19 in turn, is stabilized by folding of a short helix and its C-terminal folding core permitting PEX19 to bind to PEX3 with higher affinity than just the N-terminal interaction allows. Thus within the cell, PEX3 is stabilized by PEX19 preventing PEX3 aggregation.


Assuntos
Lipoproteínas/química , Proteínas de Membrana/química , Peroxissomos/química , Mapas de Interação de Proteínas/genética , Sequência de Aminoácidos , Humanos , Lipoproteínas/biossíntese , Lipoproteínas/ultraestrutura , Proteínas de Membrana/biossíntese , Proteínas de Membrana/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Peroxinas , Peroxissomos/genética , Conformação Proteica , Dobramento de Proteína
20.
Biotechniques ; 56(1): 36-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24447137

RESUMO

Rabbit reticulocyte lysate (RRL) is a mammalian cell-free system for protein production. However, one of the limitations of this system is its low protein yield. Inclusion of recombinant virus proteins and specific viral structures on target mRNA could enhance protein production in RRL. Here we show that simultaneous addition of influenza A virus NS1 protein and inclusion of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) in the target mRNA facilitate translation initiation and increase protein yield over 10-fold, improving the translation capacity of RRL.


Assuntos
Sistema Livre de Células , Biossíntese de Proteínas , Reticulócitos/metabolismo , Animais , RNA Mensageiro/genética , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas não Estruturais Virais/genética
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