Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 100
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Science ; 370(6518): 856-860, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33082293

RESUMO

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Neuropilina-1/metabolismo , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Betacoronavirus/genética , COVID-19 , Células CACO-2 , Feminino , Células HEK293 , Interações entre Hospedeiro e Microrganismos , Humanos , Pulmão/metabolismo , Masculino , Nanopartículas Metálicas , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neuropilina-1/química , Neuropilina-1/genética , Neuropilina-1/imunologia , Neuropilina-2/metabolismo , Mucosa Olfatória/metabolismo , Mucosa Olfatória/virologia , Pandemias , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios Proteicos , Mucosa Respiratória/metabolismo , SARS-CoV-2 , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/química
2.
Science ; 370(6518): 861-865, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33082294

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.


Assuntos
Betacoronavirus/fisiologia , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , COVID-19 , Células CACO-2 , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Furina/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
3.
Annu Rev Biochem ; 89: 21-43, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32569520

RESUMO

My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same time, our aim has been to employ cell biological approaches to expand the fundamental understanding of animal viruses and their pathogenic lifestyles. We have studied mechanisms of host cell entry and the uncoating of incoming viruses as well as the synthesis, folding, maturation, and intracellular movement of viral proteins and molecular assemblies. I have had the privilege to work in institutions in four different countries. The early years in Finland (the University of Helsinki) were followed by 6 years in Germany (European Molecular Biology Laboratory), 16 years in the United States (Yale School of Medicine), and 16 years in Switzerland (ETH Zurich).


Assuntos
Calnexina/genética , Calreticulina/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/genética , Picornaviridae/genética , Proteínas Virais/genética , Virologia/história , Animais , Calnexina/química , Calnexina/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica , História do Século XX , História do Século XXI , Humanos , Vírus da Influenza A/metabolismo , Picornaviridae/metabolismo , Dobramento de Proteína , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Internalização do Vírus
4.
Nat Microbiol ; 4(4): 578-586, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30692667

RESUMO

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , beta Carioferinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Ribonucleoproteínas/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Replicação Viral
5.
J Mol Biol ; 430(13): 1853-1862, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29709571

RESUMO

Research over a period of more than half a century has provided a reasonably accurate picture of mechanisms involved in animal virus entry into their host cells. Successive steps in entry include binding to receptors, endocytosis, passage through one or more membranes, targeting to specific sites within the cell, and uncoating of the genome. For some viruses, the molecular interactions are known in great detail. However, as more viruses are analyzed, and as the focus shifts from tissue culture to in vivo experiments, it is evident that viruses display considerable redundancy and flexibility in receptor usage, endocytic mechanism, location of penetration, and uncoating mechanism. For many viruses, the picture is still elusive because the interactions that they engage in rely on sophisticated adaptation to complex cellular functions and defense mechanisms.


Assuntos
Fenômenos Fisiológicos Virais , Animais , Endocitose , Humanos , Polissacarídeos/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus
7.
Proc Natl Acad Sci U S A ; 113(50): E8069-E8078, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27834731

RESUMO

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.


Assuntos
Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/química , Caveolina 1/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Cavéolas/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência
8.
Annu Rev Cell Dev Biol ; 32: 197-222, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27298089

RESUMO

Transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex is highly selective. As a general rule, such transport is limited to soluble and membrane-associated secretory proteins that have reached properly folded and assembled conformations. To secure the efficiency, fidelity, and control of this crucial transport step, cells use a combination of mechanisms. The mechanisms are based on selective retention of proteins in the ER to prevent uptake into transport vesicles, on selective capture of proteins in COPII carrier vesicles, on inclusion of proteins in these vesicles by default as part of fluid and membrane bulk flow, and on selective retrieval of proteins from post-ER compartments by retrograde vesicle transport.


Assuntos
Via Secretória , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Degradação Associada com o Retículo Endoplasmático , Humanos , Transporte Proteico , Vesículas Transportadoras/metabolismo
9.
PLoS Pathog ; 12(3): e1005508, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27030971

RESUMO

Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that one of the PI3K subunits, p110γ, is in fact critically required for mediating the host's antiviral response. PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus. We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions. The defect in lung dendritic cells leads to deficient CD8+ T cell priming, which is associated with higher viral titers and more severe disease course during the infection. We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Pulmão/virologia , Infecções por Orthomyxoviridae/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Epiteliais/virologia , Pulmão/imunologia , Ativação Linfocitária/imunologia , Camundongos , Replicação Viral/fisiologia
10.
J Vis Exp ; (109): e53909, 2016 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-27077390

RESUMO

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.


Assuntos
Vírus da Influenza A , Vírion/isolamento & purificação , Virologia/métodos , Internalização do Vírus , Capsídeo , Eletroforese em Gel de Poliacrilamida , Proteínas Virais/isolamento & purificação
11.
Elife ; 5: e13841, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27008177

RESUMO

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Culina/metabolismo , Endocitose , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte Biológico , Linhagem Celular , Humanos , Vírus da Influenza A/fisiologia , Internalização do Vírus
12.
Traffic ; 17(4): 351-68, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26650385

RESUMO

Human cytomegalovirus (HCMV) is an important and widespread pathogen in the human population. While infection by this ß-herpesvirus in endothelial, epithelial and dendritic cells depends on endocytosis, its entry into fibroblasts is thought to occur by direct fusion of the viral envelope with the plasma membrane. To characterize individual steps during entry in primary human fibroblasts, we employed quantitative assays as well as electron, fluorescence and live cell microscopy in combination with a variety of inhibitory compounds. Our results showed that while infectious entry was pH- and clathrin-independent, it required multiple, endocytosis-related factors and processes. The virions were found to undergo rapid internalization into large vacuoles containing internalized fluid and endosome markers. The characteristics of the internalization process fulfilled major criteria for macropinocytosis. Moreover, we found that soon after addition to fibroblasts the virus rapidly triggered the formation of circular dorsal ruffles in the host cell followed by the generation of large macropinocytic vacuoles. This distinctive form of macropinocytosis has been observed especially in primary cells but has not previously been reported in response to virus stimulation.


Assuntos
Citomegalovirus/fisiologia , Fibroblastos/virologia , Pinocitose , Internalização do Vírus , Células Cultivadas , Citomegalovirus/patogenicidade , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos
13.
Traffic ; 16(8): 814-31, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25869659

RESUMO

The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.


Assuntos
Fagocitose , Fagossomos/metabolismo , Vaccinia virus/patogenicidade , Endossomos/metabolismo , Endossomos/virologia , Células HeLa , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Fagossomos/virologia , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Vaccinia virus/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
14.
BMC Genomics ; 15: 1162, 2014 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-25534632

RESUMO

BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.


Assuntos
Genômica/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Linhagem Celular , Biblioteca Gênica , Genômica/normas , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Curva ROC , Reprodutibilidade dos Testes
15.
Science ; 346(6208): 473-7, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25342804

RESUMO

During cell entry, capsids of incoming influenza A viruses (IAVs) must be uncoated before viral ribonucleoproteins (vRNPs) can enter the nucleus for replication. After hemagglutinin-mediated membrane fusion in late endocytic vacuoles, the vRNPs and the matrix proteins dissociate from each other and disperse within the cytosol. Here, we found that for capsid disassembly, IAV takes advantage of the host cell's aggresome formation and disassembly machinery. The capsids mimicked misfolded protein aggregates by carrying unanchored ubiquitin chains that activated a histone deacetylase 6 (HDAC6)-dependent pathway. The ubiquitin-binding domain was essential for recruitment of HDAC6 to viral fusion sites and for efficient uncoating and infection. That other components of the aggresome processing machinery, including dynein, dynactin, and myosin II, were also required suggested that physical forces generated by microtubule- and actin-associated motors are essential for IAV entry.


Assuntos
Capsídeo/metabolismo , Histona Desacetilases/fisiologia , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Internalização do Vírus , Animais , Linhagem Celular Tumoral , Núcleo Celular/virologia , Complexo Dinactina , Dineínas/metabolismo , Técnicas de Inativação de Genes , Desacetilase 6 de Histona , Histona Desacetilases/genética , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/genética , Influenza Humana/metabolismo , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Ribonucleoproteínas/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Replicação Viral
16.
Cell Host Microbe ; 16(3): 403-11, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25211080

RESUMO

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.


Assuntos
Infecções por Alphavirus/virologia , Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Vírus da Floresta de Semliki/fisiologia , Transativadores/metabolismo , Replicação Viral , Infecções por Alphavirus/genética , Infecções por Alphavirus/metabolismo , Proteínas de Transporte/genética , Interações Hospedeiro-Patógeno , Humanos , RNA Helicases , Vírus da Floresta de Semliki/genética , Transativadores/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Liberação de Vírus
17.
Artigo em Inglês | MEDLINE | ID: mdl-25085912

RESUMO

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.


Assuntos
Endocitose/fisiologia , Células Eucarióticas/microbiologia , Modelos Biológicos , Internalização do Vírus , Aderência Bacteriana , Células Eucarióticas/citologia , Redes e Vias Metabólicas
18.
J Virol ; 88(22): 13029-46, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25165113

RESUMO

UNLABELLED: Influenza A virus (IAV) uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the prefusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to a pH below 6.5. The second step is triggered by a pH of <6.0 and by the influx of K(+) ions. It causes additional changes in M1 as well as a loss of stability in the viral ribonucleoprotein bundle. Our results indicate that both the switch from Na(+) to K(+) in maturing endosomes and the decreasing pH are needed to prime IAV cores for efficient uncoating and infection of the host cell. IMPORTANCE: The entry of IAV involves several steps, including endocytosis and fusion at late endosomes. Entry also includes disassembly of the viral core, which is composed of the viral ribonucleoproteins and the RNA genome. We have found that the uncoating process of IAV is initiated long before the core is delivered into the cytosol. M2, an ion channel in the viral membrane, is activated when the virus passes through early endosomes. Here, we show that protons entering the virus through M2 cause a conformational change in the matrix protein, M1. This weakens interactions between M1 and the viral ribonucleoproteins. A second change was found to occur when the virus enters late endosomes. The preacidified core is then exposed to a high concentration of K(+), which affects the interactions between the ribonucleoproteins. Thus, when cores are finally delivered to the cytosol, they are already partially destabilized and, therefore, uncoating competent and infectious.


Assuntos
Endossomos/metabolismo , Endossomos/virologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Potássio/metabolismo , Proteínas da Matriz Viral/metabolismo , Desenvelopamento do Vírus/efeitos dos fármacos , Animais , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos
19.
PLoS Pathog ; 10(5): e1004162, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24874089

RESUMO

A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.


Assuntos
Papillomavirus Humano 16 , Mitose/fisiologia , Membrana Nuclear/metabolismo , Proteínas Oncogênicas Virais/genética , Interferência de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Células Cultivadas , DNA Viral/genética , Papillomavirus Humano 16/genética , Humanos
20.
J Virol ; 88(15): 8565-78, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24850728

RESUMO

UNLABELLED: The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3(+) endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3(+) virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE: Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.


Assuntos
Inativação Gênica , Interações Hospedeiro-Patógeno , RNA Interferente Pequeno/análise , Vírus Uukuniemi/fisiologia , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Internalização do Vírus , Endossomos/química , Endossomos/virologia , Células Epiteliais/virologia , Testes Genéticos , Células HeLa , Humanos , Proteínas de Membrana Lisossomal/análise , RNA Interferente Pequeno/genética , Fatores de Tempo , Proteína 3 Associada à Membrana da Vesícula/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...