Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Traffic ; 20(2): 152-167, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30548142

RESUMEN

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein-Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX-TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine-rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co-ordinate nuclear and cytoplasmic processes.


Asunto(s)
Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 4/metabolismo , Señales de Exportación Nuclear , Proteínas de la Matriz Viral/química , Animales , Chlorocebus aethiops , Células HeLa , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 4/fisiología , Humanos , Células Vero , Proteínas de la Matriz Viral/metabolismo , Replicación Viral
2.
PLoS Pathog ; 13(4): e1006273, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28403220

RESUMEN

Human cytomegalovirus (HCMV) is a widely distributed herpesvirus that causes significant morbidity in immunocompromised hosts. Inhibitors of viral DNA replication are available, but adverse effects limit their use. Alternative antiviral strategies may include inhibition of entry. We show that soluble derivatives of the platelet-derived growth factor receptor alpha (PDGFR-alpha), a putative receptor of HCMV, can inhibit HCMV infection of various cell types. A PDGFR-alpha-Fc fusion protein binds to and neutralizes cell-free virus particles at an EC50 of 10-30 ng/ml. Treatment of particles reduced both attachment to and fusion with cells. In line with the latter, PDGFR-alpha-Fc was also effective when applied postattachment. A peptide scan of the extracellular domain of PDGFR-alpha identified a 40mer peptide that inhibits infection at an EC50 of 1-2 nmol/ml. Both, peptide and fusion protein, were effective against various HCMV strains and are hence promising candidates for the development of novel anti-HCMV therapies.


Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/terapia , Citomegalovirus/efectos de los fármacos , Péptidos/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Internalización del Virus/efectos de los fármacos , Antivirales/aislamiento & purificación , Línea Celular , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/virología , Células Endoteliales/virología , Fibroblastos/virología , Humanos , Péptidos/aislamiento & purificación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Recombinantes de Fusión , Virión
3.
Int J Mol Sci ; 19(10)2018 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-30279342

RESUMEN

Tetraspanins are suggested to regulate the composition of cell membrane components and control intracellular transport, which leaves them vulnerable to utilization by pathogens such as human papillomaviruses (HPV) and cytomegaloviruses (HCMV) to facilitate host cell entry and subsequent infection. In this study, by means of cellular depletion, the cluster of differentiation (CD) tetraspanins CD9, CD63, and CD151 were found to reduce HPV16 infection in HeLa cells by 50 to 80%. Moreover, we tested recombinant proteins or peptides of specific tetraspanin domains on their effect on the most oncogenic HPV type, HPV16, and HCMV. We found that the C-terminal tails of CD63 and CD151 significantly inhibited infections of both HPV16 and HCMV. Although CD9 was newly identified as a key cellular factor for HPV16 infection, the recombinant CD9 C-terminal peptide had no effect on infection. Based on the determined half-maximal inhibitory concentration (IC50), we classified CD63 and CD151 C-terminal peptides as moderate to potent inhibitors of HPV16 infection in HeLa and HaCaT cells, and in EA.hy926, HFF (human foreskin fibroblast) cells, and HEC-LTT (human endothelial cell-large T antigen and telomerase) cells for HCMV, respectively. These results indicate that HPV16 and HCMV share similar cellular requirements for their entry into host cells and reveal the necessity of the cytoplasmic CD151 and CD63 C-termini in virus infections. Furthermore, this highlights the suitability of these peptides for functional investigation of tetraspanin domains and as inhibitors of pathogen infections.


Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Papillomavirus Humano 16/fisiología , Tetraspaninas/antagonistas & inhibidores , Citomegalovirus/efectos de los fármacos , Células HeLa , Papillomavirus Humano 16/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Masculino , Péptidos/farmacología , Tetraspaninas/química , Tetraspaninas/metabolismo , Internalización del Virus
4.
Genome Res ; 24(6): 906-19, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24668909

RESUMEN

Gene expression is regulated in a context-dependent, cell-type-specific manner. Condition-specific transcription is dependent on the presence of transcription factors (TFs) that can activate or inhibit its target genes (global context). Additional factors, such as chromatin structure, histone, or DNA modifications, also influence the activity of individual target genes (individual context). The role of the global and individual context for post-transcriptional regulation has not systematically been investigated on a large scale and is poorly understood. Here we show that global and individual context dependency is a pervasive feature of microRNA-mediated regulation. Our comprehensive and highly consistent data set from several high-throughput technologies (PAR-CLIP, RIP-chip, 4sU-tagging, and SILAC) provides strong evidence that context-dependent microRNA target sites (CDTS) are as frequent and functionally relevant as constitutive target sites (CTS). Furthermore, we found the global context to be insufficient to explain the CDTS, and that flanking sequence motifs provide individual context that is an equally important factor. Our results demonstrate that, similar to TF-mediated regulation, global and individual context dependency are prevalent in microRNA-mediated gene regulation, implying a much more complex post-transcriptional regulatory network than is currently known. The necessary tools to unravel post-transcriptional regulations and mechanisms need to be much more involved, and much more data will be needed for particular cell types and cellular conditions in order to understand microRNA-mediated regulation and the context-dependent post-transcriptional regulatory network.


Asunto(s)
Silenciador del Gen , Genoma Humano , MicroARNs/genética , Línea Celular Tumoral , Secuencia Conservada , Redes Reguladoras de Genes , Humanos , MicroARNs/metabolismo , Motivos de Nucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo
5.
J Virol ; 90(14): 6430-42, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27147745

RESUMEN

UNLABELLED: Human cytomegalovirus (HCMV), a betaherpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins that mediate binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown. In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells by use of a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five proviral host factors from different protein families and eight antiviral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel proviral host factor and was analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either broad or narrow cell tropism were equally impaired. Infection of CD151-depleted cells by a fluorescent virus with differentially labeled capsid and envelope proteins revealed a role of CD151 in viral penetration but not in adsorption to the cell. In conclusion, the tetraspanin CD151 has emerged as a novel host factor in HCMV entry and as a putative antiviral target. IMPORTANCE: At present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with putative roles in this process were identified. The tetraspanin CD151 was discovered as a previously unrecognized proviral host factor for HCMV and was found to support viral penetration into the target cells. The findings of this study shed light on the cellular contribution during the initial steps of HCMV infection and open a new direction in HCMV research.


Asunto(s)
Citomegalovirus , Fibroblastos/virología , Células Endoteliales de la Vena Umbilical Humana/virología , Tetraspanina 24/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Células Cultivadas , Fibroblastos/metabolismo , Eliminación de Gen , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , Tetraspanina 24/antagonistas & inhibidores , Tetraspanina 24/genética
6.
Biochem Soc Trans ; 45(2): 489-497, 2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-28408489

RESUMEN

Members of the tetraspanin family have been identified as essential cellular membrane proteins in infectious diseases by nearly all types of pathogens. The present review highlights recently published data on the role of tetraspanin CD151, CD81, and CD63 and their interaction partners in host cell entry by human cytomegalo- and human papillomaviruses. Moreover, we discuss a model for tetraspanin assembly into trafficking platforms at the plasma membrane. These platforms might persist during intracellular viral trafficking.


Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Infecciones por Papillomavirus/metabolismo , Tetraspaninas/metabolismo , Proteínas Virales/metabolismo , Membrana Celular/metabolismo , Citomegalovirus/fisiología , Humanos , Modelos Moleculares , Papillomaviridae/fisiología , Tetraspanina 24/química , Tetraspanina 24/metabolismo , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Tetraspanina 30/química , Tetraspanina 30/metabolismo , Tetraspaninas/química , Internalización del Virus
7.
Proc Natl Acad Sci U S A ; 110(34): 13994-9, 2013 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-23918356

RESUMEN

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed in different tissues. However, in neither case is it known whether the isoforms fulfill different functions or polyadenylate distinct subsets of pre-mRNAs. Here we show that the three canonical nuclear PAPS isoforms in Arabidopsis are functionally specialized owing to their evolutionarily divergent C-terminal domains. A strong loss-of-function mutation in PAPS1 causes a male gametophytic defect, whereas a weak allele leads to reduced leaf growth that results in part from a constitutive pathogen response. By contrast, plants lacking both PAPS2 and PAPS4 function are viable with wild-type leaf growth. Polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs depends specifically on PAPS1 function. The resulting reduction in SAUR activity in paps1 mutants contributes to their reduced leaf growth, providing a causal link between polyadenylation of specific pre-mRNAs by a particular PAPS isoform and plant growth. This suggests the existence of an additional layer of regulation in plant and possibly vertebrate gene expression, whereby the relative activities of canonical nuclear PAPS isoforms control de novo synthesized poly(A) tail length and hence expression of specific subsets of mRNAs.


Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Arabidopsis/enzimología , Regulación de la Expresión Génica de las Plantas/fisiología , Polinucleotido Adenililtransferasa/metabolismo , Precursores del ARN/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Genotipo , Análisis por Micromatrices , Mutación/genética , Hojas de la Planta/crecimiento & desarrollo , Polinucleotido Adenililtransferasa/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad por Sustrato
8.
Traffic ; 13(10): 1326-34, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22708827

RESUMEN

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α- and ß-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences.


Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Técnicas Citológicas , Dimerización , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Señales de Localización Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Sirolimus/química , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/metabolismo
9.
Proc Natl Acad Sci U S A ; 106(10): 3710-5, 2009 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-19237566

RESUMEN

Borna disease virus (BDV) is a neurotropic enveloped RNA virus that causes a noncytolytic, persistent infection of the central nervous system in mammals. BDV belongs to the order Mononegavirales, which also includes the negative-strand RNA viruses (NSVs) Ebola, Marburg, vesicular stomatitis, rabies, mumps, and measles. BDV-M, the matrix protein (M-protein) of BDV, is the smallest M-protein (16.2 kDa) among the NSVs. M-proteins play a critical role in virus assembly and budding, mediating the interaction between the viral capsid, envelope, and glycoprotein spikes, and are as such responsible for the structural stability and individual form of virus particles. Here, we report the 3D structure of BDV-M, a full-length M-protein structure from a nonsegmented RNA NSV. The BDV-M monomer exhibits structural similarity to the N-terminal domain of the Ebola M-protein (VP40), while the surface charge of the tetramer provides clues to the membrane association of BDV-M. Additional electron density in the crystal reveals the presence of bound nucleic acid, interpreted as cytidine-5'-monophosphate. The heterologously expressed BDV-M copurifies with and protects ssRNA oligonucleotides of a median length of 16 nt taken up from the expression host. The results presented here show that BDV-M would be able to bind RNA and lipid membranes simultaneously, expanding the repertoire of M-protein functionalities.


Asunto(s)
Virus de la Enfermedad de Borna/química , ARN Viral/química , Proteínas de la Matriz Viral/química , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Nucleótidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Electricidad Estática , Propiedades de Superficie
10.
Chem Biol ; 15(5): 427-37, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18482695

RESUMEN

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.


Asunto(s)
Caenorhabditis elegans/citología , Microfluídica/instrumentación , Animales , Emulsiones , Humanos , Miniaturización
11.
PLoS One ; 12(1): e0169580, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28060895

RESUMEN

Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses.


Asunto(s)
Alphaherpesvirinae/genética , Citomegalovirus/fisiología , Expresión Génica , Genes Reporteros , Proteínas Inmediatas-Precoces/metabolismo , Luciferasas/genética , Regiones Promotoras Genéticas , Animales , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Línea Celular , Citomegalovirus/ultraestructura , Genoma Viral , Humanos , Proteínas Inmediatas-Precoces/inmunología , Tropismo Viral , Virión/ultraestructura , Replicación Viral
12.
J Virol Methods ; 235: 182-189, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27326666

RESUMEN

For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging.


Asunto(s)
Citomegalovirus/genética , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Virología/métodos , Animales , Copépodos/enzimología , Células Endoteliales/virología , Fibroblastos/virología , Genes Reporteros , Genoma Viral , Humanos , Luciferasas/química , Luciferasas/aislamiento & purificación , Glicoproteínas de Membrana , Ratones , Mutagénesis , Proteínas del Envoltorio Viral/genética , Replicación Viral
13.
Biotechniques ; 59(3): 127-36, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26345505

RESUMEN

Infection of vascular endothelial cells (ECs) is assumed to contribute to dissemination of human cytomegalovirus (HCMV). Investigation of virus-host interactions in ECs such as human umbilical vein endothelial cells (HUVECs) is limited due to the low maximal passage numbers of these primary cells. We tested a conditionally immortalized EC line (HEC-LTT) and a permanent cell line (EA.hy926) for their susceptibility to HCMV infection. Both cell lines resembled HUVECs in that they allowed for entry and immediate early protein expression of highly endotheliotropic HCMV strains but not of poorly endotheliotropic strains, rendering them suitable for analysis of the viral entry mechanism in ECs. The late phase of viral replication and release, however, was supported by growth-controlled HEC-LTT cells but not by EA.hy926 cells. HEC-LTT cells support both the early and late phase of viral replication and release infectious progeny virus at titers comparable to primary HUVECs; thus, the HEC-LTT cell line is a cell culture model representing the full viral replicative cycle of HCMV in ECs. The implementation of permanent HEC-LTT and EA.hy926 cell lines in HCMV research will facilitate long-term approaches that are not feasible in primary HUVECs.


Asunto(s)
Citomegalovirus/patogenicidad , Células Endoteliales/citología , Células Endoteliales/virología , Procesos de Crecimiento Celular , Línea Celular/virología , Células Cultivadas , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Regulación Viral de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Virales/metabolismo , Replicación Viral
14.
PLoS One ; 10(6): e0131614, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26121620

RESUMEN

While it is well established that human cytomegalovirus (HCMV) upregulates many cellular proteins and incorporates several of them into its virion, little is known about the functional relevance of such virus-host interactions. Two cellular proteins, Grb2 and DDX3, gained our interest as they appeared enriched in virion particles and this incorporation depended on the viral tegument protein pp65, suggesting a functional relevance. We therefore tested whether the level of these proteins is altered upon HCMV infection and whether they support viral replication. Immunoblotting analyses of cellular fractions showed increased levels of both proteins in infected cells with a maximum at 2 d p.i. and a reduction of the soluble Grb2 fraction. Knockdown of either gene by transfection of siRNAs reduced viral spread not only of the cell culture adapted HCMV strain TB40/E but also of recent clinical isolates. Apparently, Grb2 and DDX3 are proviral cellular factors that are upregulated in infected cells.


Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , ARN Helicasas DEAD-box/metabolismo , Proteína Adaptadora GRB2/metabolismo , Línea Celular , ARN Helicasas DEAD-box/genética , Proteína Adaptadora GRB2/genética , Técnicas de Silenciamiento del Gen , Humanos , Fosfoproteínas/metabolismo , Unión Proteica , Provirus/genética , Provirus/metabolismo , Regulación hacia Arriba , Proteínas de la Matriz Viral/metabolismo , Virión , Ensamble de Virus , Replicación Viral
15.
Methods Mol Biol ; 1064: 183-200, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23996258

RESUMEN

miRNAs have in recent years emerged as novel players in virus-host interactions. While individual miRNAs are capable of regulating many targets simultaneously, not much is known about the role of distinct host or viral miRNAs in the context of infection. Analysis of the function of a miRNA is often hampered by the complexity of virus-host interactions and the enormous changes in the host cell during infection. Many viral miRNAs as for example from Kaposi sarcoma-associated Herpesvirus (KSHV) are probably exclusively expressed in latent infection. This might lead to a steady-state situation with offense and defense mechanisms counteracting each other. Cellular miRNAs involved in defense against pathogens on the other hand might be suppressed in infection. A cell culture system allowing for constitutive expression of individual miRNAs at high levels is a useful tool to enhance miRNA-specific functions and to uncouple viral miRNA function from other infection-related mechanisms. Here, a protocol is described to generate stable cell lines for constitutive expression of single cellular or viral miRNA precursors in absence of infection. The procedure comprises cloning of the precursor sequence, generation of the lentiviral expression vector, transduction of the cells of interest, selection for polyclonal cell lines, and isolation of monoclonal cell lines by limiting dilution.


Asunto(s)
Expresión Génica , MicroARNs/genética , Línea Celular , Clonación Molecular , Vectores Genéticos/genética , Humanos , Lentivirus/genética , MicroARNs/metabolismo , Transducción Genética
16.
Methods Mol Biol ; 1064: 171-81, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23996257

RESUMEN

Quantification of infectious virus is crucial to many experimental approaches in virological research. A broadly used and facile technique is the so-called "plaque assay" which provides precise information on the absolute quantity of infectivity in a given volume. Due to advances in the understanding of viral gene expression, transactivator-promoter pairs have been identified which can be used in transgenic cell lines as reporters of viral infection. Even though such "cellular reporter assay" systems are mostly restricted to relative quantification, they are attractive tools which can complement or replace the conventional plaque assay. Cellular reporter assays become especially interesting in state-of-the-art high-throughput screening approaches, as for instance RNAi and compound library screens, since they are often compatible with small-scale and automated experimentation. In this chapter, a regular plaque assay as well as a cellular reporter assay employing a luciferase reporter gene are described. As an example, HSV-1 infectivity is assessed with both methods yielding complementary information. Advantages and disadvantages of the two techniques and possible applications are discussed.


Asunto(s)
Expresión Génica , Genes Reporteros , Herpesvirus Humano 1/fisiología , Luciferasas/genética , Ensayo de Placa Viral , Animales , Antivirales/farmacología , Línea Celular , Herpesvirus Humano 1/efectos de los fármacos , Humanos , Luciferasas/metabolismo
17.
Wiley Interdiscip Rev RNA ; 2(6): 787-801, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21976283

RESUMEN

RNA silencing is emerging as a novel layer of regulation of virus-host interaction. Since individual small RNAs can probably repress dozens if not hundreds of target mRNA molecules, and transcripts, on the other hand, may be recognized by multiple regulatory small RNAs, a dense and complex interaction network of microRNAs (miRNAs) and their targets arises. A comprehensive analysis of miRNA functions thus not only requires systematic approaches employing high-throughput technologies but also calls for the development of improved experimental technologies and a profound bioinformatic analysis. Integration of complementary approaches will enhance our understanding of the mutual regulation of virus and host. Focusing on herpesviruses, we here describe currently available technologies and summarize present results obtained by high-throughput approaches. These techniques can be broadly applied to other virus families and pathways employing other classes of small regulatory RNAs and therefore are powerful universal tools for research on virus-host interaction.


Asunto(s)
Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Virus/genética , Virus/patogenicidad , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , MicroARNs/metabolismo , Modelos Biológicos , Interferencia de ARN , ARN Viral/genética , ARN Viral/metabolismo , Virosis/genética
18.
Curr Biol ; 21(12): 1009-17, 2011 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-21658947

RESUMEN

BACKGROUND: Unlike animals, higher plants do not establish a germ line in embryo development but form haploid germ cells from diploid somatic cells late in their life cycle. However, despite its prime importance, little is known about how this transition is regulated. RESULTS: Here, we show that the WUSCHEL (WUS) gene, initially identified as a stem cell regulator in the shoot meristem, is required for megasporogenesis and thus ultimately for the formation of female generative cells. WUS functions in this process by indirectly activating the expression of the WINDHOSE1 (WIH1) and WIH2 genes that encode small peptides found in plants and fungi, but not in animals. WIH genes function together with the tetraspanin-type protein TORNADO2 (TRN2)/EKEKO in promoting megasporogenesis. CONCLUSIONS: Together, our studies identify a pathway promoting germ cell formation from somatic precursor cells.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Linaje de la Célula , Genes de Plantas , Células Germinativas de las Plantas , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Datos de Secuencia Molecular , Reproducción , Homología de Secuencia de Aminoácido , Transcripción Genética
19.
Cell Host Microbe ; 7(4): 324-334, 2010 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-20413099

RESUMEN

The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , Virología/métodos , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , MicroARNs/genética , Análisis por Micromatrices , ARN Viral/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA