Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
PLoS Pathog ; 19(3): e1011224, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36996041

RESUMO

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.


Assuntos
Aedes , Culicidae , Dengue , Flavivirus , Animais , Humanos , Flavivirus/genética , RNA Subgenômico , Saliva/metabolismo , Regiões 3' não Traduzidas , Replicação Viral , RNA Viral/genética , RNA Viral/metabolismo
2.
Nucleic Acids Res ; 49(17): 10034-10045, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34428287

RESUMO

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.


Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Fibronectinas/genética , Interferon Tipo I/imunologia , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células A549 , Linhagem Celular Tumoral , Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferon Tipo I/genética , Poli I-C/imunologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética , Regulação para Cima/genética
3.
RNA ; 24(6): 803-814, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29572260

RESUMO

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.


Assuntos
Regiões 3' não Traduzidas , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/prevenção & controle , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos , Dengue/genética , Dengue/virologia , Genoma Viral , Células HEK293 , Humanos , Proteínas de Ligação a RNA/genética
4.
RNA Biol ; 17(3): 366-380, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31829086

RESUMO

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNß transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNß activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNß induction. Consistently, ectopic expression of RIG-I activates stronger IFNß induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Interferon Tipo I/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Fosforilação , Poli I-C/genética , Poli I-C/metabolismo , Proteínas de Ligação a RNA/genética , Infecções por Respirovirus/metabolismo , Vírus Sendai/patogenicidade
5.
Anal Chem ; 89(11): 6065-6075, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28457123

RESUMO

As therapeutic recombinant fusion proteins become more widely applicable for the treatment of various types of diseases, there is an increased demand for universal methods such as liquid chromatography (LC)-mass spectrometry (MS) for the determination of their pharmacokinetic properties, particularly their catabolism. The most common approach of analyzing proteins by LC-MS is to digest them into peptides, which can serve as surrogates of the protein. Alternatively, we have developed a novel high-resolution mass spectrometry (HRMS) based approach for analyzing large-molecule proteins at the intact level in biological samples without digestion. We established an immunoaffinity capture LC-HRMS method to quantify the intact parent molecule while simultaneously identifying catabolites for recombinant fusion proteins. We describe this method using dulaglutide, a glucagon-like peptide 1 (GLP1)-Fc fusion protein. Two proteolytic sites within the GLP1 peptide sequence of dulaglutide were identified using this novel LC-HRMS analysis in vivo in mice. These proteolytic sites were identified with the intact molecule being quantified simultaneously. Together with the trypsin digestion based LC-MS/MS analysis using surrogate peptides from different domains of the analyte, an insightful understanding of the pharmacokinetics and in vivo biotransformation of dulaglutide was obtained. Thus, this method enables simultaneous acquisition of both intact drug concentration and important catabolite information for this recombinant fusion protein, providing valuable insight into the integrity of the molecule and its catabolism in vivo. This is critical for designing and screening novel protein therapeutics and for understanding their pharmacokinetics and pharmacodynamics. With continuing advancement of LC-HRMS and software, this method can be very beneficial in drug discovery and development.


Assuntos
Descoberta de Drogas/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Animais , Biotransformação , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Peptídeos Semelhantes ao Glucagon/farmacocinética , Fragmentos Fc das Imunoglobulinas , Camundongos , Proteínas/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/farmacocinética
6.
mBio ; 13(1): e0019622, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35189699

RESUMO

Infection with dengue virus (DENV) induces vast rearrangements of the endoplasmic reticulum, which allows the compartmentalization of viral RNA replication and particle assembly. Both processes occur in concert with viral and cellular proteins. Prior studies from our group suggest that the host RNA-binding protein (RBP) Y-box binding protein 1 (YBX1) is required for a late step in the DENV replication cycle. Here we report that YBX1 interacts with the viral nucleocapsid, distributes to DENV assembly sites and is required for efficient assembly of intracellular infectious virions and their secretion. Genetic ablation of YBX1 decreased the spatial proximity between capsid and envelope, increased the susceptibility of envelope to proteinase K mediated degradation, resulted in the formation of rough empty-looking particles, and decreased the secretion of viral particles. We propose a model wherein YBX1 enables the interaction between the viral nucleocapsid with the structural protein E, which is required for proper assembly of intracellular virus particles and their secretion. IMPORTANCE The global incidence of dengue virus (DENV) infections has steadily increased over the past decades representing an enormous challenge for public health. During infection, DENV viral RNA interacts with numerous host RNA binding proteins (RBPs) that aid viral replication and thus constitute potential molecular targets to curb infection. We recently reported that Y-box-binding protein 1 (YBX1) interacts with DENV RNA and is required at a late step of the replication cycle. Here we describe the molecular mechanism by which YBX1 mediates DENV infection. We show that YBX1 interacts with the viral nucleocapsid, distributes to DENV assembly sites and is required for efficient assembly of intracellular infectious virions. These results provide important insights into DENV assembly, revealing novel functions of host RBPs during viral infection and opening new avenues for antiviral intervention.


Assuntos
Vírus da Dengue , Nucleocapsídeo , Montagem de Vírus , Proteína 1 de Ligação a Y-Box , Dengue , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Humanos , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Vírion/metabolismo , Replicação Viral , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
7.
Biomater Sci ; 2: 693-702, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24955239

RESUMO

Synthetically sulfated hyaluronic acid (HA) has been shown to bind proteins with high affinity through electrostatic interactions. While HA-based hydrogels have been used widely in recent years for drug delivery and tissue engineering applications, incorporation of sulfated HA into these networks to attenuate the release of proteins has yet to be explored. Here, we developed sulfated and methacrylate-modified HA macromers and incorporated them into HA hydrogels through free radical-initiated crosslinking. The sulfated HA macromers bound a heparin-binding protein (i.e., stromal cell-derived factor 1-α, SDF-1α) with an affinity comparable to heparin and did not alter the gelation behavior or network mechanics when copolymerized into hydrogels at low concentrations. Further, these macromers were incorporated into electrospun nanofibrous hydrogels to introduce sulfate groups into macroporous scaffolds. Once incorporated into either uniform or fibrous HA hydrogels, the sulfated HA macromers significantly slowed encapsulated SDF-1α release over 12 days. Thus, these macromers provide a useful way to introduce heparin-binding features into radically-crosslinked hydrogels to alter protein interactions for a range of applications.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA