RESUMEN
Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK-DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand-dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor-ligand interactions in triggering the antidengue response of primary human NK cells.
Asunto(s)
Células Dendríticas/inmunología , Virus del Dengue/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón Tipo I/inmunología , Células Asesinas Naturales/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Comunicación Celular/inmunología , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Células Dendríticas/virología , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Regulación de la Expresión Génica , Granzimas/genética , Granzimas/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Evasión Inmune , Interferón Tipo I/genética , Células Asesinas Naturales/virología , Perforina/genética , Perforina/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.
Asunto(s)
Flavivirus/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Microscopía Fluorescente/métodos , Microscopía/métodos , Flavivirus/aislamiento & purificación , Imagenología Tridimensional , Microscopía de Fuerza Atómica/métodos , Proteínas Virales/química , Proteínas Virales/ultraestructuraRESUMEN
Dengue virus (DENV) capsid (C) protein is one of the three structural proteins that form a mature virus. The main challenge impeding the study of this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively charged, stability and solubility properties. Here, we report a strategy to construct, express, biotinylate and purify non-truncated, full-length DENV C protein. A 6× His tag and a biotin acceptor peptide (BAP) were cloned at the N-terminus of C protein using overlapping extension-polymerase chain reaction method for site-specific biotinylation. The final construct was inserted into pET28a plasmid and BL-21 (CodonPlus) expression competent cell strain was selected as there are 12% rare codons in the C protein sequence. Strikingly, we found that our recombinant proteins with BAP were biotinylated endogenously with high efficiency in Escherichia coli BL-21 strains. To purify this His-tagged C protein, nickel-nitriloacetic acid affinity chromatography was first carried out under denaturing condition. After stepwise dialysis and concurrent refolding, ion exchange-fast protein liquid chromatography was performed to further separate the residual contaminants. To obtain C protein with high purity, a final round of purification with size exclusion chromatography was carried out and a single peak corresponding to C protein was attained. With this optimized sequential purification protocol, we successfully generated pure biotinylated full-length DENV C protein. The functionality of this purified non-truncated DENV C protein was examined and it was suitable for structural and molecular studies.
Asunto(s)
Proteínas de la Cápside/aislamiento & purificación , Virus del Dengue/química , Secuencia de Aminoácidos , Biotinilación , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Dengue/virología , Virus del Dengue/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-alpha. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-alpha/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-alpha/C protein interaction in the context of flavivirus life-cycle.