Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.
Bioorthogonal chemistry provides one of the possibilities to modify various biomolecules in their native environment. The combination of Click chemistry with the BONCAT method (bioorthogonal non-canonical amino acid tagging) is widely used for tagging and analysis of newly synthesized proteins, which are clearly distinguishable from the pre-existing protein pool. However, the commonly used procedure results in low quality 2D electrophoretic profiles. We put a lot of effort into obtaining clear results using a standard Click protocol, with a negligible effect. Here we describe a Click-on-membrane approach which we successfully used not only to monitor de novo protein synthesis but also to detect newly synthesized RNA.
In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.
- MeSH
- down regulace MeSH
- fagocytóza MeSH
- genová ontologie MeSH
- kultivované buňky MeSH
- mapy interakcí proteinů MeSH
- myši inbrední C57BL MeSH
- peritoneální makrofágy metabolismus MeSH
- proteom metabolismus MeSH
- proteomika * MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.
- MeSH
- genetická transkripce MeSH
- klíšťová encefalitida genetika metabolismus virologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- prekurzory RNA MeSH
- proteosyntéza genetika MeSH
- RNA ribozomální genetika metabolismus MeSH
- RNA-polymerasa I genetika metabolismus MeSH
- viry klíšťové encefalitidy fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity.
- MeSH
- analýza spermatu veterinární MeSH
- lipidy chemie MeSH
- motilita spermií fyziologie MeSH
- rejnokovití fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Vector-borne diseases constitute 17% of all infectious diseases in the world; among the blood-feeding arthropods, ticks transmit the highest number of pathogens. Understanding the interactions between the tick vector, the mammalian host and the pathogens circulating between them is the basis for the successful development of vaccines against ticks or the tick-transmitted pathogens as well as for the development of specific treatments against tick-borne infections. A lot of effort has been put into transcriptomic and proteomic analyses; however, the protein-carbohydrate interactions and the overall glycobiology of ticks and tick-borne pathogens has not been given the importance or priority deserved. Novel (bio)analytical techniques and their availability have immensely increased the possibilities in glycobiology research and thus novel information in the glycobiology of ticks and tick-borne pathogens is being generated at a faster pace each year. This review brings a comprehensive summary of the knowledge on both the glycosylated proteins and the glycan-binding proteins of the ticks as well as the tick-transmitted pathogens, with emphasis on the interactions allowing the infection of both the ticks and the hosts by various bacteria and tick-borne encephalitis virus.
- MeSH
- Anaplasma patogenita MeSH
- Borrelia patogenita MeSH
- glykomika metody MeSH
- glykosylace MeSH
- interakce hostitele a patogenu fyziologie MeSH
- klíště mikrobiologie fyziologie virologie MeSH
- lektiny metabolismus MeSH
- nemoci přenášené klíšťaty patofyziologie MeSH
- polysacharidy metabolismus MeSH
- proteomika MeSH
- sacharidy fyziologie MeSH
- viry klíšťové encefalitidy patogenita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Tick-borne encephalitis virus (TBEV) is a member of the genus Flavivirus. It can cause serious infections in humans that may result in encephalitis/meningoencephalitis. Although several studies have described the involvement of specific genes in the host response to TBEV infection in the central nervous system (CNS), the overall network remains poorly characterized. Therefore, we investigated the response of DAOY cells (human medulloblastoma cells derived from cerebellar neurons) to TBEV (Neudoerfl strain, Western subtype) infection to characterize differentially expressed genes by transcriptome analysis. Our results revealed a wide panel of interferon-stimulated genes (ISGs) and pro-inflammatory cytokines, including type III but not type I (or II) interferons (IFNs), which are activated upon TBEV infection, as well as a number of non-coding RNAs, including long non-coding RNAs. To obtain a broader view of the pathways responsible for eliciting an antiviral state in DAOY cells we examined the effect of type I and III IFNs and found that only type I IFN pre-treatment inhibited TBEV production. The cellular response to TBEV showed only partial overlap with gene expression changes induced by IFN-β treatment - suggesting a virus-specific signature - and we identified a group of ISGs that were highly up-regulated following IFN-β treatment. Moreover, a high rate of down-regulation was observed for a wide panel of pro-inflammatory cytokines upon IFN-β treatment. These data can serve as the basis for further studies of host-TBEV interactions and the identification of ISGs and/or lncRNAs with potent antiviral effects in cases of TBEV infection in human neuronal cells.
- MeSH
- aktivace transkripce MeSH
- cytokiny genetika imunologie MeSH
- interakce hostitele a patogenu MeSH
- interferony genetika imunologie MeSH
- klíšťová encefalitida genetika imunologie virologie MeSH
- lidé MeSH
- neurony imunologie virologie MeSH
- viry klíšťové encefalitidy genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. CONCLUSIONS/SIGNIFICANCE: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.
- MeSH
- buňky A549 MeSH
- DEAD box protein 58 metabolismus MeSH
- epidemický výskyt choroby MeSH
- fylogeneze MeSH
- genom virový * MeSH
- infekce virem zika virologie MeSH
- interakce hostitele a patogenu MeSH
- interferon typ I antagonisté a inhibitory biosyntéza genetika MeSH
- lidé MeSH
- replikace viru MeSH
- RNA virová genetika izolace a purifikace MeSH
- Vero buňky MeSH
- virus zika genetika izolace a purifikace patogenita fyziologie MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Brazílie epidemiologie MeSH
A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.
- MeSH
- biosyntéza peptidů genetika imunologie MeSH
- buněčné linie MeSH
- fylogeneze MeSH
- genom virový * MeSH
- glioblastom virologie MeSH
- klíště virologie MeSH
- klíšťová encefalitida virologie MeSH
- lidé MeSH
- meduloblastom virologie MeSH
- mutace MeSH
- neuroblastom virologie MeSH
- otevřené čtecí rámce * MeSH
- viry klíšťové encefalitidy genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
