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
- Klíčová slova
- Sputnik,
- MeSH
- COVID-19 prevence a kontrola MeSH
- lidé MeSH
- vakcíny proti COVID-19 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- populární práce MeSH
- rozhovory MeSH
As opposed to pathogens passively circulating in the body fluids of their host, pathogenic species within the Spirochetes phylum are able to actively coordinate their movement in the host to cause systemic infections. Based on the unique morphology and high motility of spirochetes, we hypothesized that their surface adhesive molecules might be suitably adapted to aid in their dissemination strategies. Designing a system that mimics natural environmental signals, which many spirochetes face during their infectious cycle, we observed that a subset of their surface proteins, particularly Decorin binding protein (Dbp) A/B, can strongly enhance the motility of spirochetes in the extracellular matrix of the host. Using single-molecule force spectroscopy, we disentangled the mechanistic details of DbpA/B and decorin/laminin interactions. Our results show that spirochetes are able to leverage a wide variety of adhesion strategies through force-tuning transient molecular binding to extracellular matrix components, which concertedly enhance spirochetal dissemination through the host.
- MeSH
- bakteriální adheze * MeSH
- bakteriální adheziny genetika metabolismus MeSH
- Borrelia burgdorferi genetika metabolismus patogenita MeSH
- dekorin metabolismus MeSH
- extracelulární matrix metabolismus mikrobiologie MeSH
- interakce hostitele a patogenu MeSH
- kinetika MeSH
- klíště mikrobiologie MeSH
- králíci MeSH
- laminin metabolismus MeSH
- lymeská nemoc metabolismus mikrobiologie MeSH
- pohyb MeSH
- vazba proteinů MeSH
- zobrazení jednotlivé molekuly MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- vakcinace * dějiny MeSH
- vakcíny proti COVID-19 MeSH
- vakcíny farmakologie imunologie klasifikace MeSH
- vyvíjení léků MeSH
- Publikační typ
- rozhovory MeSH
- Geografické názvy
- Československo MeSH
DNA methylation at the fifth position of cytosine (5mC) and at the sixth position of adenine (6 mA) plays an important role in the regulation of the gene expression and, in eukaryotes, is essential for normal development. For Ixodes ricinus, the most common European arthropod vector of human and animal pathogens, the DNA methylation profile and the role of DNA methylation in tick development are still under discussion. Our goal was to analyze the status of I. ricinus DNA methylation at different life stages and identify enzymes that produce this type of DNA modification. We found that 5mC and 6mA are present in I. ricinus genomic DNA at all life stages. In the transcriptome of I. ricinus, we identified the sequences of the putative IrDNMT1, IrDNMT3, and IrDAMT enzymes, and bioinformatic analysis and three-dimensional modeling predicted their DNA methylation activity. This confirms that I. ricinus possesses a complete DNA methylation toolkit. Our results suggest that DNA methylation is important for the physiology and transstadial development of ticks.
- MeSH
- epigeneze genetická * MeSH
- genetická transkripce MeSH
- klíště enzymologie genetika růst a vývoj MeSH
- larva genetika růst a vývoj MeSH
- methyltransferasy chemie genetika MeSH
- molekulární konformace MeSH
- nymfa genetika růst a vývoj MeSH
- ovum růst a vývoj MeSH
- proteiny členovců chemie genetika MeSH
- regulace genové exprese * MeSH
- transkriptom * MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A highly virulent strain (Hypr) of tick-borne encephalitis virus (TBEV) was serially subcultured in the mammalian porcine kidney stable (PS) and Ixodes ricinus tick (IRE/CTVM19) cell lines, producing three viral variants. These variants exhibited distinct plaque sizes and virulence in a mouse model. Comparing the full-genome sequences of all variants, several nucleotide changes were identified in different genomic regions. Furthermore, different sequential variants were revealed to co-exist within one sample as quasispecies. Interestingly, the above-mentioned nucleotide changes found within the whole genome sequences of the new variants were present alongside the nucleotide sequence of the parental strain, which was represented as a minority quasispecies. These observations further imply that TBEV exists as a heterogeneous population that contains virus variants pre-adapted to reproduction in different environments, probably enabling virus survival in ticks and mammals.
- MeSH
- buněčné linie MeSH
- fyziologická adaptace genetika MeSH
- genetická variace MeSH
- genom virový MeSH
- klíště cytologie virologie MeSH
- klíšťová encefalitida virologie MeSH
- ledviny cytologie virologie MeSH
- mutace MeSH
- myši MeSH
- prasata MeSH
- quasispecies * MeSH
- virulence MeSH
- viry klíšťové encefalitidy genetika patogenita fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
