Flavivirus assembly is driven by the envelope glycoproteins pre-membrane (prM) and envelope (E) in the neutral pH environment of the endoplasmic reticulum. Newly budded, spiky particles are exported through the Golgi apparatus, where mildly acidic pH induces a major surface rearrangement. The glycoproteins reorganize into (prM/E)\₂ complexes at the surface of smooth particles, with prM trapped at the E dimer interface, thereby exposing a furin cleavage site (FCS) for proteolytic maturation into infectious virions. Here, we show that in the absence of furin, immature tick-borne flavivirus particles-tick-borne encephalitis virus, Langat virus, and Louping ill virus-remain fully infectious and pathogenic in female BALB/c mice, in contrast to mosquito-borne flaviviruses such as Usutu, West Nile, and Zika viruses. We further show that the FCS in tick-borne viruses remains exposed at neutral pH, allowing furin at the surface of target cells to activate viral fusogenicity, while mosquito-borne counterparts require acidic re-exposure. Mutations increasing the dynamic behavior of the E dimer mimic the mosquito-borne phenotype, with retracted FCS at neutral pH and loss of infectivity. Our multidisciplinary approach-combining virological assays, targeted mutagenesis, structural modeling, and molecular dynamics simulations-highlights the role of E dimer dynamics in regulating flavivirus maturation and infectivity.

• Publikační typ
• časopisecké články MeSH

Electron microscopy stands as a cornerstone in unraveling the intricate dynamics of viral infections, with its high-resolution capabilities offering invaluable insights into the interactions between viruses and the infected cells. Here, we present a comprehensive methodology designed to explore the three-dimensional interactions specifically between tick-borne encephalitis virus (TBEV) and host cells. This approach allows to study all stages of viral lifecycle, including replication, budding, maturation, and host cell defense mechanisms. The methodology encompasses a range of techniques, commencing with sample preparation using high-pressure freezing, followed by freeze substitution, epoxy embedding, and ultrathin sectioning. Subsequently, we employ electron tomography in conjunction with image processing and analysis techniques to unravel the intricate nuances of TBEV-host cell interactions.

• Klíčová slova
• Electron tomography, Tick-borne encephalitis virus, Transmission electron microscopy,
• MeSH
• buněčné linie MeSH
• interakce hostitele a patogenu * MeSH
• klíšťová encefalitida * virologie   MeSH
• lidé MeSH
• mrazová substituce MeSH
• replikace viru MeSH
• tomografie elektronová * metody   MeSH
• viry klíšťové encefalitidy * fyziologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

• MeSH
• Flavivirus fyziologie   MeSH
• klíšťová encefalitida virologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• proteiny virového obalu * chemie   metabolismus   MeSH
• virion MeSH
• viry klíšťové encefalitidy * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny virového obalu * MeSH

Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III (EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.

• Klíčová slova
• CP: Immunology, CP: Microbiology, escape mutant, monoclonal antibody, neutralization, tick-borne encephalitis, tick-borne encephalitis virus,
• MeSH
• epitopy MeSH
• klíšťová encefalitida * MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• protilátky virové MeSH
• viry klíšťové encefalitidy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• protilátky virové MeSH

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.

• Klíčová slova
• ATPase, RNA helicase, crystal structure, enzyme kinetics, flavivirus, molecular dynamics, nonstructural protein 3, tick-borne encephalitis virus, viral protein,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy * metabolismus   MeSH
• dvouvláknová RNA metabolismus   MeSH
• fosfáty metabolismus   MeSH
• jednovláknová DNA * metabolismus   MeSH
• lidé MeSH
• RNA-helikasy * metabolismus   MeSH
• virové nestrukturální proteiny * metabolismus   MeSH
• viry klíšťové encefalitidy * enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy * MeSH
• dvouvláknová RNA MeSH
• fosfáty MeSH
• jednovláknová DNA * MeSH
• NS3 protein, flavivirus MeSH Prohlížeč
• RNA-helikasy * MeSH
• virové nestrukturální proteiny * MeSH

The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• virologie * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Česká republika MeSH

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

• MeSH
• antivirové látky * farmakologie   terapeutické užití   MeSH
• G-kvadruplexy * MeSH
• klíšťová encefalitida farmakoterapie   genetika   MeSH
• lidé MeSH
• ligandy MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• viry klíšťové encefalitidy * účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• ligandy MeSH
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH

The aim of this review is to follow the history of studies on endemiv arboviruses and the diseases they cause which were detected in the Czech lands (Bohemia, Moravia and Silesia (i.e., the Czech Republic)). The viruses involve tick-borne encephalitis, West Nile and Usutu flaviviruses; the Sindbis alphavirus; Ťahyňa, Batai, Lednice and Sedlec bunyaviruses; the Uukuniemi phlebovirus; and the Tribeč orbivirus. Arboviruses temporarily imported from abroad to the Czech Republic have been omitted. This brief historical review includes a bibliography of all relevant papers.

• Klíčová slova
• arthropods, birds, mammals, mosquitoes, ticks,
• MeSH
• arbovirové infekce dějiny   MeSH
• arboviry fyziologie   MeSH
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• přehledy MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.

• MeSH
• analýza přežití MeSH
• epitopy imunologie   MeSH
• imunoglobulin G aplikace a dávkování   imunologie   MeSH
• klíšťová encefalitida imunologie   prevence a kontrola   virologie   MeSH
• kohortové studie MeSH
• kultivované buňky MeSH
• lidé MeSH
• monoklonální protilátky aplikace a dávkování   genetika   imunologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• neutralizující protilátky aplikace a dávkování   genetika   imunologie   MeSH
• proteiny virového obalu genetika   imunologie   MeSH
• protilátky virové aplikace a dávkování   genetika   imunologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• viry klíšťové encefalitidy účinky léků   imunologie   fyziologie   MeSH
• zkřížené reakce imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• epitopy MeSH
• imunoglobulin G MeSH
• monoklonální protilátky MeSH
• neutralizující protilátky MeSH
• proteiny virového obalu MeSH
• protilátky virové MeSH

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

• MeSH
• glykoproteiny chemie   metabolismus   MeSH
• glykosylace MeSH
• klíšťata virologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteiny virového obalu chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• viry klíšťové encefalitidy růst a vývoj   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykoproteiny MeSH
• proteiny virového obalu MeSH