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.

• 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

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

• MeSH
• antivirové látky farmakologie   MeSH
• COVID-19 farmakoterapie   MeSH
• fluorescenční barviva chemie   MeSH
• inhibitory proteas farmakologie   MeSH
• koronavirové proteasy podobné papainu antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• lidé MeSH
• preklinické hodnocení léčiv MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• RNA-helikasy antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• RNA-viry enzymologie   MeSH
• rychlé screeningové testy metody   MeSH
• SARS-CoV-2 enzymologie   MeSH
• serinové endopeptidasy chemie   genetika   metabolismus   MeSH
• virové nestrukturální proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Tick-borne encephalitis virus (TBEV) is a medically important representative of the Flaviviridae family. The TBEV genome encodes a single polyprotein, which is co/post-translationally cleaved into three structural and seven non-structural proteins. Of the non-structural proteins, NS5, contains an RNA-dependent RNA polymerase (RdRp) domain that is highly conserved and is responsible for the genome replication. Screening for potential antivirals was done using a hybrid receptor and ligand-based pharmacophore search likely targeting the RdRp domain. For the identification of pharmacophores, a mixture of small probe molecules and nucleotide triphosphates were used. The ligand/receptor interaction screenings of structures from the ZINC database resulted in five compounds. Zinc 3677 and 7151 exhibited lower cytotoxicity and were tested for their antiviral effect against TBEV in vitro. Zinc 3677 inhibited TBEV at micromolar concentrations. The results indicate that Zinc 3677 represents a good target for structure-activity optimizations leading potentially to a discovery of effective TBEV antivirals.

• MeSH
• antivirové látky farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• klíšťata virologie   MeSH
• klíšťová encefalitida virologie   MeSH
• lidé MeSH
• replikace viru účinky léků   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• virové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy účinky léků   enzymologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH