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.

• Keywords
• SARS-CoV-2, TBEV, discovery, drug, flavivirus, high-throughput screening, papain-like, protease, virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 Drug Treatment MeSH
• Fluorescent Dyes chemistry   MeSH
• Protease Inhibitors pharmacology   MeSH
• Coronavirus Papain-Like Proteases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• RNA Helicases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• RNA Viruses enzymology   MeSH
• High-Throughput Screening Assays methods   MeSH
• SARS-CoV-2 enzymology   MeSH
• Serine Endopeptidases chemistry   genetics   metabolism   MeSH
• Viral Nonstructural Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH
• Fluorescent Dyes MeSH
• Protease Inhibitors MeSH
• Coronavirus Papain-Like Proteases MeSH
• NS3 protein, flavivirus MeSH Browser
• papain-like protease, SARS-CoV-2 MeSH Browser
• RNA Helicases MeSH
• Serine Endopeptidases MeSH
• Viral Nonstructural Proteins MeSH

Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2'-deoxy-2'-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d-nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2.

• Keywords
• 2′-Fluoronucleoside, Nucleoside phosphonate, Prodrug, Tetradialdose d-nucleoside, Triphosphate,
• Publication type
• Journal Article MeSH

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.

• Keywords
• Flavivirus, Inhibitor, RNA-dependent RNA polymerase, Remdesivir,
• MeSH
• Adenosine Triphosphate analogs & derivatives   chemistry   pharmacology   MeSH
• Antiviral Agents chemistry   pharmacology   MeSH
• Betacoronavirus drug effects   enzymology   MeSH
• COVID-19 MeSH
• COVID-19 Drug Treatment MeSH
• Flavivirus drug effects   enzymology   MeSH
• Inhibitory Concentration 50 MeSH
• Coronavirus Infections drug therapy   virology   MeSH
• Humans MeSH
• Pandemics MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   metabolism   MeSH
• RNA Viruses drug effects   enzymology   MeSH
• SARS-CoV-2 MeSH
• Pneumonia, Viral drug therapy   virology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Antiviral Agents MeSH
• GS-441524 triphosphate MeSH Browser
• RNA-Dependent RNA Polymerase MeSH

We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.

• Keywords
• Crystal structure, Primase, RNA, SARS-CoV-2,
• MeSH
• Betacoronavirus chemistry   MeSH
• DNA Primase chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Coronavirus RNA-Dependent RNA Polymerase MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Multiprotein Complexes MeSH
• RNA metabolism   MeSH
• SARS-CoV-2 MeSH
• Binding Sites MeSH
• Viral Nonstructural Proteins chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primase MeSH
• Coronavirus RNA-Dependent RNA Polymerase MeSH
• Multiprotein Complexes MeSH
• NS8 protein, SARS-CoV-2 MeSH Browser
• NSP7 protein, SARS-CoV-2 MeSH Browser
• RNA MeSH
• Viral Nonstructural Proteins MeSH

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

• Keywords
• BCX4430, attenuation, drug resistance, galidesivir, mutation, tick-borne encephalitis virus,
• MeSH
• Adenine analogs & derivatives   chemistry   pharmacology   MeSH
• Adenosine analogs & derivatives   MeSH
• Alleles MeSH
• Drug Resistance, Microbial MeSH
• Antiviral Agents chemistry   pharmacology   MeSH
• Cell Line MeSH
• Genotype MeSH
• Encephalitis, Tick-Borne drug therapy   virology   MeSH
• Disease Models, Animal MeSH
• Mutation * MeSH
• Mice MeSH
• Pyrrolidines chemistry   pharmacology   MeSH
• Amino Acid Substitution * MeSH
• Drug Resistance, Viral * MeSH
• Viral Nonstructural Proteins genetics   MeSH
• Encephalitis Viruses, Tick-Borne drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Adenosine MeSH
• Antiviral Agents MeSH
• galidesivir MeSH Browser
• Pyrrolidines MeSH
• Viral Nonstructural Proteins MeSH

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.

• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• Intracellular Membranes metabolism   MeSH
• Scattering, Small Angle MeSH
• Membrane Proteins metabolism   MeSH
• Protein Multimerization * MeSH
• 14-3-3 Proteins metabolism   MeSH
• rab GTP-Binding Proteins metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Membrane Proteins MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Browser
• 14-3-3 Proteins MeSH
• rab GTP-Binding Proteins MeSH
• rab11 protein MeSH Browser
• Recombinant Proteins MeSH

Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

• MeSH
• Adenosine analogs & derivatives   chemistry   pharmacology   MeSH
• Magnesium chemistry   MeSH
• Zika Virus Infection genetics   virology   MeSH
• Protein Conformation drug effects   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Nucleosides chemistry   MeSH
• Nucleotides chemistry   MeSH
• Polyphosphates chemistry   MeSH
• Virus Replication genetics   MeSH
• RNA-Dependent RNA Polymerase chemistry   genetics   MeSH
• RNA chemistry   genetics   MeSH
• Molecular Docking Simulation MeSH
• Viral Nonstructural Proteins chemistry   genetics   MeSH
• Zika Virus chemistry   genetics   pathogenicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Magnesium MeSH
• NITD008 MeSH Browser
• Nucleosides MeSH
• Nucleotides MeSH
• Polyphosphates MeSH
• RNA-Dependent RNA Polymerase MeSH
• RNA MeSH
• triphosphoric acid MeSH Browser
• Viral Nonstructural Proteins MeSH

7-Deazapurine (pyrrolo[2,3-d]pyrimidine) nucleosides are important analogues of biogenic purine nucleosides with diverse biological activities. Replacement of the N7 atom with a carbon atom makes the five-membered ring more electron rich and brings a possibility of attaching additional substituents at the C7 position. This often leads to derivatives with increased base-pairing in DNA or RNA or better binding to enzymes. Several types of 7-deazapurine nucleosides with potent cytostatic or cytotoxic effects have been identified. The most promising are 7-hetaryl-7-deazaadenosines, which are activated in cancer cells by phosphorylation and get incorporated both to RNA (causing inhibition of proteosynthesis) and to DNA (causing DNA damage). Mechanism of action of other types of cytostatic nucleosides, 6-hetaryl-7-deazapurine and thieno-fused deazapurine ribonucleosides, is not yet known. Many 7-deazaadenosine derivatives are potent inhibitors of adenosine kinases. Many types of sugar-modified derivatives of 7-deazapurine nucleosides are also strong antivirals. Most important are 2'-C-methylribo- or 2'-C-methyl-2'-fluororibonucleosides with anti-HCV activities (several compounds underwent clinical trials). Some underexplored areas of potential interest are also outlined.

• Keywords
• antivirals, cytostatics, deazapurines, nucleosides, nucleotides,
• MeSH
• Antiviral Agents chemical synthesis   chemistry   pharmacology   MeSH
• A549 Cells MeSH
• Hep G2 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Nucleosides chemical synthesis   chemistry   pharmacology   MeSH
• Antineoplastic Agents chemical synthesis   chemistry   pharmacology   MeSH
• Purines chemistry   MeSH
• Drug Design MeSH
• Drug Screening Assays, Antitumor MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• 7-deazapurine MeSH Browser
• Antiviral Agents MeSH
• Nucleosides MeSH
• Antineoplastic Agents MeSH
• Purines MeSH

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

• Keywords
• antiviral agents, antiviral therapy, escape mutant, tick-borne encephalitis virus, tick-borne pathogens,
• Publication type
• Journal Article MeSH