Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Virology, antiviral agents, antiviral therapy, escape mutant, tick-borne, encephalitis virus, tick-borne pathogens, C VIRUS-REPLICATION, NUCLEOSIDE/NUCLEOTIDE ANALOG INHIBITORS, INFECTIOUS CDNA-CLONE, RNA-POLYMERASE, ACTIVE-SITE, ENCEPHALITIS, DENGUE, NUCLEOTIDE, RESISTANCE, MINOCYCLINE, tick-borne encephalitis virus, 06 Biological Sciences, 07 Agricultural and Veterinary Sciences, 11 Medical and Health Sciences, 30 Agricultural, veterinary and food sciences, 31 Biological sciences, 32 Biomedical and clinical sciences

Abstract:

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 outcome, 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 to 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 (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 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 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, 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 could be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.