Publication detail

Escape of tick-borne flavivirus from 2'-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness

Eyer, L., Kondo, H., Zouharova, D., Hirano, M., Valdés, J.J., Muto, M., Kastl, T., Kobayashi, S., Haviernik, J., Igarashi, M., Kariwa, H., Vaculovicova, M., Cerny, J., Kizek, R., Kröger, A., Lienenklaus, S., Dejmek, M., Nencka, R., Palus, M., Salat, J., De Clercq, E., Yoshii, K., Ruzek, D.

Original Title

Escape of tick-borne flavivirus from 2'-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness

English Title

Escape of tick-borne flavivirus from 2'-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness

Type

journal article

Language

en

Original 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 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.

English 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 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.

Keywords

antiviral agents; antiviral therapy; escape mutant; tick-borne encephalitis virus; tick-borne pathogens

Released

01.11.2017

Pages from

1

Pages to

20

Pages count

20