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.

Central European Institute of Technology Brno University of Technology Brno Czech Republic

Central Laboratories Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Brno Czech Republic

Department of Virology Veterinary Research Institute Brno Czech Republic

Institute for Laboratory Animal Science Hannover Medical School Hannover Germany

Institute for Medical Microbiology Otto von Guericke University Magdeburg Magdeburg Germany

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Laboratory of Public Health Graduate School of Veterinary Medicine Hokkaido University Sapporo Japan

Rega Institute for Medical Research KU Leuven Leuven Belgium

Research Center for Zoonosis Control Hokkaido University Sapporo Japan

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De Clercq E, Li G. 2016. Approved antiviral drugs over the past 50 years. Clin Microbiol Rev 29:695–747. doi:10.1128/CMR.00102-15. PubMed DOI PMC

Boldescu V, Behnam MAM, Vasilakis N, Klein CD. 2017. Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond. Nat Rev Drug Discov 16:565–586. doi:10.1038/nrd.2017.33. PubMed DOI PMC

Cihlar T, Ray AS. 2010. Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine. Antiviral Res 85:39–58. doi:10.1016/j.antiviral.2009.09.014. PubMed DOI

Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, Schultz M, Davis MN, Kayali Z, Reddy KR, Jacobson IM, Kowdley KV, Nyberg L, Subramanian GM, Hyland RH, Arterburn S, Jiang D, McNally J, Brainard D, Symonds WT, McHutchison JG, Sheikh AM, Younossi Z, Gane EJ. 2013. Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med 368:1878–1887. doi:10.1056/NEJMoa1214853. PubMed DOI

Schinazi R, Halfon P, Marcellin P, Asselah T. 2014. HCV direct-acting antiviral agents: the best interferon-free combinations. Liver Int 34(Suppl 1):S69–S78. doi:10.1111/liv.12423. PubMed DOI PMC

Sofia MJ, Chang W, Furman PA, Mosley RT, Ross BS. 2012. Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase. J Med Chem 55:2481–2531. doi:10.1021/jm201384j. PubMed DOI

Hercík K, Kozak J, Šála M, Dejmek M, Hřebabecký H, Zborníková E, Smola M, Ruzek D, Nencka R, Boura E. 2017. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase. Antiviral Res 137:131–133. doi:10.1016/j.antiviral.2016.11.020. PubMed DOI

Potisopon S, Ferron F, Fattorini V, Selisko B, Canard B. 2017. Substrate selectivity of dengue and Zika virus NS5 polymerase towards 2′-modified nucleotide analogues. Antiviral Res 140:25–36. doi:10.1016/j.antiviral.2016.12.021. PubMed DOI

Olsen DB, Eldrup AB, Bartholomew L, Bhat B, Bosserman MR, Ceccacci A, Colwell LF, Fay JF, Flores OA, Getty KL, Grobler JA, LaFemina RL, Markel EJ, Migliaccio G, Prhavc M, Stahlhut MW, Tomassini JE, MacCoss M, Hazuda DJ, Carroll SS. 2004. A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrob Agents Chemother 48:3944–3953. doi:10.1128/AAC.48.10.3944-3953.2004. PubMed DOI PMC

Chen YL, Yokokawa F, Shi PY. 2015. The search for nucleoside/nucleotide analog inhibitors of dengue virus. Antiviral Res 122:12–19. doi:10.1016/j.antiviral.2015.07.010. PubMed DOI

Eyer L, Nencka R, Huvarová I, Palus M, Joao Alves M, Gould EA, De Clercq E, Rùžek D. 2016. Nucleoside inhibitors of Zika virus. J Infect Dis 214:707–711. doi:10.1093/infdis/jiw226. PubMed DOI

Zmurko J, Marques RE, Schols D, Verbeken E, Kaptein SJ, Neyts J. 2016. The viral polymerase inhibitor 7-deaza-2′-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model. PLoS Negl Trop Dis 10:e0004695. doi:10.1371/journal.pntd.0004695. PubMed DOI PMC

Eyer L, Valdés JJ, Gil VA, Nencka R, Hřebabecký H, Šála M, Salát J, Černý J, Palus M, De Clercq E, Rùžek D. 2015. Nucleoside inhibitors of tick-borne encephalitis virus. Antimicrob Agents Chemother 59:5483–5493. doi:10.1128/AAC.00807-15. PubMed DOI PMC

Eyer L, Šmídková M, Nencka R, Neča J, Kastl T, Palus M, De Clercq E, Rùžek D. 2016. Structure-activity relationships of nucleoside analogues for inhibition of tick-borne encephalitis virus. Antiviral Res 133:119–129. doi:10.1016/j.antiviral.2016.07.018. PubMed DOI

Dumpis U, Crook D, Oksi J. 1999. Tick-borne encephalitis. Clin Infect Dis 28:882–890. doi:10.1086/515195. PubMed DOI

Heinz FX, Mandl CW. 1993. The molecular biology of tick-borne encephalitis virus. Review article. APMIS 101:735–745. doi:10.1111/j.1699-0463.1993.tb00174.x. PubMed DOI

Růzek D, Dobler G, Donoso Mantke O. 2010. Tick-borne encephalitis: pathogenesis and clinical implications. Travel Med Infect Dis 8:223–232. doi:10.1016/j.tmaid.2010.06.004. PubMed DOI

Poveda E, Wyles DL, Mena A, Pedreira JD, Castro-Iglesias A, Cachay E. 2014. Update on hepatitis C virus resistance to direct-acting antiviral agents. Antiviral Res 108:181–191. doi:10.1016/j.antiviral.2014.05.015. PubMed DOI

Menéndez-Arias L, Álvarez M, Pacheco B. 2014. Nucleoside/nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism of action and resistance. Curr Opin Virol 8:1–9. doi:10.1016/j.coviro.2014.04.005. PubMed DOI

Lauring AS, Andino R. 2010. Quasispecies theory and the behavior of RNA viruses. PLoS Pathog 6:e1001005. doi:10.1371/journal.ppat.1001005. PubMed DOI PMC

Lauring AS, Frydman J, Andino R. 2013. The role of mutational robustness in RNA virus evolution. Nat Rev Microbiol 11:327–336. doi:10.1038/nrmicro3003. PubMed DOI PMC

Berger KL, Scherer J, Ranga M, Sha N, Stern JO, Quinson AM, Kukolj G. 2015. Baseline polymorphisms and emergence of drug resistance in the NS3/4A protease of hepatitis C virus genotype 1 following treatment with faldaprevir and pegylated interferon alpha 2a/ribavirin in phase 2 and phase 3 studies. Antimicrob Agents Chemother 59:6017–6025. doi:10.1128/AAC.00932-15. PubMed DOI PMC

Lienenklaus S, Cornitescu M, Zietara N, Łyszkiewicz M, Gekara N, Jabłónska J, Edenhofer F, Rajewsky K, Bruder D, Hafner M, Staeheli P, Weiss S. 2009. Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo. J Immunol 183:3229–3236. doi:10.4049/jimmunol.0804277. PubMed DOI

Madadkar-Sobhani A, Guallar V. 2013. PELE web server: atomistic study of biomolecular systems at your fingertips. Nucleic Acids Res 41(Web Server issue):W322–W328. doi:10.1093/nar/gkt454. PubMed DOI PMC

Borrelli KW, Vitalis A, Alcantara R, Guallar V. 2005. PELE: protein energy landscape exploration. A novel Monte Carlo based technique. J Chem Theory Comput 1:1304–1311. doi:10.1021/ct0501811. PubMed DOI

Bressanelli S, Tomei L, Rey FA, De Francesco R. 2002. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol 76:3482–3492. doi:10.1128/JVI.76.7.3482-3492.2002. PubMed DOI PMC

Cohen J. 1988. Statistical power analysis for the behavioral sciences. Erlbaum, Hillsdale, NJ.

Valdés JJ, Gil VA, Butterill PT, Rùžek D. 2016. An all-atom, active site exploration of antiviral inhibitors that target Flaviviridae polymerases. J Gen Virol 97:2552–2565. doi:10.1099/jgv.0.000569. PubMed DOI

Valdés JJ, Butterill PT, Rùžek D. 2017. Flaviviridae viruses use a common molecular mechanism to escape nucleoside analogue inhibitors. Biochem Biophys Res Commun 2017:S0006-291X(17)30535-1. doi:10.1016/j.bbrc.2017.03.068. PubMed DOI

Hayasaka D, Gritsun TS, Yoshii K, Ueki T, Goto A, Mizutani T, Kariwa H, Iwasaki T, Gould EA, Takashima I. 2004. Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of tick-borne encephalitis virus. J Gen Virol 85:1007–1018. doi:10.1099/vir.0.19668-0. PubMed DOI

Yoshii K, Ikawa A, Chiba Y, Omori Y, Maeda J, Murata R, Kariwa H, Takashima I. 2009. Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus. J Virol Methods 161:173–176. doi:10.1016/j.jviromet.2009.05.016. PubMed DOI

Weber E, Finsterbusch K, Lindquist R, Nair S, Lienenklaus S, Gekara NO, Janik D, Weiss S, Kalinke U, Överby AK, Kröger A. 2014. Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses. J Virol 88:12202–12212. doi:10.1128/JVI.01215-14. PubMed DOI PMC

Růzek D, Salát J, Palus M, Gritsun TS, Gould EA, Dyková I, Skallová A, Jelínek J, Kopecký J, Grubhoffer L. 2009. CD8+ T-cells mediate immunopathology in tick-borne encephalitis. Virology 384:1–6. doi:10.1016/j.virol.2008.11.023. PubMed DOI

Palus M, Vojtíšková J, Salát J, Kopecký J, Grubhoffer L, Lipoldová M, Demant P, Rùžek D. 2013. Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system. J Neuroinflammation 10:77. doi:10.1186/1742-2094-10-77. PubMed DOI PMC

Mishra MK, Basu A. 2008. Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis. J Neurochem 105:1582–1595. doi:10.1111/j.1471-4159.2008.05238.x. PubMed DOI

Atrasheuskaya AV, Fredeking TM, Ignatyev GM. 2003. Changes in immune parameters and their correction in human cases of tick-borne encephalitis. Clin Exp Immunol 131:148–154. doi:10.1046/j.1365-2249.2003.02050.x. PubMed DOI PMC

Mishra MK, Dutta K, Saheb SK, Basu A. 2009. Understanding the molecular mechanism of blood-brain barrier damage in an experimental model of Japanese encephalitis: correlation with minocycline administration as a therapeutic agent. Neurochem Int 55:717–723. doi:10.1016/j.neuint.2009.07.006. PubMed DOI

Dutta K, Mishra MK, Nazmi A, Kumawat KL, Basu A. 2010. Minocycline differentially modulates macrophage mediated peripheral immune response following Japanese encephalitis virus infection. Immunobiology 215:884–893. doi:10.1016/j.imbio.2009.12.003. PubMed DOI

Kumar R, Basu A, Sinha S, Das M, Tripathi P, Jain A, Kumar C, Atam V, Khan S, Singh AS. 2016. Role of oral minocycline in acute encephalitis syndrome in India—a randomized controlled trial. BMC Infect Dis 16:67. doi:10.1186/s12879-016-1385-6. PubMed DOI PMC

Schul W, Liu W, Xu HY, Flamand M, Vasudevan SG. 2007. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. J Infect Dis 195:665–674. doi:10.1086/511310. PubMed DOI

Arnold JJ, Sharma SD, Feng JY, Ray AS, Smidansky ED, Kireeva ML, Cho A, Perry J, Vela JE, Park Y, Xu Y, Tian Y, Babusis D, Barauskus O, Peterson BR, Gnatt A, Kashlev M, Zhong W, Cameron CE. 2012. Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides. PLoS Pathog 8:e1003030. doi:10.1371/journal.ppat.1003030. PubMed DOI PMC

Wu R, Smidansky ED, Oh HS, Takhampunya R, Padmanabhan R, Cameron CE, Peterson BR. 2010. Synthesis of a 6-methyl-7-deaza analogue of adenosine that potently inhibits replication of polio and dengue viruses. J Med Chem 53:7958–7966. doi:10.1021/jm100593s. PubMed DOI PMC

Klumpp K, Kalayanov G, Ma H, Le Pogam S, Leveque V, Jiang WR, Inocencio N, De Witte A, Rajyaguru S, Tai E, Chanda S, Irwin MR, Sund C, Winqist A, Maltseva T, Eriksson S, Usova E, Smith M, Alker A, Najera I, Cammack N, Martin JA, Johansson NG, Smith DB. 2008. 2′-Deoxy-4′-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2′-alpha-hydroxyl groups. J Biol Chem 283:2167–2175. doi:10.1074/jbc.M708929200. PubMed DOI

Klumpp K, Lévêque V, Le Pogam S, Ma H, Jiang WR, Kang H, Granycome C, Singer M, Laxton C, Hang JQ, Sarma K, Smith DB, Heindl D, Hobbs CJ, Merrett JH, Symons J, Cammack N, Martin JA, Devos R, Nájera I. 2006. The novel nucleoside analog R1479 (4′-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 281:3793–3799. doi:10.1074/jbc.M510195200. PubMed DOI

Migliaccio G, Tomassini JE, Carroll SS, Tomei L, Altamura S, Bhat B, Bartholomew L, Bosserman MR, Ceccacci A, Colwell LF, Cortese R, De Francesco R, Eldrup AB, Getty KL, Hou XS, LaFemina RL, Ludmerer SW, MacCoss M, McMasters DR, Stahlhut MW, Olsen DB, Hazuda DJ, Flores OA. 2003. Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 278:49164–49170. doi:10.1074/jbc.M305041200. PubMed DOI

Flint M, McMullan LK, Dodd KA, Bird BH, Khristova ML, Nichol ST, Spiropoulou CF. 2014. Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses. Antimicrob Agents Chemother 58:3206–3216. doi:10.1128/AAC.02393-14. PubMed DOI PMC

Xu HT, Hassounah SA, Colby-Germinario SP, Oliveira M, Fogarty C, Quan Y, Han Y, Golubkov O, Ibanescu I, Brenner B, Stranix BR, Wainberg MA. 2017. Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors. J Antimicrob Chemother 72:727–734. doi:10.1093/jac/dkw514. PubMed DOI PMC

Mateo R, Nagamine CM, Kirkegaard K. 2015. Suppression of drug resistance in dengue virus. mBio 6:e01960-15. doi:10.1128/mBio.01960-15. PubMed DOI PMC

Curti E, Jaeger J. 2013. Residues Arg283, Arg285, and Ile287 in the nucleotide binding pocket of bovine viral diarrhea virus NS5B RNA polymerase affect catalysis and fidelity. J Virol 87:199–207. doi:10.1128/JVI.06968-11. PubMed DOI PMC

Steitz TA, Steitz JA. 1993. A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci U S A 90:6498–6502. doi:10.1073/pnas.90.14.6498. PubMed DOI PMC

Dobson BM, Procter DJ, Hollett NA, Flesch IE, Newsome TP, Tscharke DC. 2014. Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice. Virology 456–457:145–156. doi:10.1016/j.virol.2014.03.020. PubMed DOI

Mateu CG, Recalde MP, Artuso MC, Hermida G, Linero FN, Scolaro LA, Damonte EB, Pujol CA, Carlucci MJ. 2011. Emergence of herpes simplex virus-1 syncytial variants with altered virulence for mice after selection with a natural carrageenan. Sex Transm Dis 38:555–561. PubMed

Rubio MP, López-Bueno A, Almendral JM. 2005. Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor. J Virol 79:11280–11290. doi:10.1128/JVI.79.17.11280-11290.2005. PubMed DOI PMC

McCown MF, Rajyaguru S, Le Pogam S, Ali S, Jiang WR, Kang H, Symons J, Cammack N, Najera I. 2008. The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors. Antimicrob Agents Chemother 52:1604–1612. doi:10.1128/AAC.01317-07. PubMed DOI PMC

Pospisil L, Jandasek L, Pesek J. 1954. Isolation of new strains of meningoencephalitis virus in the Brno region during the summer of 1953. Lek List 9:3–5. PubMed

Kozuch O, Mayer V. 1975. Pig kidney epithelial (PS) cells: a perfect tool for the study of flaviviruses and some other arboviruses. Acta Virol 19:498. PubMed

De Madrid AT, Porterfield JS. 1969. A simple micro-culture method for the study of group B arboviruses. Bull World Health Organ 40:113–121. PubMed PMC

Růzek D, Gritsun TS, Forrester NL, Gould EA, Kopecký J, Golovchenko M, Rudenko N, Grubhoffer L. 2008. Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus. Virology 374:249–255. doi:10.1016/j.virol.2008.01.010. PubMed DOI

Takashima I, Morita K, Chiba M, Hayasaka D, Sato T, Takezawa C, Igarashi A, Kariwa H, Yoshimatsu K, Arikawa J, Hashimoto N. 1997. A case of tick-borne encephalitis in Japan and isolation of the virus. J Clin Microbiol 35:1943–1947. PubMed PMC

Yoshii K, Igarashi M, Ito K, Kariwa H, Holbrook MR, Takashima I. 2011. Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5. Virus Res 155:61–68. doi:10.1016/j.virusres.2010.08.023. PubMed DOI

Schrödinger LLC. 2016. Release, 2016-3. Maestro. Schrödinger LLC, New York, NY.

Jorgensen WL, Tirado-Rives J. 1988. The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. J Am Chem Soc 110:1657–1666. doi:10.1021/ja00214a001. PubMed DOI

Giannotti MI, Cabeza de Vaca I, Artés JM, Sanz F, Guallar V, Gorostiza P. 2015. Direct measurement of the nanomechanical stability of a redox protein active site and its dependence upon metal binding. J Phys Chem B 119:12050–12058. doi:10.1021/acs.jpcb.5b06382. PubMed DOI

Läärä E. 2009. Statistics: reasoning on uncertainty, and the insignificance of testing null. Ann Zool Fennici 46:138–157.

Zuur AF, Ieno EN, Elphick CS. 2010. A protocol for data exploration to avoid common statistical problems. Methods Ecol Evol 1:3–14. doi:10.1111/j.2041-210X.2009.00001.x. DOI

Humphrey W, Dalke A, Schulten K. 1996. VMD: visual molecular dynamics. J Mol Graph 14:33–38. doi:10.1016/0263-7855(96)00018-5. PubMed DOI

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