An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor Galidesivir (BCX4430) and also attenuates the virus for mice

Abstract

19The adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has 20 entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under 21 clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also 22 inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other 23 medically important flaviviruses. Until now, studies of this antiviral agent have not yielded 24 resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of 25 TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell 26 culture. Stochastic molecular simulations indicate that the steric freedom caused by the 27 E460D substitution increases close electrostatic interactions between the inhibitor and the 28 interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an 29 incorrect/modified nucleotide. Galidesivir-resistant TBEV exhibited no cross-resistance to 30 structurally different antiviral nucleoside analogues, such as 7-deaza-2´-C-methyladenosine, 31 2´-C-methyladenosine and 4'-azido-aracytidine. Although, the E460D substitution led only to 32 a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly 33 attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. 34Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, 35 flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to 36 flavivirus neurovirulence. 37 38 attenuation 40 41 Importance 42Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections 43 in large areas of Europe and Asia and for which there is currently no specific therapy. We 44 have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, 45which is under clinical development for treatment of Ebola virus infection, has a strong 46 antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant 47 mutants to understand how the drug works. Here, we produced TBEV mutants resistant to 48 Galidesivir and found that the resistance is caused by a single amino acid substitution in an 49 3 active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for 50 replication of viral RNA genome. Although, this substitution led only to a subtle decrease in 51 viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse 52 model. Our results contribute to understanding the molecular basis of Galidesivir antiviral 53 activity. 54 4 55 Introduction 56Flaviviruses (family Flaviviridae, genus Flavivirus) cause widespread human morbidity 57 and mortality throughout the world. These viruses are typically transmitted to humans by 58 mosquito or tick vectors. Tick-borne encephalitis virus (TBEV) is a typical flavivirus 59 transmitted by Ixodes spp. ticks. TBEV is a causative agent of tick...