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

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' --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' --methylated nucleoside derivatives, but no cross-resistance was seen to other nucleoside analogs, such as 4' -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' --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' --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. This study found that the nucleoside analog 7-deaza-2' --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' --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.

FDA-Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses in Vero Cells

“…In the HCV subgenomic replicon system, dasabuvir inhibits genotype 1a and 1b replicons with EC 50 values of 7.7 and 1.8 nM, respectively [30]. In our in vitro assays, dasabuvir had a micromolar EC 50 , which was comparable to other small molecule inhibitors that showed effectiveness in laboratory animals infected with VBFs [25][26][27]39,40]. The antiviral activity of dasabuvir was demonstrated regardless if applied before infection, at the time of infection, or post-infection ( Figures 2B and 4, Supplementary Figure S1).…”

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

“…Similarly, the plaque morphology of the E460D mutant and wild-type virus were almost identical to each other; large, clear, and round plaques reflected the rapid and aggressive spread of both mutant and wild-type viruses in PS cell cultures. Thus, the E460D TBEV mutant differs from the recently isolated S603T TBEV mutant resistant to 2=-C-methylated nucleosides; the S603T mutant exerted significantly decreased replication capacity in PS cells and a completely different plaque morphology (small, turbid plaques) compared to the wild-type virus (8). Our results demonstrate that antiviral resistance developed against two structurally different nucleoside analogues with the same mechanism of action can result in different effects on viral replication capacity in cell culture.…”

“…The replication of the E460D mutant seems to be slower compared to the wild-type (as indicated by the comparison of the growth curves of these two viruses in cell culture) and, it is therefore likely that the virus is rapidly eliminated by the innate immunity soon after the inoculation. Attenuation of in vivo replication have previously been reported for drug-resistant RNA or DNA viral mutants, e.g., for TBEV resistance to 2=-C-methylated nucleosides (8), chikungunya virus resistance to T-705 (30), ribavirin resistance to porcine reproductive and respiratory syndrome virus (31), vaccinia virus resistance to acyclic nucleoside phosphonates (32), and pleconaril resistance to coxsackievirus (33).…”

“…The location of the resistance-associated mutations within the viral RdRp active site is essential to understand the mechanism of action of galidesivir; this compound prevents the binding of subsequent nucleotides to the RdRp active site, being considered a nonobligate chain terminator of viral RNA synthesis (10,24). Single amino acid changes within the RdRp were previously identified in flaviviruses resistant to structurally different nucleoside analogues, as exemplified by the mutations S600T, S603T, S604T, and S282T conferring a high-level resistance to the 2=-C-methylated nucleosides in dengue virus, TBEV, Zika virus, and hepatitis C virus, respectively (8,(25)(26)(27)(28). In Alkhurma haemorrhagic fever virus, the mutation S603T was associated with additional amino acid substitutions located in the NS5 RdRp active site, particularly with C666S and M644V (29).…”

“…Among the different strategies aimed at inhibiting virus or cell components involved in TBEV replication, the viral nonstructural NS5 protein, an RNA-dependent RNA polymerase (RdRp), has become an attractive target for specific and effective inhibition of viral replication, with limited measurable effect on the host cells (5). Several molecules, mainly nucleoside analogues, were found to be potent inhibitors of the TBEV NS5 polymerase activity (6)(7)(8)(9). The mechanism of action of these nucleoside analogues is based on their initial metabolization to the active triphosphate (nucleotide) form by cellular kinases and subsequent incorporation into the nascent genome by the RdRp, leading to premature chain termination (5).…”