C-methylated nucleoside antivirals is mediated by a single conservative mutation in N

54 Pages·2017·5.03 MB·English

JVI Accepted Manuscript Posted Online 16 August 2017
J. Virol. doi:10.1128/JVI.01028-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Title: Escape of tick-borne flavivirus from 2’-C-methylated nucleoside antivirals is mediated
2 by a single conservative mutation in NS5 that has a dramatic effect on viral fitness
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4 Ludek Eyer,a,b Hirofumi Kondo,c Darina Zouharova,a Minato Hirano,c James J. Valdés,a,b Memi
5 Muto,c Tomas Kastl,a Shintaro Kobayashi,c Jan Haviernik,a Manabu Igarashi,d Hiroaki Kariwa,c
6 Marketa Vaculovicova,e,f Jiri Cerny,a,b Rene Kizek,g Andrea Kröger,h Stefan Lienenklaus,i Milan
7 Dejmek,j Radim Nencka,j Martin Palus,a,b Jiri Salat,a Erik De Clercq,k Kentaro Yoshii,c# and
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8 Daniel Ruzeka,b#* o
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10 (a) Department of Virology, Veterinary Research Institute, Brno, Czech Republic d
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11 (b) Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske
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12 Budejovice, Czech Republic m

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13 (c) Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido t
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14 University, Sapporo, Japan //
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15 (d) Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan i.a
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16 (e) Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech
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17 Republic g
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18 (f) Central European Institute of Technology, Brno University of Technology, Brno, n

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19 Czech Republic p
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20 (g) Central Laboratories, Faculty of Pharmacy, University of Veterinary and il 6
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21 Pharmaceutical Sciences, Brno, Czech Republic 0
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22 (h) Institute for Medical Microbiology, Otto-von-Guericke University Magdeburg, 9

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23 Magdeburg, Germany
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24 (i) Institute for Laboratory Animal Science, Hannover Medical School, Hannover, e
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25 Germany
26 (j) Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences,
27 Prague, Czech Republic
28 (k) Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
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30 # Shared senior co-authorship
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32 Running title: Escape of TBEV from antivirals
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34 * Address correspondence to: Daniel Ruzek, Veterinary Research Institute, Hudcova 70, CZ-
35 62100 Brno, Czech Republic, e-mail: [email protected], phone: [phone]; fax:
36 [phone].
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38 Word count: abstract (249), main text (7,534)
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39 Abstract
40 Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in
41 humans. Despite its high medical relevance, no specific antiviral therapy is currently
42 available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2´-C-
43 methyladenosine (7-deaza-2´-CMA), substantially improved disease outcome, increased
44 survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected
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45 with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2´-CMA, two w
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46 drug-resistant TBEV clones were generated and characterized. The two clones shared a a
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47 signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase
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48 (RdRp) domain. This mutation conferred resistance to various 2´-C-methylated nucleoside

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49 derivatives, but no cross-resistance was seen to other nucleoside analogs, such as 4´-C- :
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50 azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular i.a
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51 dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that .o
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52 coordinates the position of a metal ion cofactor as 2´-C-methylated nucleoside analogs o
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53 approach the active site. To investigate its phenotype, the S603T mutation was introduced p
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54 into a recombinant TBEV (Oshima-IC) generated from an infectious cDNA clone and into a ,

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55 TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants 9

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56 were replication-impaired, showing reduced growth and small plaque size in mammalian cell
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57 culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These s
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58 results indicate that TBEV resistance to 2´-C-methylated nucleoside inhibitors is conferred by
59 a single conservative mutation that causes a subtle atomic effect within the active site of
60 viral NS5 RdRp and is associated with strong attenuation of the virus.
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62 Importance
63 This study found that the nucleoside analog 7-deaza-2´-C-methyladenosine (7-deaza-2´-
64 CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that
65 causes severe human neuroinfections in large areas of Europe and Asia and for which there
66 is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-
67 deaza-2´-CMA resulted in significantly higher survival rates, reduced the severity of
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68 neurological signs of the disease. Thus, this compound shows promise for further w
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69 development as an anti-TBEV drug. It is important to generate drug-resistant mutants to a
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70 understand how the drug works and to develop guidelines for patient treatment. We
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71 generated TBEV mutants that were resistant not only to 7-deaza-2´-CMA but also to a broad

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72 range of other 2´-C-methylated antiviral medications. Our findings suggest that combination :
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73 therapy could be used to improve treatment and reduce the emergence of drug-resistant i.a
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74 viruses during nucleoside analog therapy for TBEV infection. .o
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75 o
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76 Introduction
77 Nucleosides and nucleotide analogs represent an important class of therapeutic antiviral
78 agents that are commonly used to treat many life-threatening viral diseases [1,2]. In
79 particular, nucleoside/tide derivatives are a backbone of treatment for HIV/AIDS and for
80 chronic hepatitis B infection [3], with the nucleotide derivative sofosbuvir acting to
81 revolutionize the treatment of chronic hepatitis C (HCV) infection [4,5]. The constant
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82 struggle to develop novel nucleoside/tide analogs as potential antiviral agents against HCV w
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83 has yielded numerous interesting derivatives [6] that are potential treatments for diseases a
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84 caused by other members of the Flaviviridae family. Indeed, 2′-methyl nucleoside derivatives
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85 act as polymerase reaction terminators [7,8] and are active against HCV; they also have a

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86 profound antiviral effect against several medically important flaviviruses. For instance, 7- :
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87 deaza-2´-C-methyladenosine (7-deaza-2´-CMA), also known as MK-608 (Fig 1A), a 2′-methyl i.a
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88 nucleoside analog originally developed by Merck Research Laboratories as an inhibitor of .o
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89 HCV [9], has strong antiviral effects against mosquito-borne members of the genus o
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90 Flavivirus, such as dengue virus [10] and Zika virus [11,12]. Recently we found that 7-deaza- p
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91 2´-CMA has inhibitory activity in vitro against tick-borne encephalitis virus (TBEV), the most ,

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92 medically important tick-borne flavivirus [13,14]. 9

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93 TBEV is the causative agent of tick-borne encephalitis (TBE), a potentially deadly
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94 neuroinfection that is prevalent in large areas of Europe and northern Asia. Despite available s
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95 effective TBEV vaccines, the number of TBE cases continues to increase in Europe, and the
96 risk areas continue to expand [15,16]. The severity of TBE clinical manifestations ranges from
97 mild flu-like infection to meningitis, encephalitis, and meningoencephalitis/radiculitis, with a
98 10% to 20% risk of long-lasting or permanent neuropsychiatric sequelae [17]. Fatality rates
99 range from <2% in Europe to up to 20% in Russia [17]. No specific antiviral therapy is
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100 currently available; thus, there is an urgent need for efficient drugs to treat patients with
101 TBE [17]. Currently, 2′-methyl nucleoside derivatives, and 7-deaza-2´-CMA in particular,
102 represent the lead candidates for further development of antivirals and their prodrug forms
103 as effective therapeutics for this disease [13,14].
104 Unfortunately, antiviral therapy based on chemical inhibitors of viral replication is often
105 accompanied by the rapid emergence of drug-resistant “escape” mutants, substantially
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106 complicating therapy. Experience with the treatment of HIV, HBV, and HCV shows that w
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107 resistance can develop rapidly in literally all of the direct-acting antiviral agents [18,19]. In a
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108 the case of RNA viruses, including flaviviruses, the development of drug resistance is mainly
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109 related to the high mutation rates caused by the low fidelity of viral RNA-dependent RNA

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110 polymerases, which introduce 10-4 to 10-6 errors per nucleotide. This, together with a high p:
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111 viral replication rate, can generate highly variable viral populations that are termed i.a
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112 quasispecies. Viral quasispecies represent dynamic mutant networks in which individual .o
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113 sequences are continuously diversified and regenerated by mutations, allowing the o
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114 population to quickly adapt to changes in the environment such as the presence of an p
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115 antiviral drug [20,21]. Drug-resistant variants can rapidly become the dominant part of the ,

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116 viral population and lead to therapy failure. However, drug-resistant variants often also have 9

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117 a lower replicative capacity or “fitness” than wild-type viruses [22]. If the therapy is stopped,
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118 the more virulent wild-type virus can again become dominant [22]. Thus, it is crucial to s
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119 identify specific mutations in all antiviral agents that lead to drug-resistance, to characterize
120 the replicative capacity of the resistant virus, and to evaluate the risk of potential
121 therapeutic failure.
122 Here we evaluated the therapeutic effects of 7-deaza-2´-CMA in a lethal mouse model
123 of TBE. We found that 7-deaza-2´-CMA treatment significantly reduced the development of
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124 signs of neuroinfection, reduced viral titers in the brain, and increased survival after the
125 infection. We also generated a 7-deaza-2´-CMA-resistant TBEV variant by serial passage of
126 the virus with increasing concentrations of the drug in vitro. The fitness of the drug-resistant
127 virus was characterized both in vitro as well as in vivo in a mouse model. Whole-genome
128 sequencing was used to identify a specific mutation associated with drug resistance and the
129 phenotype change, and the effect of the identified mutation was confirmed by reverse
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130 genetics. The results revealed that a single conservative amino acid substitution in the w
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131 sequence of the TBEV RNA-dependent RNA polymerase conferred resistance to a broad a
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132 spectrum of 2’-methylated nucleoside inhibitors and strongly reduced viral replication
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133 fitness and viral neuroinvasiveness. In the absence of 7-deaza-2´-CMA, there was a rapid

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134 reversion to the wild-type genotype and phenotype in vitro but not in vivo. Taken together, :
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135 these results have important implications for the development of drugs based on 2’- i.a
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136 methylated nucleoside analogs as therapy for infections caused by flaviviruses. .o
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138 Results p
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139 7-deaza-2´-CMA is effective in treating a lethal TBEV infection in a mouse model ,

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140 The antiviral effects of 7-deaza-2´-CMA (Fig 1A) were examined in vivo using a mouse model 9

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141 of lethal TBEV infection. Characteristic clinical signs of infection, such as ruffled fur, slowing
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142 of activity, asthenia, lethargy, tremor, and mild or complete paralysis of the limbs in TBEV- s
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143 infected untreated adult BALB/c mice typically appeared on days 8 to 11 post-infection (p.i.).
144 Subcutaneous infection with 103 PFU was 100% lethal, with a mean survival time of 10±1.4
145 days p.i. (Fig 1B).
146 Treatment of TBEV-infected mice with 5 or 15 mg/kg 7-deaza-2´-CMA once a day
147 (initiated at the time of virus inoculation and ceased on day 17 p.i.) resulted in survival rates
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148 of 35% and 50% (p<0.001; Fig 1B), respectively, and was associated with mean survival times
149 of 12.5±0.7 and 13±1.4 days p.i., respectively. Treatment of TBEV-infected mice with 25
150 mg/kg of 7-deaza-2´-CMA that was initiated at the time of infection, administered twice
151 daily, and ceased on day 17 p.i., resulted in a significantly higher survival rate of 60%
152 (p<0.0001) (Fig 1B) and significantly reduced the development of clinical signs of
153 neuroinfection (p<0.001; Fig 1D). The mean survival time of infected and treated mice was
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154 approximately 6.5 days longer than that of infected and mock-treated control mice. No w
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155 apparent toxicity or other side effects were seen in mice treated with the highest dose of 7- a
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156 deaza-2´-CMA, i.e., 25 mg/kg twice a day, during the entire 28-day monitoring period. Viral
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157 titers were significantly reduced in the brains of treated TBEV-infected mice compared to

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158 those of mock-treated TBEV-infected mice (p<0.01; Fig 1C). :
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159 Mortality following TBEV infection is the result of both direct virus-mediated damage i.a
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160 and immunopathology, so we tested whether treatment with a combination of 7-deaza-2´- .o
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161 CMA plus minocycline, a drug with immunomodulatory properties, would increase the o
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162 survival of TBEV-infected mice. TBEV-infected mice were treated with 7-deaza-2´-CMA two p
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163 times a day (25 mg/kg/dose) and/or with minocycline (45 mg/kg/day). Treatment with ,

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164 minocycline alone resulted in significantly higher survival (p<0.001; Fig 1E) and prolonged 9

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165 the mean survival time of the infected mice (11.8±1.7 days). However, the survival rates and
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166 mean survival times after infection were comparable in mice treated with the drug s
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167 combination as in mice treated only with 7-deaza-2´-CMA, so the combination therapy
168 showed no additive effects.
169 To monitor the antiviral effects of therapy with 7-deaza-2´-CMA in mice before the
170 development of the first clinical signs of neuroinfection, we developed a bioluminescence
171 mouse model of TBEV infection based on IFNβ+/Δβ-luc mice [23]. This model allowed us to
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172 continuously monitor the development of the TBEV infection and the effectiveness of
173 antiviral therapy early in the infection (Fig 1F) by visualizing the host’s response to the virus.
174 Monitoring reporter activity in uninfected control mice revealed a background-level
175 luminescent signal that was stable over the 7-day experimental period. In TBEV-infected
176 mice, 2- to 3-times higher levels of luminescence signal were detected as early as day 1 p.i. A
177 further increase in the luminescence signal was observed in the brain, lymph nodes, and
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178 peritoneal cavity (probably in the spleen and liver) on day 3 p.i. On day 4 p.i., the w
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179 bioluminescent signal levels were approximately 5-times higher when compared to a
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180 uninfected controls. On day 7 p.i., when the mice showed the first clinical signs of infection,
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181 the signal was 7-times higher in the untreated TBEV-infected mice than in the uninfected

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182 controls (Fig 1F). :
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183 Treatment of TBEV-infected IFNβ+/Δβ-luc mice with 7-deaza-2´-CMA (25 mg/kg/two times i.a
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184 a day) significantly reduced the bioluminescence signals at all investigated intervals. A low .o
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185 bioluminescent signal was detected primarily in the peritoneal cavity and/or lymph nodes; o
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186 the signals in brain had low values that were close to background (Fig 1F, G). p
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187 ,

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188 TBEV resistance to 7-deaza-2´-CMA is associated with a single mutation in the NS5 gene 9

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189 The generation of drug-resistant mutants is important for understanding how a drug works
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190 at the molecular level and can be helpful clinically in terms of guiding treatment. To select s
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191 viruses that were resistant to 7-deaza-2´-CMA, TBEV was serially passaged in porcine kidney
192 stable (PS) cells in the presence of increasing concentrations of 7-deaza-2´-CMA (Fig 2A).
193 Two drug-resistant clones were independently obtained after 12 passages with a maximal
194 concentration of 50 µM 7-deaza-2´-CMA. Whole-genome sequence analysis (Sanger method)
195 of the passaged viruses revealed a single mutation at amino acid position 603 in the NS5
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196 protein that changed a serine residue to a threonine (Fig 2B). This corresponded to
197 nucleotide substitution ACA→TCA in the NS5 gene. The same substitution was detected in
198 both drug-resistant clones, but not in wild-type virus that was passaged in the absence of
199 the drug. Sequencing of viruses after each passage revealed that the virus acquired the
200 S603T mutation at passage No. 4 and that it was retained until the end of the experiment
201 (Fig 2C). The S603T mutation mapped to the active site of the RNA-dependent RNA-
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202 polymerase domain of the NS5 protein (Fig 2D). w
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203 a
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204 The S603T TBEV mutant is resistant to a broad spectrum of 2´-C-methylated nucleoside
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205 inhibitors

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206 To test whether the S603T TBEV mutant affects sensitivity to other nucleoside inhibitors, we :
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207 tested a broad spectrum of diverse nucleoside inhibitors to determine their inhibitory effects i.a
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208 on TBEV-infected PS cells in vitro. A series of 2'-C-methyl-substituted nucleosides, 4´-C- .o
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209 azidocytidine, and RO-9187 was tested for inhibitory activity against both the S603T TBEV o
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210 mutant and the wild-type TBEV. The sensitivity of the S603 TBEV mutant to 7-deaza-2´-CMA p
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211 (EC50 value >50 µM) was 46-fold lower than that of the wild-type virus (EC50 value 1.07 ± 0.03 ,
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212 µM) (Table 1). The S603T TBEV mutant showed high levels of cross-resistance to other 2´-C- 9

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213 methyl modified nucleosides, such as 2´-C-methyladenosine, 2´-C-methylguanosine, 2´-C-
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214 methylcytidine, and 2´-C-methyluridine; their inhibitory effect was reduced dramatically by s
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215 8- to 30-fold in the S603T TBEV mutant compared to the wild-type virus (Fig 2E). The
216 inhibitory effects of 4´-C-azidocytidine and its stereochemical counterpart, RO-9187, were
217 not affected in the S603T TBEV mutant, indicating an absence of cross-resistance between
218 4´-C-azido modified and 2´-C-methylated nucleosides (Fig 2E).
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