Escape of Tick-Borne Flavivirus from 2′-C-Methylated Nucleoside Antivirals Is Mediated by a

20 Pages·2017·3.32 MB·English

VACCINES AND ANTIVIRAL AGENTS
crossm
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
D
o
w
LudekEyer,a,bHirofumiKondo,cDarinaZouharova,aMinatoHirano,c
n
JamesJ.Valdés,a,bMemiMuto,cTomasKastl,aShintaroKobayashi,c lo
JanHaviernik,aManabuIgarashi,dHiroakiKariwa,cMarketaVaculovicova,e,f a
d
JiriCerny,a,bReneKizek,gAndreaKröger,hStefanLienenklaus,iMilanDejmek,j e
RadimNencka,jMartinPalus,a,bJiriSalat,aErikDeClercq,kKentaroYoshii,c d

f
DanielRuzeka,b r
o
m
DepartmentofVirology,VeterinaryResearchInstitute,Brno,CzechRepublica;InstituteofParasitology,Biology

CentreoftheCzechAcademyofSciences,CeskeBudejovice,CzechRepublicb;LaboratoryofPublicHealth, h
t
GraduateSchoolofVeterinaryMedicine,HokkaidoUniversity,Sapporo,Japanc;ResearchCenterforZoonosis t
p
Control,HokkaidoUniversity,Sapporo,Japand;DepartmentofChemistryandBiochemistry,MendelUniversity :
/
/
inBrno,Brno,CzechRepublice;CentralEuropeanInstituteofTechnology,BrnoUniversityofTechnology,Brno, jv
CzechRepublicf;CentralLaboratories,FacultyofPharmacy,UniversityofVeterinaryandPharmaceutical i.
a
Sciences,Brno,CzechRepublicg;InstituteforMedicalMicrobiology,OttovonGuerickeUniversityMagdeburg, s
Magdeburg,Germanyh;InstituteforLaboratoryAnimalScience,HannoverMedicalSchool,Hannover, m
Germanyi;InstituteofOrganicChemistryandBiochemistry,CzechAcademyofSciences,Prague,Czech .o
Republicj;RegaInstituteforMedicalResearch,KULeuven,Leuven,Belgiumk rg
/

o
ABSTRACT Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal n

neuroinfection in humans. Despite its high medical relevance, no specific antiviral Received19June2017 Accepted1August A
2017 p
therapy is currently available. Here we demonstrate that treatment with a nucleoside r
analog, 7-deaza-2=-C-methyladenosine (7-deaza-2=-CMA), substantially improved dis- AAuccgeupstte2d01m7anuscriptpostedonline16 il 2
,
ease outcomes, increased survival, and reduced signs of neuroinfection and viral ti-
CitationEyerL,KondoH,ZouharovaD,Hirano 2
ters in the brains of mice infected with a lethal dose of TBEV. To investigate the M,ValdésJJ,MutoM,KastlT,KobayashiS, 0
1
mechanism of action of 7-deaza-2=-CMA, two drug-resistant TBEV clones were gener- HaviernikJ,IgarashiM,KariwaH,Vaculovicova 9
ated and characterized. The two clones shared a signature amino acid substitution, M,CernyJ,KizekR,KrögerA,LienenklausS, b
DejmekM,NenckaR,PalusM,SalatJ,DeClercq y
S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This muta- E,YoshiiK,RuzekD.2017.Escapeoftick-borne g
tion conferred resistance to various 2=-C-methylated nucleoside derivatives, but no flavivirusfrom2=-C-methylatednucleoside ue
cross-resistance was seen with other nucleoside analogs, such as 4=-C-azidocytidine antiviralsismediatedbyasingleconservative s
mutationinNS5thathasadramaticeffecton t
and 2=-deoxy-2=-beta-hydroxy-4=-azidocytidine (RO-9187). All-atom molecular dynam- viralfitness.JVirol91:e01028-17.https://doi
ics simulations revealed that the S603T RdRp mutant repels a water molecule that .org/10.1128/JVI.01028-17.
EditorTerenceS.Dermody,Universityof
coordinates the position of a metal ion cofactor as 2=-C-methylated nucleoside ana-
PittsburghSchoolofMedicine
logs approach the active site. To investigate its phenotype, the S603T mutation was
Copyright©2017AmericanSocietyfor
introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious Microbiology.AllRightsReserved.
cDNAcloneandintoaTBEVrepliconthatexpressesareporterluciferasegene(Oshima- AddresscorrespondencetoDanielRuzek,
REP-luc2A). The mutants were replication impaired, showing reduced growth and a [email protected].
K.Y.andD.R.areco-seniorauthorsofthis
small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness
article.
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
associatedwithstrongattenuationofthevirus.
IMPORTANCE This study found that the nucleoside analog 7-deaza-2=-C-methyl-
adenosine (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
November2017 Volume91 Issue21 e01028-17 JournalofVirology jvi.asm.org 1
Eyeretal. JournalofVirology
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 ther-
apy may be used to improve treatment and reduce the emergence of drug-resistant
virusesduringnucleosideanalogtherapyforTBEVinfection.
KEYWORDS antiviralagents,antiviraltherapy,escapemutant,tick-borne
encephalitisvirus,tick-bornepathogens
D
Nucleoside and nucleotide analogs represent an important class of therapeutic o
w
antiviral agents that are commonly used to treat many life-threatening viral n
diseases(1,2).Inparticular,nucleoside/tidederivativesareabackboneoftreatment lo
a
forHIV/AIDSandforchronichepatitisBvirus(HBV)infection(3),withthenucleotide d
e
derivative sofosbuvir acting to revolutionize the treatment of chronic hepatitis C d

virus(HCV)infection(4,5).Theconstantstruggletodevelopnovelnucleoside/tide f
r
o
analogsaspotentialantiviralagentsagainstHCVhasyieldednumerousinteresting m
derivatives(6)thatarepotentialtreatmentsfordiseasescausedbyothermembers
h
of the Flaviviridae family. Indeed, 2=-methyl nucleoside derivatives act as polymer- tt
p
asereactionterminators(7,8)andareactiveagainstHCV;theyalsohaveaprofound :/
/
antiviral effect againstseveralmedicallyimportantflaviviruses.Forinstance,7-deaza- jv
i.
2=-C-methyladenosine (7-deaza-2=-CMA; also known as MK-608) (Fig. 1A ), a 2=-methyl a
s
nucleoside analog originally developed as an inhibitor of HCV by Merck Research m
Laboratories (9), has strong antiviral effects against mosquito-borne members of the .o
r
genusFlavivirus,suchasdenguevirus(DENV)(10)andZikavirus(11,12).Werecently g
/
foundthat7-deaza-2=-CMAhasinhibitoryactivityinvitroagainsttick-borneencepha- o
n
litisvirus(TBEV),themostmedicallyimportanttick-borneflavivirus(13,14).

A
TBEV is the causative agent of tick-borne encephalitis (TBE), a potentially deadly p
neuroinfection that is prevalent in large areas of Europe and northern Asia. Despite ril
available effective TBEV vaccines, the number of TBE cases continues to increase in 2
,

Europe, and the risk areas continue to expand (15, 16). The severities of TBE clinical 2
0
manifestations range from a mild flu-like infection to meningitis, encephalitis, and 1
9
meningoencephalitis/radiculitis, with a 10% to 20% risk of long-lasting or permanent
b
neuropsychiatricsequelae(17).Fatalityratesrangefrom(cid:2)2%inEuropetoupto20% y

inRussia(17).Nospecificantiviraltherapyiscurrentlyavailable;thus,thereisanurgent g
u
needforefficientdrugstotreatpatientswithTBE(17).Currently,2=-methylnucleoside e
s
derivatives,and7-deaza-2=-CMAinparticular,representtheleadcandidatesforfurther t
development of antivirals and their prodrug forms as effective therapeutics for this
disease(13,14).
Unfortunately, antiviral therapy based on chemical inhibitors of viral replication is
often accompanied by the rapid emergence of drug-resistant “escape” mutants, sub-
stantially complicating therapy. Experience with the treatment of HIV, HBV, and HCV
showsthatresistancecandeveloprapidlywithliterallyallofthedirect-actingantiviral
agents (18, 19). In the case of RNA viruses, including flaviviruses, the development of
drugresistanceismainlyrelatedtothehighmutationratescausedbythelowfidelity
ofviralRNA-dependentRNApolymerases(RdRps),whichintroduce10(cid:3)4to10(cid:3)6error
per nucleotide. This, together with a high viral replication rate, can generate highly
variable viral populations that are termed quasispecies. Viral quasispecies represent
dynamic mutant networks in which individual sequences are continuously diversified
andregeneratedbymutations,allowingthepopulationtoadaptquicklytochangesin
the environment, such as the presence of an antiviral drug (20, 21). Drug-resistant
variants can rapidly become the dominant part of the viral population and lead to
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 2
EscapeofTBEVfromAntivirals JournalofVirology
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FIG17-Deaza-2=-CMAiseffectiveattreatinglethalTBEVinfectioninamousemodel.(A)Structureof7-deaza-2=-CMA.(B)
GroupsofadultBALB/cmicewereinfectedwithalethaldoseofTBEVandtreatedintraperitoneallywiththeindicateddoses
of7-deaza-2=-CMAormocktreatedwithPBS.Survivalwasmonitoreddailyfor28days.(C)GroupsofadultBALB/cmicewere
infectedwithalethaldoseofTBEVandtreatedwiththeindicateddoseof7-deaza-2=-CMA,minocycline,acombinationof
(Continuedonnextpage)
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 3
Eyeretal. JournalofVirology
therapy failure. However, drug-resistant variants often also have a lower replicative
capacity or “fitness” than wild-type viruses (22). If the therapy is stopped, the more
virulentwild-typeviruscanagainbecomedominant(22).Thus,itiscrucialtoidentify
specificmutationsinallantiviralagentsthatleadtodrugresistance,tocharacterizethe
replicative capacity of the resistant virus, and to evaluate the risk of potential thera-
peuticfailure.
Here we evaluated the therapeutic effects of 7-deaza-2=-CMA in a lethal mouse
model of TBE. We found that 7-deaza-2=-CMA treatment significantly reduced the
developmentofsignsofneuroinfection,reducedviraltitersinthebrain,andincreased
survivalaftertheinfection.Wealsogenerateda7-deaza-2=-CMA-resistantTBEVvariant
by serial passage of the virus with increasing concentrations of the drug in vitro. The
fitnessofthedrug-resistantviruswascharacterizedbothinvitroandinvivoinamouse
model.Whole-genomesequencingwasusedtoidentifyaspecificmutationassociated
with drug resistance and the phenotype change, and the effect of the identified D
mutation was confirmed by reverse genetics. The results revealed that a single con- o
w
servative amino acid substitution in the sequence of the TBEV RNA-dependent RNA n
polymerase conferred resistance to a broad spectrum of 2=-methylated nucleoside lo
a
inhibitorsandstronglyreducedviralreplicationfitnessandviralneuroinvasiveness.In d
e
theabsenceof7-deaza-2=-CMA,therewasarapidreversiontothewild-typegenotype d

and phenotype in vitro but not in vivo. Taken together, these results have important f
r
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implicationsforthedevelopmentofdrugsbasedon2=-methylatednucleosideanalogs m
astherapyforinfectionscausedbyflaviviruses.
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RESULTS :
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7-Deaza-2=-CMAiseffectiveattreatinglethalTBEVinfectioninamousemodel. jv
The antiviral effects of 7-deaza-2=-CMA (Fig. 1A) were examined in vivo by use of a i.a
s
mousemodeloflethalTBEVinfection.Characteristicclinicalsignsofinfection,suchas m
ruffledfur,slowingofactivity,asthenia,lethargy,tremor,andmildorcompleteparalysis .
o
ofthelimbs,typicallyappearedinTBEV-infecteduntreatedadultBALB/cmiceondays rg
8to11postinfection(p.i.).Subcutaneousinfectionwith103PFUwas100%lethal,with /
o
ameansurvivaltimeof10(cid:4)1.4daysp.i.(Fig.1B). n

A
TreatmentofTBEV-infectedmicewith5and15mg/kgofbodyweightof7-deaza-
p
2re=s-CuMlteAdoinncseuarvdivaayl(riantietisatoefd3a5t%thaentdim5e0%of(vPiru(cid:2)s0in.0o0c1u)la(tFioign. 1anBd), creesapseecdtiovnelyd,aayn1d7wp.ai.s) ril 2
,
associatedwithmeansurvivaltimesof12.5(cid:4)0.7and13(cid:4)1.4daysp.i.,respectively. 2
0
TreatmentofTBEV-infectedmicewith25mg/kgof7-deaza-2=-CMAthatwasinitiated 1
atthetimeofinfection,administeredtwicedaily,andceasedonday17p.i.resultedin 9

b
asignificantlyhighersurvivalrate(60%)(P(cid:2)0.0001)(Fig.1B)andsignificantlyreduced y
the development of clinical signs of neuroinfection (P (cid:2) 0.001) (Fig. 1D). The mean g
u
survivaltimeofinfectedanddrug-treatedmicewasapproximately6.5dayslongerthan e
s
that of infected and mock-treated control mice. No apparent toxicity or other side t
effects were seen in mice treated with the highest dose of 7-deaza-2=-CMA, i.e., 25
mg/kg twice a day, during the entire 28-day monitoring period. Viral titers were
significantlyreducedinthebrainsoftreatedTBEV-infectedmicecomparedtothosein
thebrainsofmock-treatedTBEV-infectedmice(P(cid:2)0.01)(Fig.1C).
MortalityfollowingTBEVinfectionistheresultofbothdirectvirus-mediateddam-
age and immunopathology, so we tested whether treatment with a combination of
FIG1Legend(Continued)
7-deaza-2=-CMAandminocycline,orPBS.Onday8p.i.,thebrainsofthemicewerecollectedandhomogenized,andtheviral
titersweredeterminedusingaplaqueassay.(D)Illnesssignswerescoredasfollows:0,nosymptoms;1,ruffledfur;2,slowing
ofactivityorhunchedposture;3,astheniaormildparalysis;4,lethargy,tremor,orcompleteparalysisofthelimbs;and5,death.
(E)GroupsofadultBALB/cmicewereinfectedwithalethaldoseofTBEVandtreatedintraperitoneallywithminocycline,with
a combination of minocycline and 7-deaza-2=-CMA, or with PBS. Survival was monitored daily for 28 days. (F and G)
IFN(cid:2)(cid:5)/Δ(cid:2)-lucmicewereinfectedwithalethaldoseofTBEVandtreatedwith7-deaza-2=-CMAormocktreatedwithPBS.The
bioluminescencesignalindicatingtheIFN-(cid:2)responsetovirusinfectionwasscannedusingalive-animalimagingdevice(F)and
quantified(G).*,P(cid:2)0.05;**,P(cid:2)0.01;***,P(cid:2)0.001;****,P(cid:2)0.0001.
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 4
EscapeofTBEVfromAntivirals JournalofVirology
7-deaza-2=-CMA plus minocycline, a drug with immunomodulatory properties, would
increase the survival of TBEV-infected mice. TBEV-infected mice were treated with
7-deaza-2=-CMAtwotimesaday(25mg/kg/dose)and/orwithminocycline(45mg/kg/
day). Treatment with minocycline alone resulted in a significantly higher survival rate
(P(cid:2)0.001)(Fig.1E)andprolongedthemeansurvivaltimeoftheinfectedmice(11.8(cid:4)
1.7 days). However, the survival rates and mean survival times after infection were
comparable for mice treated with the drug combination and mice treated only with
7-deaza-2=-CMA,sothecombinationtherapyshowednoadditiveeffects.
Tomonitortheantiviraleffectsoftherapywith7-deaza-2=-CMAinmicebeforethe
development of the first clinical signs of neuroinfection, we developed a biolumines-
cence mouse model of TBEV infection based on IFN(cid:2)(cid:5)/Δ(cid:2)-luc mice (23). This model
allowed us to continuously monitor the development of TBEV infection and the
effectiveness of antiviral therapy early in infection (Fig. 1F) by visualizing the host’s
responsetothevirus.Monitoringreporteractivityinuninfectedcontrolmicerevealed D
a background-level luminescence signal that was stable over the 7-day experimental o
w
period. In TBEV-infected mice, 2 to 3 times higher levels of luminescence signal were n
detected as early as day 1 p.i. A further increase in the luminescence signal was lo
a
observedinthebrain,lymphnodes,andperitonealcavity(probablyinthespleenand d
e
liver)onday3p.i.Onday4p.i.,thebioluminescencesignallevelswereapproximately d

5timeshigherthanthoseofuninfectedcontrols.Onday7p.i.,whenthemiceshowed f
r
o
the first clinical signs of infection, the signal was 7 times higher in the untreated m
TBEV-infectedmicethanintheuninfectedcontrols(Fig.1F).
h
TreatmentofTBEV-infectedIFN(cid:2)(cid:5)/Δ(cid:2)-lucmicewith7-deaza-2=-CMA(25mg/kgtwo tt
p
times a day) significantly reduced the bioluminescence signals at all investigated :/
/
intervals.Alowbioluminescencesignalwasdetectedprimarilyintheperitonealcavity jv
i.
and/or lymph nodes; the signals in the brain had low values that were close to the a
s
backgroundlevel(Fig.1FandG). m
TBEVresistanceto7-deaza-2=-CMAisassociatedwithasinglemutationinthe .o
NS5 gene. The generation of drug-resistant mutants is important for understanding rg
/
how a drug works at the molecular level and can be helpful clinically in terms of o
n
guiding treatment. To select viruses that were resistant to 7-deaza-2=-CMA, TBEV was

A
serially passaged in porcine kidney stable (PS) cells in the presence of increasing p
concentrations of 7-deaza-2=-CMA (Fig. 2A). Two drug-resistant clones were indepen- ril
dentlyobtainedafter12passageswithamaximalconcentrationof50(cid:3)M7-deaza- 2
,

2=-CMA. Whole-genome sequence analysis (Sanger method) of the passaged viruses 2
0
revealedasinglemutation,ataminoacidposition603intheNS5protein,thatchanged 1
aserineresiduetoathreonine(Fig.2B).ThiscorrespondedtoaTCG¡ACGnucleotide 9
b
substitution in the NS5 gene. The same substitution was detected in both drug- y

resistant clones but not in the wild-type virus passaged in the absence of the drug. g
u
Sequencing of viruses after each passage revealed that the virus acquired the S603T e
s
mutation at passage 4 and that the mutation was retained until the end of the t
experiment (Fig. 2C). The S603T mutation mapped to the active site of the RNA-
dependent RNA polymerase domain of the NS5 protein (Fig. 2D).
The S603T TBEV mutant is resistant to a broad spectrum of 2=-C-methylated
nucleoside inhibitors. To test whether the S603T mutation affects the sensitivity of
TBEVtoothernucleosideinhibitors,wetestedabroadspectrumofnucleosideinhib-
itorstodeterminetheirinhibitoryeffectsonTBEV-infectedPScellsinvitro.Aseriesof
2=-C-methyl-substituted nucleosides, 4=-C-azidocytidine, and RO-9187 were tested
forinhibitoryactivityagainstboththeS603TTBEVmutantandwild-typeTBEV.The
sensitivity of the S603T TBEV mutant to 7-deaza-2=-CMA (50% effective concentra-
tion [EC ] of(cid:6) 50 (cid:3)M) was 46-fold lower than that of the wild-type virus (EC (cid:7)
50 50
1.07 (cid:4) 0.03 (cid:3)M) (Table 1). The S603T TBEV mutant showed high levels of cross-
resistancetoother2=-C-methyl-modifiednucleosides,suchas2=-C-methyladenosine,
2=-C-methylguanosine,2=-C-methylcytidine,and2=-C-methyluridine;theirinhibitoryef-
fectswerereduceddramatically(8-to30-fold)intheS603TTBEVmutantcomparedto
those against the wild-type virus (Fig. 2E). The inhibitory effects of 4=-C-azidocytidine
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Eyeretal. JournalofVirology
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FIG2TBEVresistanceto7-deaza-2=-CMAisassociatedwithasinglemutationintheNS5gene.(A)Diagramshowingtheselectionprocessforthegeneration 2
ofaTBEVstrainresistantto7-deaza-2=-CMA.TBEVwasseriallypassagedinporcinekidneystable(PS)cellsinthepresenceofincreasingconcentrationsof 0
1
7-deaza-2=-CMA.(BandC)Whole-genomesequenceanalysisofthepassagedvirusrevealedasinglemutation,ataminoacidposition603intheNS5protein, 9
thatchangedaserineresiduetoathreonine.(D)TheS603TmutationwasmappedtotheactivesiteoftheRNA-dependentRNApolymerasedomainofthe
b
NS5protein.Structuraldifferencesbetweenthewild-type(S603)andmutated(T603)TBEVNS5polymeraseswereexamined.(a)Modelsofthewild-typeand y
mutatedTBEVNS5polymeraseswerecreatedbyhomologymodelingusingthestructuresoftheWestNilevirus(WNV)(PDBentry2HFZ),denguevirus(DENV) g
(PDBentries2J7U,4V0Q,and4V0R),andJapaneseencephalitisvirus(JEV)(PDBentry4K6M)polymerasesinMODELLER.(b)Closerviewofthepolymeraseactive u
e
sites.(c)Differencesintheactivesitestructures,showingchangesinthehydrogenbondnetwork.(E)Thesensitivityorresistanceofwild-typeTBEV,theS603T
s
TBEVmutant,andtherevertanttoabroadspectrumofdiversenucleosideinhibitorswasevaluatedinTBEV-infectedPScellsinvitro.TheS603TTBEVmutant t
showedhighlevelsofresistancetoabroadspectrumof2=-C-methylatednucleosideinhibitors.
and its stereochemical counterpart, RO-9187, were not affected in the S603T TBEV
mutant, indicating an absence of cross-resistance between 4=-C-azido-modified and
2=-C-methylatednucleosides(Fig.2E).
TBEVNS5S603Tescapemechanismfrom7-deaza-2=-CMAasdeterminedusing
all-atom molecular dynamics simulation. All-atom stochastic software (24, 25) was
used to determine how the NS5 polymerase from the S603T TBEV mutant helps the
virustoescape2=-C-methylatednucleosidetriphosphates(NTPs).Thestartingposition
for each NTP (namely, ATP, 2=-C-methyladenosine, 2=-C-methylcytidine, and 7-deaza-
2=-CMA) was (cid:8)18 Å from the polymerase active site of the wild type and the S603T
TBEV mutant via the NTP tunnel. Measurements were obtained for the root mean
squaredeviation(RMSD)fromtheinitialwild-typepositionforthefollowingresponse
variables: NTP, Mn2(cid:5) ion cofactor, and water molecule. A few water molecules were
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 6
EscapeofTBEVfromAntivirals JournalofVirology
TABLE1Sensitivitiesofwild-typeandS603TmutantTBEVstrainsto2=-C-methylatedand
4=-C-azido-modifiednucleosides
EC ((cid:2)M)a(foldincreasecomparedto
50
wild-typevalue)
Compound Wildtype S603Tmutant
7-Deaza-2=-C-methyladenosine 1.07(cid:4)0.03 (cid:6)50((cid:6)46.7)
2=-C-Methyladenosine 1.69(cid:4)0.19 (cid:6)50((cid:6)29.5)
2=-C-Methylguanosine 1.98(cid:4)0.12 (cid:6)50((cid:6)25.3)
2=-C-Methylcytidine 2.24(cid:4)0.27 (cid:6)50((cid:6)22.3)
2=-C-Methyluridine 5.79(cid:4)0.15 (cid:6)50((cid:6)8.6)
4=-C-Azidocytidine 5.45(cid:4)0.03 5.36(cid:4)0.21(0.98)
RO-9187 0.99(cid:4)0.00 1.13(cid:4)0.21(1.14)
aDeterminedfromthreeindependentexperiments.EC swerecalculatedasconcentrationsgivinga50%
50
reductionoftheviraltiterbyusingtheReed-Muenchmethod.
D
o
selectedbasedonthefollowing2criteria(sinceover20watermoleculeswereincluded w
n
in the simulations): (i) the water molecule must be in close proximity to the NTP and lo
substrate binding residues, i.e., the catalytic, priming, and interrogating residues (26); a
d
and(ii)thewatermoleculemusthaveanRMSDof(cid:6)0.5Åfromitsinitialpositionfor e
d
allNTPsandstrains.
f
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Two-way analysis of variance (ANOVA) was performed (Table 2). The explanatory o
variables(NTPandstrain)werebothsignificant(P(cid:2)0.05)foreachresponsevariable, m

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TABLE2Descriptivestatisticsfortwo-wayANOVAwithRMSDdata /jv
Moleculeandsource Sumof Mean i.a
ofvariation df squares square Fvalue Pvalue (cid:3)2a s
m
NTPN1
.
NTP 3 4,033.08 1,344.36 203.54 (cid:2)0.0001 0.211 o
r
Strain 1 382.39 382.39 57.89 (cid:2)0.0001 0.020 g
NTP(cid:9)strain 3 1,112.68 370.89 56.15 (cid:2)0.0001 0.058 o/
Error 2,064 13,632.73 6.61 n

A
Watermolecule1 p
SNtTraPin 31 1181.31.128 61.1034.28 912,7.3332.35 (cid:2)(cid:2)00..00000011 00..036920b ril 2
NTP(cid:9)strain 3 23.96 7.99 122.15 (cid:2)0.0001 0.083 ,
2
Error 2,064 134.97 0
1
9
Watermolecule2
NTP 3 197.36 65.79 40.17 (cid:2)0.0001 0.039 by
Strain 1 1,512.26 1,512.26 923.40 (cid:2)0.0001 0.296b g
NTP(cid:9)strain 3 13.45 4.49 2.74 0.04 0.003 u
Error 2,064 3,380.22 1.64 e
s
t
Watermolecule3
NTP 3 349.81 116.60 67.72 (cid:2)0.0001 0.070
Strain 1 397.69 397.69 230.97 (cid:2)0.0001 0.080
NTP(cid:9)strain 3 679.31 226.44 131.51 (cid:2)0.0001 0.136
Error 2,064 3,553.90 1.72
Mn2(cid:5)ionA
NTP 3 385.21 128.40 171.78 (cid:2)0.0001 0.062
Strain 1 4,006.99 4,006.99 5,360.78 (cid:2)0.0001 0.649b
NTP(cid:9)strain 3 240.55 80.18 107.27 (cid:2)0.0001 0.038
Error 2,064 1,542.76 0.75
Mn2(cid:5)ionB
NTP 3 133.12 44.37 74.31 (cid:2)0.0001 0.062
Strain 1 128.06 128.06 214.44 (cid:2)0.0001 0.060
NTP(cid:9)strain 3 643.12 214.37 358.99 (cid:2)0.0001 0.301
Error 2,064 1,232.52 0.60
aCohen’seffectsizeguidelineisasfollows:small(cid:7)0.01,medium(cid:7)0.06,andlarge(cid:7)0.14.
bLargeeffectsize(forstrain)betweenwild-typeandS603TmutantTBEVNS5polymerases.
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Eyeretal. JournalofVirology
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FIG3TBEVNS5S603Tmutantmechanismofescapefrom7-deaza-2=-CMAasdeterminedbyall-atommoleculardynamicssimulation.Thebarchartsshow r
g
meanRMSDvalueswithstandarderrors(yaxes)foreachNTP(xaxes).Valuesforthetwowatermolecules(AandB)andtheMn2(cid:5)ion(C)withlargeeffect /

sizes(Table2)areshownforthewildtype(graybars)andtheS603TTBEVpolymerasemutant(blackbars).(D)TBEVpolymerasestructure,withthethumb, o
finger,andpalmdomainslabeled.TheBandFmotifsarehighlighted(cyan)andlabeledtoshowtheorientationoftheNTPtunnel(arrow).Theinsetshows n

theactivesiteturned90°,whichshowsthepositioningpatternsofthetwowatermoleculesandtheMn2(cid:5)ion;denseclustersareshownindarkercolors.The A
interrogatingR473residueofmotifF,theS603residueofmotifB,andtheactivesitecenterofmass(COM)areshown.Thearrowsindicatethedirectionof p
r
migration,withthelargestRMSDshownforthewildtype(WT)andtheTBEVS603Tmutant.Thelegendinthetopleftcorneroftheinsetindicatesthemeaning il
of the colors for wild-type TBEV and the S603T TBEV mutant for the two water molecules and the Mn2(cid:5) ion. 2=-CMA, 2=-C-methyladenosine; 2=-CMC, 2
,
2=-C-methylcytidine;7-DMA,7-deaza-2=-C-methyladenosine;TP,triphosphate.
2
0
1
9

b
sothemagnitudeoftheeffectsizewascalculatedasetasquared((cid:4)2)andwasguided y

byCohen’sruleofthumb(27)(Table2).Althoughsmalltolargeeffectsizeswerenoted g
u
for the explanatory variable NTP and for the interaction between NTP and strain, we e
s
restrictedourinterpretationtoresponsevariableswithlargeeffectsizescausedbythe t
strains.Figure3Ashowsthatonewatermoleculewasfartherfromitsinitialpositionin
the S603T TBEV mutant strain than in wild-type TBEV. In contrast, the second water
molecule(Fig.3B)showedahigherdeviationinthewild-typevirusthaninthemutant
strain.
TwometalionsareboundwithintheRdRpcatalyticsite,withoneion(A)chelating
with 3 catalytic aspartic residues and the other (B) chelating with 2 aspartic residues
(26).FlavivirusRdRpsarestructurallyhomologoustootherFlaviviridaeRdRps(28),and
their all-atom mechanism of escape from antiviral drugs is similar to that of the
drug-resistantRdRpmutantofhepatitisCvirus(29).Theconcertedpositioningofthe
2watermoleculesandtheMn2(cid:5)Aionduringthe7-deaza-2=-CMAexplorationofTBEV
polymeraseinbothstrainsisshowninFig.3D.Thefirstwatermoleculeisbetweenthe
S603residueandtheconservedarginine,whichisbothaninterrogatingandapriming
residue (26). The additional hydrophobic methyl group of threonine repels the water
towardthearginine,leadingtothehigherdeviationintheS603TTBEVmutant(Fig.3A).
ThesecondwatermoleculeisinproximitytotheopeningoftheNTPtunnelandnear
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 8
EscapeofTBEVfromAntivirals JournalofVirology
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FIG4TheS603TTBEVmutanthasanattenuatedphenotype.(A)(Top)TheplaquemorphologyoftheS603TTBEVmutantwasassayedinaculture
g
ofporcinekidneystable(PS)cellsandcomparedtothatofthewild-typevirus.(Bottom)Imagesoftheplaqueswerescanned,andtheplaque u
areas(B)andplaquediameters(C)ofthewild-typevirusandtheS603TTBEVmutantwerecompared.(D)ThegrowthkineticsoftheS603TTBEV e
s
mutantwascomparedwiththegrowthkineticsofthewildtypeandageneticrevertanttoassessthereplicationefficacyofthemutantinPScells. t
TheneuroinvasivenessoftheS603TTBEVmutantwasstudiedinBALB/cmiceandwascomparedtothatofthewild-typevirus.AdultBALB/cmice
wereinfectedsubcutaneouslywith103PFUofeithervirus,andsurvival(E)andtheclinicalscoresforneuroinfectionsymptoms(F)weremonitored
for28days.*,P(cid:2)0.05;**,P(cid:2)0.01;***,P(cid:2)0.001.
theMn2(cid:5)Aion.Thepositioningofthehydrophobicmethylgroupofthreonineinthe
mutantpreventsthesecondwatermoleculefrommigratingtowardthepositioningof
theMn2(cid:5)Aion,allowingittomaintainalowerRMSDthanthatforthewildtype(Fig.
3B).Thesecondwild-typeTBEVwatermoleculeformslargeclustersattheMn2(cid:5)Aion
position, which may cause greater ion displacement than that in the S603T TBEV
mutant(Fig.3C).
ThegrowthfitnessoftheS603TTBEVmutantisattenuatedincellculture.To
characterizethephenotypeofthedrug-resistantmutant,theplaquemorphologyand
growth properties of the S603T TBEV mutant were assayed in PS cell cultures and
compared to those of the wild-type virus (Fig. 4A). Wild-type virus produced plaques
thatrangedfrom269to1,333(cid:3)mindiameter(mean,730(cid:4)216(cid:3)m)andfrom0.148
November2017 Volume91 Issue21 e01028-17 jvi.asm.org 9
Eyeretal. JournalofVirology
to5.578mm2inarea(mean,1.814(cid:4)1.052mm2);theplaquesweremostlylarge,clear,
round,andregularinshape(Fig.4AtoC).Duringpassageoftheviruswithincreasing
concentrations of 7-deaza-2=-CMA, the wild-type-sized plaques gradually disappeared
and were increasingly replaced by small and turbid plaques that ranged from 160 to
1,170(cid:3)mindiameter(mean,540(cid:4)207(cid:3)m)andfrom0.08to4.3mm2inarea(mean,
1.051(cid:4)0.207mm2)(Fig.4AtoC).Thesmall-plaquephenotypebecamethedominant
phenotypeintheviralpopulationstartingatpassage5,andthiswasmaintainedduring
allsubsequentpassages.
The growth kinetics of the S603T TBEV mutant were compared to those of the
wild-typevirustoassessthereplicationefficacyofthemutantinPScells.Thewild-type
virusreachedapeaktiterof7.2(cid:9)105PFU/mlonday2p.i.Startingonday4p.i.,the
titerdeclinedslightlyandwas8.3(cid:9)104PFU/mlonday7p.i.(Fig.4D).Incontrast,the
replication capacity of the drug-resistant virus (from the final stock after the 50 (cid:3)M
selection)wassignificantlylowerthanthatofthewildtype.ThetitersoftheS603TTBEV D
mutantwereapproximately103-foldlowerforupto3daysp.i.Ondays4to6p.i.,the o
w
titersoftheS603TTBEVmutantwere10-to100-foldlowerthanthewild-typetiters;on n
day 7 p.i., the difference in titers between the mutant and wild-type viruses was less lo
a
pronounced,andbothhadtitersofabout104PFU/ml(Fig.4D). d
e
TheneuroinvasivenessoftheS603TTBEVmutantishighlyattenuatedinmice. d

TheneuroinvasivenessoftheS603TTBEVmutantwasstudiedandcomparedtothatof f
r
thewild-typevirusinBALB/cmice.AdultBALB/cmice(n(cid:7)10pergroup)wereinfected om
subcutaneously with 103 PFU of either virus, and clinical signs of neuroinfection and
h
survival were monitored for 28 days. Mice infected with the wild-type virus showed tt
p
severeclinicalsignsofinfection(ruffledfur,hunchedposture,andhindlegparalysis), :/
/
with90%mortalityandameansurvivaltimeof10(cid:4)1days(Fig.4E).Miceinfectedwith jv
the S603T TBEV mutant showed significantly lower mortality (20%) (P (cid:2) 0.001) and a i.a
s
significantlylongermeansurvivaltime(21(cid:4)2.8days)(P(cid:2)0.001)thanthoseofmice m
infectedwiththewild-typevirus(Fig.4E).Therewerealsoconsiderabledifferencesin .o
theonsetofdiseasesignsandclinicalscoringoutcomes(P(cid:2)0.0001)(Fig.4F). rg
/
BrainswerecollectedfrommicethatdidnotsurviveinfectionwiththeS603TTBEV o
n
mutant,viralRNAwasisolatedandamplifiedbyPCR,andtheNS5genewassequenced

A
toexcludepossiblereversionoftheS603Tmutationtothewild-typegenotypeduring p
replicationinthemouse.WefoundthatTBEVcarryingtheS603Tmutationwaspresent ril
inallofthemousebrainsweinvestigated,indicatingthatnoreversemutationoccurred 2
,

spontaneouslyduringtheinfection(Table3).However,afewadditionalmutationsthat 2
0
led to amino acid substitutions were detected in the NS5 gene of the drug-resistant 1
9
mutantfollowingpropagationinmice(Table3).
b
RapidreversionoftheS603Tmutanttothewild-typegenotypeinvitrointhe y

absence of 7-deaza-2=-CMA. The stability of the S603T mutant genotype was inves- g
u
tigated during serial passages in PS cells in the absence of 7-deaza-2=-CMA. After 5 e
s
passages, the S603T mutant genotype reverted to wild type. This correlated with a t
reemergence of the wild-type phenotype (i.e., large plaques and gross cytopathic
effectsinPScellculturesandgrowthkineticsinPScellsthatwerecomparabletothose
of the wild-type virus) (Fig. 4D). Moreover, the revertant was sensitive to all 2=-C-
methylatednucleosidestested(Fig.2E).Thesensitivityofthevirusto4=-C-azidocytidine
andRO-9187wasnotaffectedduringviruspassaging(Fig.2E).
Site-directed mutagenesis confirms the role of the S603T mutation in drug
resistanceandvirusattenuation.ToexaminethedirecteffectsoftheS603Tmutation
on TBEV characteristics, the mutation was introduced into a recombinant TBEV strain
(Oshima-IC) generated from an infectious cDNA clone and a TBEV replicon that ex-
presses a reporter luciferase gene (Oshima-REP-luc2A) (30, 31). In the absence of
7-deaza-2=-CMA, the introduced S603T mutation rapidly reverted to the parental
sequence (Ser) in the recovered recombinant virus from cells transfected with the
Oshima-ICS603TmRNA.TheS603Tmutantviruswasrecoveredonlyinthepresenceof
7-deaza-2=-CMA, and the virus titer was (cid:2)2 (cid:9) 103 PFU/ml, while the titer was (cid:6)106
PFU/mlincellstransfectedwithOshima-ICwild-typemRNA.Thefocussizeofthecells
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