Results

T-705 inhibits CHIKV RNAsynthesis.CHIKV-infected cells were not treated (NT) ortreated with T-705 for 1 to 6 hours asschematically depicted (A). At 5.5h p.i. cellulartranscription was inhibited with actinomycin D,followed by specific labeling of CHIKV RNAsynthesis with 3H-uridine for 1 hour starting at6h post infection. Uninfected control cells wereincluded to demonstrate the specificity of themetabolic labeling for viral RNA synthesis. B:Total amount of CHIKV genomic andsubgenomic RNA at 7h p.i. in cells that wereeither untreated (NT) or treated with T-705 forthe duration indicated above the lanes.Detection was done by hybridization with aprobe specific for CHIKV positive-strand RNA.18S ribosomal RNA (detected by hybridization)was used as loading control. C: Incorporationof 3H-uridine into CHIKV RNA D: Quantitationof the total amount of CHIKV RNA (circles) andCHIKV RNA synthesis activity (squares) at 7hp.i. in cells treated with T-705 for the durationindicated on the x-axis, and compared to thevalues of untreated infected control cells(100%).

Inhibition of Chikungunya virus replication by T-705 (favipiravir) and identification of resistance associated mutations in the RNA-

dependent RNA polymerase

Jochmans D1, Segura Guerrero N1,5, Delang L1, Pastorino B2, Querat G2, Dallmeier K1, Bello F5, Tas A4, Snijder E4, de Lamballerie X2, Martina B3, Froeyen M1, Neyts J1, van Hemert M4, Leyssen P1

1 University of Leuven, Belgium; 2 Université de la Méditerranée, Marseille, France; 3 Erasmus Medical Center, Rotterdam, The Netherlands; 4 Leiden University Medical Center, The

Netherlands; 5 Universidad del Rosario, Bogota, Colombia

IntroductionChikungunya virus (CHIKV) is a mosquito-borne,emerging human pathogen that causes adebilitating, often persistent arthralgia. T-705(favipiravir), a nucleotide prodrug, is indevelopment for the treatment of influenza and hasdemonstrated antiviral activity against differentRNA viruses (Furuta et al., Antiviral Res. (2009) 82,95–

102). Recently it was shown that inhibition ofinfluenza replication is due to lethal mutagenesis(Baranovich et al., J. Virol. (2013) 87, 3741-51).

Here, we describe the antiviral activity of T-705 onCHIKV replication.

Chemical structure of T-705 and its activemetabolite T-705 ribofuranosyl triphosphate

ConclusionWe provided for the first time unequivocal proofthat the nsP4 protein of CHIKV is involved in themechanism of action of T-705. Metabolic labelingexperiments confirmed a direct effect of T-705 onCHIKV RNA synthesis. The K291R mutation in thensP4 of CHIKV was demonstrated to beresponsible for an increase in resistance to theantiviral effect of the T-705. This position in theRdRp appears to be the primary interaction site forthe compound or more likely the 5’triphosphate ofthe nucleoside metabolite of T-705. This studyopens up the possibility for an in-depth study of theprecise molecular mechanism by which T-705 notonly inhibits CHIKV replication but many other RNAviruses that are susceptible to the antiviral effect ofT-705. To our knowledge this is the first report thatprovides direct evidence (following characterizationof drug-resistant virus) that the viral polymerase is,in the context of the intact cell, the molecular targetof the compound.

AcknowledgementsThis research was funded by the European Union FP7

Program under SILVER grant agreement n°260644.

T-705 inhibits CHIKV

replication in vitro.The antiviral effect of T-705 was determined inparallel with that of chloroquine in vitro. Vero cellswere infected with the indicated virus strains inthe presence of different concentrations ofcompound. After 48 h viral RNA in the SN wasquantified using RT-qPCR and EC50s werecalculated.

Virus Strain

EC50 (µM)

T-705 Chloroquine

899 (lab) 5.9 ± 3.3 13 ± 1.2

899 (lab) 4.7 ± 1.5 15 ± 2.7

LR2006-Opy1 (lab) 9.8 ± 0.1 ND

Venturini (Italy 2008) 12 ± 0.3 ND

Bianchi (Italy 2008) 6.1 ± 0.1 ND

Congo 95 (2011) 1.9 ± 1.1 ND

EC

50

(µM

)

Wil

d-t

yp

e

nsP

4_

K2

91

R

nsP

3_

Op

al5

24

W

nsP

3_

Op

al5

24

W_

nsP

4_

K2

91

R

nsP

2_

K4

9R

-E

62

2G

nsP

3_

Op

al5

24

W_

nsP

4_

K2

91

RT-705 89 ± 8 150 ± 5 63 ± 8 154 ± 46 337 ± 193

FC NA 1.7 0.7 1.7 3.8

Resistance phenotype of reverse-engineered CHIKV mutantsAverage and standard deviation were calculated fromthe data obtained from at least 2 independentexperiments;

FC = fold change of EC50

NA = not applicable

3D-model of the binding of T-705 to CHIKV

nsP4.Homology model of CHIKV nsP4 structure (fingers = blue, palm = green,thumb = red ribbons) superimposed onto the Norwalk virus polymerasestructure (grey ribbons). The primer RNA strand has yellow carbons andribbon, the template has brown carbons and ribbon. The favipiravirtriphosphate T-705RTP has purple carbons. The mutation K291R goingfrom a hydrogen bond interaction to a steric clash, is highlighted by acolor change from green to cyan carbons. CHIKV conserved residuesD371, D466, D467 (binding the 2 Mn++ ions that also stabilize thetriphosphate group; ionic bonds are shown as red dashes) and residuesN439, S430 and D376 important for the catalytic reaction have greencarbons. Hydrogen bonds from the sugar part of T-705RTP are shown asblack dashed lines.

T-705 inhibits CHIKV inducedmortality in vivo

Survival curve of (�) 6 mice infected with 100

TCID50 of CHIKV strain S27 and that receivedplacebo treatment with PBS (BID for 7 days)and (�) 6 mice similarly infected that received

the first oral dosing of a 7-day BID 300mg/kg/day treatment schedule with T-705starting 4h after infection.0

20

40

60

80

100

0 2 4 6 8 10 12 14

% S

urv

iva

l

Days post-infection

0.1

1.0

10.0

1 10 100

Log

10

in

fect

ivit

yra

tio

tre

ate

dv

su

ntr

ea

ted

con

tro

l

Log10 concentration (µM)

chloroquine T-705

The effect of T-705 is not due to lethal mutagenesisThe graph depicts the specific infectivity, calculated asthe ratio of infectious virus yield x 10-3 to the genomecopy number, of CHIKV 899 grown in the presence of theindicated compound. For both compounds, the calculatedspecific infectivity remains between 0.5-1.0 for allconcentrations tested. These results showed that thedecrease in infectivity upon treatment was proportional tothe decrease in viral RNA content.

P Nsp1 Nsp2 Nsp3 Nsp4

A

3 - K49R E622G F354S Opal524W K291R

10 D89R K49R N198R S405P

E622G Y543C

D31G E240G F354S

N420 S471P Opal524W

K291R

B

3 - K49R E622G F354S Opal524W K291R

10 D89R K49R S405P E622G

Y543C

D31G N420D F354S

S471P

K291R

C

3 - - - K291R

10 - N198R Y543C D31G E240G F354S

N420D S471P

K291R

T-705 selects for K291R in CHIKVNsp4-RDRPThree independent selections of T-705 resistant CHIKV899 were carried out. Virus selected at 130 (P3) and 160µM T-705 (P10) were genotyped. Mutations thatappeared in at least two independent selections areshown. Those appearing in three selections areunderlined.