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1 Novel Function of Bluetongue Virus NS3 Protein in Regulation of

2 the MAPK/ERK Signaling Pathway

3

4 Authors: Cindy Kundlacz1, Marie Pourcelot1, Aurore Fablet1, Rayane Amaral Da Silva

5 Moraes1, Thibaut Léger2, Bastien Morlet2, Cyril Viarouge1, Corinne Sailleau1, Mathilde

6 Turpaud1, Axel Gorlier1, Emmanuel Breard1, Sylvie Lecollinet1, Piet A. van Rijn3,4, Stephan

7 Zientara1, Damien Vitour1,Ψ#, Grégory Caignard1,Ψ#

8

9 Affiliations: UMR VIROLOGIE, INRA, École Nationale Vétérinaire d’Alfort, ANSES,

10 Université Paris-Est, Maisons-Alfort, 94700, France1. Mass spectrometry and proteomics

11 facility, Jacques Monod Institute, UMR 7592, Paris Diderot University, CNRS, Sorbonne Paris

12 Cité, F-75205, Paris Cedex 13, France2. Department of Virology, Wageningen Bioveterinary

13 Research, PO box 65, 8200 AB, Lelystad, The Netherlands3. Department of Biochemistry,

14 Centre for Human Metabolomics, North-West University, South Africa4. These authors

15 contributed equally to this workΨ.

16

17 # Corresponding authors:

18 Grégory Caignard, UMR VIROLOGIE, Maisons-Alfort, France

19 Email: [email protected]; Phone: 33-(1)-43-96-73-75

20 Damien Vitour, UMR VIROLOGIE, Maisons-Alfort, France

21 Email: [email protected]; Phone: 33-(1)-43-96-73-30

22

23 Running title: BTV-NS3 binds BRAF and activates the MAPK/ERK pathway.

24 Abstract: 242 words. Text: 5,651 words.

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25 Abstract

26

27 Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide

28 range of wild and domestic ruminants. In this report, we showed that BTV, through its virulence

29 non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway. In

30 response to growth factors, the MAPK/ERK pathway activates cell survival, differentiation,

31 proliferation and protein translation but can also lead to the production of several inflammatory

32 cytokines. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis

33 from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein

34 kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor

35 of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by

36 BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling

37 cascade. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the

38 MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be

39 specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK

40 pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its

41 own replication. Therefore, the activation of the MAPK/ERK pathway by BTV-NS3 could

42 benefit to BTV replication by promoting its own viral protein synthesis but could also explain

43 the deleterious inflammation associated with tissue damages as already observed in severe cases

44 of BT disease. Altogether, our data provide molecular mechanisms to explain the role of BTV-

45 NS3 as a virulence factor and determinant of pathogenesis.

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46 Importance

47

48 Bluetongue Virus (BTV) is responsible of the non-contagious arthropod-borne disease

49 Bluetongue (BT) transmitted to ruminants by blood-feeding midges. Despite the fact that BTV

50 has been extensively studied, we still have little understanding of the molecular determinants

51 of BTV virulence. In this report, we found that the virulence protein NS3 interacts with BRAF,

52 a key component of the MAPK/ERK pathway. In response to growth factors, this pathway

53 promotes cell survival, increases protein translation but also contributes to the production of

54 inflammatory cytokines. We showed that BTV-NS3 enhances the MAPK/ERK pathway and

55 this activation is BRAF-dependent. Our results demonstrate, at the molecular level, how a

56 single virulence factor has evolved to target a cellular function to ensure its viral replication.

57 On the other hand, our findings could also explain the deleterious inflammation associated with

58 tissue damages as already observed in severe cases of BT disease.

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59 Introduction

60

61 Bluetongue Virus (BTV) is the etiologic agent of the non-contagious arthropod-borne

62 disease Bluetongue (BT) transmitted to ruminants by blood-feeding midges of the genus

63 Culicoides. It belongs to the Orbivirus genus within the Reoviridae family, with 27 serotypes

64 currently identified (1) and at least 6 putative new serotypes (2–7). BTV infects a broad

65 spectrum of wild and domestic ruminants even if sheep are the most sensitive species to the

66 disease. During the 20th century BTV was principally circumscribed to tropical and subtropical

67 geographical areas (8). In 2006, BTV serotype 8 (BTV-8, strain 2006) emerged in Northern

68 Europe (9) from which it rapidly spread to Central and Western Europe, causing significant

69 economic losses (mortality, morbidity, reduced production and restrictions in trade of

70 ruminants). Despite the fact that a high vaccination coverage has been achieved across many

71 European countries allowing the control of the BT disease, BTV outbreaks are still a major

72 concern for the World Organisation for Animal Health (OIE), particularly in Europe (1).

73 Clinical signs include hemorrhagic fever, ulcer in the oral cavity and upper gastrointestinal

74 tract, necrosis of the skeletal and cardiac muscle and oedema of the lungs (10). These

75 variabilities in its host range and clinical manifestations are due to several factors related both

76 to the infected hosts and the viral serotypes and strains.

77 The BTV genome is composed of 10 double-stranded RNA (dsRNA) segments

78 encoding seven structural (VP1 to VP7) and five, or possibly six, non-structural (NS1 to NS4,

79 NS3A and possibly NS5) proteins (11–13). The BT virion is an icosahedral particle organized

80 as a triple-layered capsid. Viral genomic segments are associated with replication complexes

81 containing VP1 (RNA-dependent RNA polymerase), VP4 (capping enzyme including

82 methyltransferase), VP6 (RNA-dependent ATPase and helicase) and enclosed by VP3

83 (subcore) and VP7 (core) (14). Cell attachment and viral entry involve the two structural

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84 proteins of the outer capsid VP5 and the most variable of BTV protein VP2 representing the

85 main target of neutralizing antibodies and determines the serotype specificity (15, 16). Non-

86 structural proteins contribute to the control of BTV replication (17), viral protein synthesis (18),

87 maturation and export from the infected cells (19–23). Initially described for NS4, NS3 has also

88 been shown to counteract the innate immune response, and in particular the type I interferon

89 (IFN-α/β) pathway (13, 24, 25).

90 NS3 is encoded by the segment 10 and expressed as two isoforms, NS3 and NS3A, the

91 latter being translated from an second in-frame start codon by which the first N-terminal 13

92 amino acids residues are lacking (26). NS3 proteins are glycoproteins that promote viral release

93 either through its viroporin activity (20) or by budding. This latter implies interactions between

94 NS3 and outer capsid VP2/VP5 proteins (27), and cellular proteins involved in the pathway of

95 endosomal sorting complexes required for transport (ESCRT) (TSG101 and NEDD4-like

96 ubiquitin ligase) and the calpactin light chain p11 (23, 28, 29). Altogether, these reports provide

97 molecular basis of the multifunctional role of BTV-NS3 as a virulence factor and determinant

98 of pathogenesis as also illustrated by other in vivo studies using BTV monoreassortants and

99 NS3/NS3A knockout mutants (30–32).

100 Many viruses can modulate and hijack signaling pathways related to the mitogen-

101 activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway for more

102 efficient replication (33). In response to extracellular stimuli such as growth factors, several

103 downstream components of the MAPK/ERK pathway including RAS, RAF, MEK1/2 and

104 ERK1/2 are successively activated. Then, ERK1/2 directly or indirectly regulates transcription

105 factors (e.g. Elk1) involved in cell proliferation, differentiation and survival (34, 35) but also

106 cellular factors that control mRNA translation like eukaryotic initiation factor 4E (eIF4E) (36).

107 In 2010, Mortola and colleagues were the first to show the modulation, as activation, of the

108 MAPK/ERK pathway by BTV (37). In contrast to this finding, other studies demonstrated that

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109 the phosphorylation of ERK1/2 was reduced (38) or unchanged (39) after BTV infection. The

110 discrepancy of these studies may be due to different virus infection kinetics or differences in

111 viral serotype/strain and/or cell line used. In addition, the molecular mechanisms underlying

112 the potential modulation of this pathway and its possible contribution to the BTV pathogenesis

113 remain to be established. Altogether, these data led us to investigate the functional impact of

114 BTV on the MAPK/ERK signaling pathway.

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115 Results

116

117 BTV-NS3 activates the MAPK/ERK signaling pathway

118 To address the question of BTV modulation with the MAPK/ERK pathway, we used a

119 trans-reporter gene assay that measures Elk1 activation by ERK1/2. In this system, Elk1

120 transcription factor is fused to the DNA binding domain of Gal4 (Gal4-DB) and leads to the

121 expression of the firefly luciferase reporter gene downstream of a promoter sequence containing

122 a Gal4 binding site. Upon stimulation with a growth factor like EGF, Elk1 was activated as

123 assessed by a 5-fold increase of luciferase activity compared to unstimulated HEK-293T cells

124 (Figure 1A; mock control). Cells infected with BTV at different MOIs showed a very strong

125 enhancement in this cellular pathway, even in absence of EGF stimulation (Figure 1A), and in

126 a MOI-dependent manner. Moreover, the inability of a UV-inactivated BTV to activate the

127 luciferase reporter gene indicated that the induction of MAPK/ERK was dependent on viral

128 replication or de novo viral protein expression. To determine whether viral protein(s) could be

129 involved in the MAPK/ERK activation, we tested separately all the BTV proteins in our reporter

130 assay. As shown in Figure 1B, only BTV-NS3 is able to strongly activate the MAPK/ERK

131 pathway both in presence or absence of EGF stimulation. Thus, the activation of the

132 MAPK/ERK pathway by BTV notably involves its NS3 viral protein.

133

134 BTV-NS3 interacts with BRAF

135 As a first approach to understand at molecular level how BTV-NS3 activates the

136 MAPK/ERK pathway, we undertook proteomic analyses after immunoprecipitation of NS3

137 from either BTV-infected or NS3-transfected HEK-293T cells. The whole purification protocol

138 and LC-MS/MS analyses are presented in the Materials and Methods section. These analyses

139 revealed that BTV-NS3 copurified with BRAF in both BTV-infected and NS3-transfected cells

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140 (with Mascot scores of 64 and 104, respectively) whereas BRAF was not detected in the control

141 conditions (mock infected- or empty pCI-neo-3xFLAG transfected-cells). The identified

142 peptides corresponding to BRAF are listed in Table 1.

143 BRAF, together with ARAF and CRAF, are members of the RAF kinase family that

144 play a central role in regulating the MAPK/ERK signaling pathway. Therefore, binding to

145 BRAF represents a potential molecular mechanism underlying this manipulation that we

146 decided to investigate. To validate this interaction, full-length BTV-NS3 (NS3FL) and different

147 fragments of NS3 (Figure 2A) were tested for their ability to interact with endogenous BRAF.

148 To do so, GST-tagged NS3FL or indicated fragments were expressed in HEK-293T cells and

149 purified 48 h later with glutathione-sepharose beads. As shown in Figure 2B, endogenous

150 BRAF copurified only with NS3FL.

151 Using the same luciferase assay, we showed that only full-length BTV-NS3 is able to

152 enhance Elk1 activation (Figure 2C). In contrast, the indicated fragments of NS3 were unable

153 to do so, consistently with the previous pull-down assays (Figure 2B). Altogether, these results

154 demonstrate that only the full-length BTV-NS3 interacts with BRAF and activates the

155 MAPK/ERK signaling pathway.

156

157 Phosphorylation of ERK1/2 and eIF4E is stimulated by BTV infection and in cells

158 expressing BTV-NS3

159 To further decipher the impact of BTV-NS3 on the MAPK/ERK signaling pathway, we

160 compared the phosphorylation kinetics of ERK1/2 and eIF4E in HEK-293T cells infected by

161 BTV (Figure 3A) and in cells expressing BTV-NS3 (Figure 3B). HEK-293T cells were infected

162 with BTV (MOI=0.01) and 24 h later, cells were serum-starved for 12 h before being stimulated

163 with EGF. Phosphorylation levels of ERK1/2, determined at 10, 30, 120 min, 6 h and 24 h after

164 stimulation, were markedly and reproducibly higher after BTV infection compared to mock

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165 control (Figure 3A). Interestingly, we observed that BTV also induced ERK1/2

166 phosphorylation even in absence of EGF stimulation. This is reminiscent to what was observed

167 for Elk1 activation (Figure 1A) showing a significant activation of the MAPK/ERK pathway

168 by BTV. In parallel, we also determined the phosphorylation level of the translation initiation

169 factor eIF4E, a downstream target of this pathway that is involved in the control of mRNA

170 translation. In contrast to ERK1/2, the phosphorylation level of eIF4E in BTV infected-cells

171 was increased only at later time points after EGF stimulation whereas p-eIF4E decreased in

172 mock condition (Figure 3A).

173 In parallel to the BTV infectious context, cells were transfected with 3xFLAG-tagged

174 BTV-NS3 or a control plasmid. After 24 h, cells were serum-starved and the phosphorylation

175 levels of ERK1/2 and eIF4E were measured at the same time points post-EGF stimulation as

176 before. Like BTV infection, similar phosphorylation kinetics of ERK1/2 and eIF4E were

177 observed for BTV-NS3 expression alone (Figure 3B). In conclusion, these phosphorylation

178 kinetics confirm that either BTV infection or transient expression of BTV-NS3 can both

179 activate the MAPK/ERK pathway.

180

181 BRAF intracellular localization is modified by BTV

182 Although HEK-293T are highly efficient for transfection and support BTV replication,

183 we aimed to complement our analysis by carrying out similar experiments using a cell line

184 derived from a host naturally infected by BTV. To do so, we measured phosphorylation levels

185 of ERK1/2 in a bovine kidney cell line (MDBK). MDBK cells were serum-starved and infected

186 with BTV at different MOIs for 24 h. As shown in Figure 4A, BTV increased phosphorylation

187 levels of ERK1/2 in a MOI-dependent manner, which is consistent to what was observed for

188 Elk1 activation in HEK-293T cells infected with BTV (Figure 1A).

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189 To determine the potential consequences of NS3-BRAF interaction on their own

190 subcellular localizations, we carried out fluorescence microscopy in MDBK cells. Cells were

191 serum-starved and infected with BTV (MOI=0.01). Then, BTV-NS3 and BRAF localizations

192 were analyzed at 24 h post-infection by fluorescence microscopy (Figure 4B). Firstly, in mock-

193 infected cells, BRAF is present throughout the cytosol with a sparse punctate distribution. As

194 expected, in cells infected by BTV, NS3 is localized in specific cytoplasmic structures

195 evocative of the Golgi apparatus but also at the plasma membrane. Interestingly, we also found

196 that the subcellular distribution of BTV-NS3 matched the relocalization of BRAF in BTV-

197 infected cells. These results demonstrated that BTV-NS3 alters the localization of BRAF, which

198 may contribute to the BTV-activated MAPK/ERK pathway.

199

200 U0126 inhibitor blocks the activation of MAPK/ERK by BTV and alters viral

201 replication

202 To demonstrate that enhancement of Elk1 activation by BTV-NS3 is completely

203 dependent on ERK1/2 activation, HEK-293T cells were transfected with 3xFLAG-tagged

204 BTV-NS3 or a control plasmid, and 24 h later treated with MEK1/2 inhibitor U0126 (Figure

205 5A). The U0126 molecule targets MEK1/2 that are directly activated by BRAF proteins (40).

206 As shown in Figure 5A, NS3-induced Elk1 activation was completely inhibited by U0126. To

207 confirm these results, we measured the phosphorylation levels of ERK1/2 and eIF4E in HEK-

208 293T cells treated with U0126 before infection with BTV. As observed for Elk1 activation,

209 U0126 efficiently blocked the phosphorylation of both ERK1/2 and eIF4E after BTV infection

210 (Figure 5B). Interestingly, the presence of U0126 also prevented the expression of BTV-NS3.

211 To test if this inhibitor could have an antiviral effect on BTV replication, HEK-293T cells were

212 treated with MEK1/2 inhibitor U0126 and infected with BTV (MOI=0.01). As shown in Figure

213 5C, HEK-293T cells treated with U0126 exhibited significant lower viral titers compared to the

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214 DMSO control. Altogether, these results suggest that BTV manipulates the MAPK/ERK

215 signaling pathway to increase replication efficiency.

216

217 BRAF silencing impairs BTV-activated MAPK/ERK pathway

218 To further confirm the experiments with the MAPK/ERK pharmacologic inhibitor

219 U0126, we used a gene silencing approach targeting BRAF. HEK-293T cells were transfected

220 with BRAF-specific or control non-specific siRNA before infection with BTV (Figure 6A and

221 6C) or transfection with 3xFLAG-tagged BTV-NS3 (Figure 6B and 6D). In both cases, the

222 reduction of BRAF expression led to a significant decrease of the MAPK/ERK activation as

223 assessed by the luciferase reporter gene assays and anti-pERK/1/2 immunoblotting. These

224 results support a model where BTV-NS3 interaction with BRAF enhances MAPK/ERK

225 pathway activation.

226

227 NS3 interaction with BRAF and activation of the MAPK/ERK signaling pathway

228 are characteristic of BTV

229 To address the question of the specificity of the BRAF interaction, we first compared

230 NS3 proteins from three serotypes (BTV 1, 8 and 27) in their ability to interact with endogenous

231 BRAF. GST-tagged NS3 from BTV1, 8 and 27 were expressed in HEK-293T cells and purified

232 48 h later with glutathione-sepharose beads. As shown in Figure 7A, NS3 proteins from BTV1,

233 8 and 27 have similar binding capacities for BRAF. As a consequence, BTV1-NS3 and BTV27-

234 NS3 were also able to enhance the MAPK/ERK pathway, although the activation by BTV8-

235 NS3 was stronger compared to NS3 proteins from BTV1 and 27 (Figure 7B). Then, NS3

236 proteins from other members of Orbivirus genus, such as epizootic hemorrhagic disease virus

237 (EHDV), African horse sickness virus (AHSV) and equine encephalosis virus (EEV) were also

238 tested for binding to endogenous BRAF. Only BTV-NS3 was able to co-purify with BRAF,

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239 demonstrating the specificity of this interaction (Figure 7C). Moreover, only BTV-NS3 highly

240 activates MAPK/ERK pathway, in contrast to EHDV-NS3, AHSV-NS3 and EEV-NS3 (Figure

241 7D). Thus, the interaction with BRAF and activation of the MAPK/ERK pathway are unique

242 to BTV-NS3.

243

244 BTV-NS3 inhibits the induction of IFN-α/β independently of MAPK/ERK

245 signaling

246 Our team has demonstrated the major role of BTV-NS3 in counteracting the induction

247 of the type I interferon (IFN-α/β) response (24). Interestingly, it has been reported that the

248 activation of the MAPK/ERK pathway could be associated with the inhibition of IFN-α/β

249 synthesis (41). Therefore, we asked if the activation of the MAPK/ERK pathway by BTV-NS3

250 is required for an efficient control of the IFN-α/β response. Using a luciferase gene reporter

251 assay, we tested NS3FL and its fragments for their capacity to inhibit an IFN-β specific promoter

252 downstream a stimulation with a constitutively active form of RIG-I (NΔRIG-I for N-terminal

253 CARDs of RIG-I). As shown in Figure 8A, only NS3FL fully blocked the IFN-β promoter

254 activity. Moreover, while NS3118-229 and NS3Δ118-182 fragments were not able to activate Elk1

255 as previously shown in Figure 2C, both are partially, but significantly, able to block the IFN-β

256 promoter activity (Figure 8A). As a complementary approach, we used the MEK1/2 inhibitor

257 U1026 and measure its impact on the capacity of BTV-NS3 to inhibit the IFN-β promoter

258 activity. As shown in Figure 8B, the U0126 molecule was unable to prevent the antagonist

259 function of BTV-NS3 on the induction of IFN-α/β and thus to rescue a significant activation of

260 the IFN-β promoter. In conclusion, our data demonstrate that the activation of MAPK/ERK by

261 BTV-NS3 does not contribute its antagonist activity on the IFN-α/β synthesis.

262

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263 Discussion

264

265 As obligate intracellular parasites, viruses have evolved multiples strategies to hijack

266 their host cellular machineries and control them to survive, replicate and spread. The

267 MAPK/ERK pathway contains one of the most highly conserved family of serine/threonine

268 kinases from yeast to humans, which regulates a multiplicity of cellular processes including

269 cell survival, proliferation and differentiation, as well as immune and inflammatory responses.

270 Therefore, many viruses have been shown to modulate this pathway for their own benefit (33).

271 Considering that its aberrant activation represents an important step toward carcinogenesis, the

272 modulation of the MAPK/ERK pathway has been initially described for DNA tumor viruses

273 and oncogenic retroviruses. However, non-oncogenic RNA viruses are also able to activate this

274 signaling cascade even if the molecular mechanisms underlying this manipulation, in particular

275 in term of protein-protein interactions, often remain a pending question.

276 In this report, we show that BTV activates the MAPK/ERK pathway as assessed by

277 Elk1 transactivation and phosphorylation levels of ERK1/2 and eIF4E, which is reminiscent to

278 findings of Mortola and colleagues (37), but we also give the first example of a BTV protein

279 that contributes to this positive regulation. We further demonstrate that both BTV infection and

280 NS3 expression alone also activate the MAPK/ERK pathway in the absence of external stimuli.

281 Moreover, our data provide molecular basis to this activity through the identification of BRAF

282 as a new interactor of BTV-NS3 together with the fact that BRAF silencing impairs BTV-

283 activated MAPK/ERK signaling. Intriguingly, two other studies have shown that BTV does not

284 activate the MAPK/ERK pathway (38, 39). However, their findings are not necessarily in

285 contradiction with our current data. This apparent discrepancy does not appear to depend on

286 factors related to the virus used as we have demonstrated that NS3 proteins from at least three

287 serotypes of BTV have similar abilities to bind BRAF and activate the MAPK/ERK pathway.

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288 In addition, we have confirmed this activation in both human and bovine cells. One possible

289 explanation could be that, in contrast to these previous studies, all of our experiments were

290 performed under starvation conditions, which is likely to be essential since the MAPK/ERK

291 pathway is activated in response to growth factors.

292 In contrast to BTV, NS3 proteins from EHDV, AHSV and EEV are unable to activate

293 the MAPK/ERK pathway suggesting that BTV-NS3 is likely to be functionally distinct from

294 other Orbiviruses NS3 proteins. Although BTV-NS3 shares several domains with EHDV-NS3,

295 AHSV-NS3 and EEV-NS3 (e.g. the amphipathic helix at the N terminus, the late-domain

296 motifs, the extracellular and the two transmembrane domains) (23, 42), these proteins are

297 genetically different. Indeed, the NS3 proteins of AHSV and EEV only share »30% of sequence

298 homology with BTV-NS3 whereas the protein sequence is more conserved between BTV-NS3

299 and EHDV-NS3 (57% of sequence homology), which is consistent with the fact that both BTV

300 and EHDV are transmitted to ruminants. Although we have currently no explanation for this

301 apparent specificity of BTV, these differences in protein sequence could account for their

302 capacity or incapacity to activate the MAPK/ERK pathway. Nevertheless, further investigations

303 will be needed to understand how the MAPK activation could provide an advantage for BTV

304 at molecular/cellular level in comparison to other orbiviruses.

305 Considering that ERK1/2 regulate more than 160 downstream target factors (43),

306 activation of the MAPK/ERK pathway by BTV could have several consequences on host cell

307 biology. It triggers both NF-kB, c-Jun and STAT1 transcription factors (44, 45) leading to an

308 increased expression of inflammatory factors, in particular cytokines and chemokines that

309 participate to immunity and inflammation such as IL-6 and IL-8 (46–48). Although expression

310 of cytokines and chemokines are critical for developing an efficient antiviral response, their

311 excessive production could also lead to deleterious inflammation by causing tissue damages

312 and, therefore, contributing to disease pathogenesis. Indeed, both in vivo and in vitro studies

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313 have shown that BTV-infected cells secrete numerous pro-inflammatory cytokines, including

314 IL-6 and IL-8 (49–55). A consequence of such production could be an aggravation of the

315 endothelial injury associated with an increased vascular permeability as already observed in

316 severe cases of BT disease (10, 56). Altogether, our data suggest that BTV-NS3 interaction

317 with BRAF enhances MAPK/ERK activation above normal level in infected cells, possibly

318 contributing to a deregulation of the blood vessels permeability to promote BTV replication

319 and spreading.

320 Besides its effects on immunity and inflammation, activation of the MAPK/ERK

321 pathway has considerable consequences on viral replication as assessed by experiments using

322 MEK1/2 inhibitor U0126 inhibitors. Indeed, it is now well documented that MAPK/ERK

323 pathway inhibition with U0126 highly alters the replication of several viruses such as Influenza

324 virus, Junin virus, Herpes simplex virus 1, Astrovirus, Borna disease virus, Coronavirus, human

325 parainfluenza virus type 3 and Porcine epidemic diarrhea virus (57–64). Similarly, we show in

326 this report that MAPK/ERK pathway inhibition by U0126 prevents BTV protein expression in

327 infected cells resulting in reduced viral titer. It should also be noted that the inhibition of the

328 MAPK/ERK pathway, as assessed by Elk1 transactivation and phosphorylation levels of

329 ERK1/2, was more efficient in cells treated with U0126 than those transfected with the BRAF

330 siRNA (Figure 5A compared to 6B and Figure 5B compared to 6C). These differences could

331 be due to the fact that BRAF expression was not fully blocked following gene silencing (Figures

332 6C and 6D) and this would explain why NS3 expression, as well as viral titers (data not shown),

333 are not affected in cells transfected with the BRAF siRNA. Our findings also correlate with

334 data obtained with other members of Reoviridae family. Indeed, activation of EGF signaling

335 pathway has been correlated with increased reovirus replication and spread through the

336 regulation of multiple steps of the infectious life cycle including viral uncoating and

337 disassembly, viral protein translation and generation of viral progeny (for review, see (65)).

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338 Rhesus and group A rotaviruses also activate MAPK/ERK pathway to facilitate viral replication

339 and viral uncoating, respectively (66, 67). A possible link between MAPK/ERK pathway and

340 BTV protein expression could be related to the downstream targets of this pathway which are

341 essential factors of the cellular translational machinery, like eIF4E.

342 eIF4E is believed to be the least abundant of all initiation factors. Therefore, it can be

343 considered as an excellent target to regulate protein synthesis. Phosphorylation of eIF4E is

344 mediated by MAPK interacting kinase 1 (MNK1), itself mainly activated by ERK1/2 (36).

345 However, MNK1 has also other downstream targets including eukaryotic initiation factor 4G

346 (eIF4G). Once activated, eIF4E interacts with the cap structure and brings translation initiation

347 factors together with the small ribosomal subunit via the scaffold protein eIF4G to initiate cap-

348 dependent mRNA translation (68). Since BTV mRNA are capped like their host counterparts

349 (69, 70), it may be assumed that BTV increases phosphorylation level of eIF4E, thereby

350 stimulating cap-dependent translation, to promote its own viral protein synthesis within infected

351 cells.

352 Our findings support a model where BTV-NS3 interacts with BRAF to activate

353 MAPK/ERK pathway. To the best of our knowledge, this is the first report demonstrating both

354 BRAF as a target of a viral protein and this interaction as an important step toward a viral

355 manipulation of the MAPK/ERK pathway. We also demonstrate that BTV infection leads to a

356 re-localization of BRAF either at the cell membrane or the Golgi apparatus. Initially described

357 to be mainly regulated at the plasma membrane (71, 72), it has now been clearly established

358 that the control of MAPK/ERK pathway can also occur in the Golgi apparatus (73). Thus, these

359 specific localizations could contribute to the aggregation of NS3-BRAF complexes to enhance

360 MAPK/ERK signaling. Further analyses with confocal microscopy will be needed to confirm

361 the colocalization between NS3 and BRAF and additional biochemical investigations are still

362 required to decipher how this viral protein activates BRAF and the downstream events of this

16 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

363 pathway. Altogether, NS3 interactions with BRAF represents a potential target for the

364 development of antiviral molecules against BTV.

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365 Materials and Methods

366

367 Cell lines and viral infections

368 HEK-293T and MDBK cells were maintained in Dulbecco's modified Eagle's medium

369 (DMEM; Gibco-Invitrogen) containing 10% fetal bovine serum, penicillin, and streptomycin

370 at 37°C and 5% CO2. BTV8 WT strain was amplified and titrated on BSR-T7. Inactivated virus

371 was prepared by exposing live virus to 254-nm UV as previously described (74). Serum-free

372 medium was used as an inoculum for BTV-infected cells. BTV infection was analyzed at the

373 indicated time points using a specific NS3 antibody (kindly provided by Dr. Frederick Arnaud)

374 (75) and visualized by fluorescence microscopy or western blotting.

375

376 Plasmid DNA constructs

377 ORFs-encoding sequences from BTV8 WT strain (isolated in the French Ardennes in

378 2006 (76)), BTV1 WT strain (isolated in France in 2008 (77)), BTV27 WT strain (isolated in

379 Corsica in 2014 (78)), EHDV6 WT strain (isolated in Reunion Island in 2009 (79)), AHSV4

380 WT strain (isolated in Morocco in 1990 (80)) and EEV3 WT strain (isolated in South Africa in

381 1974 (81)) were amplified by RT-PCR (Roche) from purified infected-cell RNAs.

382 Amplification was performed using ORF specific primers flanked with the following Gateway

383 cloning sites: 5′-ggggacaactttgtacaaaaaagttggc and 5′-ggggacaactttgtacaagaaagttgg. PCR

384 products were cloned by in vitro recombination into pDONR207 (Gateway system; Invitrogen).

385 ORF coding sequences were subsequently transferred by in vitro recombination from

386 pDONR207 into different Gateway-compatible destination vectors (see below) following

387 manufacturer's recommendation (LR cloning reaction, Invitrogen). In mammalian cells, GST-

388 tag and 3xFLAG-tag fusions were achieved using pDEST27 (Invitrogen) and pCI-neo-3xFLAG

389 vector, respectively (82). An expression vector pNRIG-I carrying genes for the constitutively

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390 active N-terminal CARDs of RIG-I (NRIG-I) has been used to stimulate the luciferase reporter

391 gene downstream of an IFN-β specific promoter sequence as previously described (84).

392

393 Luciferase reporter gene assay

394 HEK-293T were plated in 24-well plates (5×105 cells per well). One day later, cells

395 were transfected with either pFA2-Elk1 (0.3 µg/well; Stratagene) and pGal4-UAS-Luc

396 plasmids (0.3 µg/well; provided by Dr. Yves Jacob) or IFN-β-pGL3 (0.3 µg/well; Stratagene)

397 together with pRL-CMV reference plasmid (0.03 µg/well; Promega). Cells were

398 simultaneously co-transfected with 0.3 µg/well of the empty pCI-neo-3xFLAG expression

399 vector or encoding viral proteins as specified. 12 h after transfection, cells were serum-starved

400 for 6 h then stimulated with EGF (Sigma) at 400 ng/ml. When specified, at the time of EGF

401 stimulation, cells were infected by BTV with the indicated MOI. After 24 h, cells were lysed,

402 and both firefly and Renilla luciferase activities in the lysate were determined using the Bright-

403 Glo and Renilla-Glo Luciferase Assay System (Promega), respectively. Reporter activity was

404 calculated as the ratio of firefly luciferase activity to reference Renilla luciferase activity. All

405 graphs represent the mean, and include error bars of the standard deviation.

406

407 Statistical analyses

408 p-values are a result of unpaired two-tailed Student’s T test. Differences were

409 considered to be significant if P value <0.05 (*) or <0.005(**) or <0.0005(***).

410

411 Co-affinity purification experiments

412 To perform co-affinity purification experiments coupled to mass spectrometry analyses,

413 HEK-293T cells were either infected with BTV8 WT strain or transfected with pCI-neo-

414 3xFLAG expression vectors encoding for 3xFLAG alone or fused to BTV-NS3FL. Briefly,

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415 2×106 HEK-293T cells were dispensed in each well of a 6-well plate (3 wells per condition),

416 and 24 h later, infected (MOI=0.1) or transfected with 1 µg of each plasmid DNA (JetPRIME;

417 Polyplus). 18 h post-infection or 24 h post-transfection, cells were washed in PBS, then

418 resuspended in lysis buffer (20 mm MOPS-KOH pH7.4, 120 mm of KCl, 0.5% Igepal, 2 mm

419 β-Mercaptoethanol), supplemented with Complete Protease Inhibitor Cocktail (Roche). Cell

420 lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10

421 min. Protein extracts were incubated for 3 h on a spinning wheel at 4°C with 40 µl of Protein

422 G Sepharose beads (Roche) and 2.5 µg of the specific BTV-NS3 antibody. Beads were then

423 washed with ice-cold lysis buffer 3 times for 5 minutes on a spinning wheel.

424 For other co-affinity purification experiments, ORFs encoding NS3 or fragments were

425 transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST

426 fusion. HEK-293T cells were transfected with 300 ng of each plasmid DNA per well. Two days

427 post-transfection, cells were collected in PBS and then incubated on ice in lysis buffer for 20

428 min and clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, protein

429 extracts were incubated for 2 h at 4°C with 30 µl of glutathione-sepharose beads (Amersham

430 Biosciences) to purify GST-tagged proteins. Beads were then washed with ice-cold lysis buffer

431 3 times for 5 minutes and proteins were recovered by boiling in denaturing loading buffer

432 (Invitrogen).

433

434 LC-MS/MS analyses

435 Co-immunoprecipitation beads from two independent biological replicates were eluted

436 in Laemmli buffer and run on a 4-12 % acrylamide gel (Invitrogen) and proteins were stained

437 with Coomassie blue (Biorad). The experiment was reproduced one time to obtain two

438 independent biological replicates of the total experiment. For both experiments, three gel plugs

439 were cut for each condition. Plugs were reduced with 10 mM DTT, alkylated with 55 mM

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440 iodoacetamide (IAA) and incubated with 20 µl of 25 mM NH4HCO3 containing 12.5 µg/ml

441 sequencing-grade trypsin (Promega, France) overnight at 37°C. The resulting peptides were

442 sequentially extracted from the gel with 30 % acetonitrile, 0.1 % formic acid and 70 %

443 acetonitrile. Digests were pooled (three per condition) according to the different experimental

444 conditions. Peptides mixtures were analyzed by a Q-Exactive Plus coupled to a Nano-LC

445 Proxeon 1000 (both from Thermo Scientific). Peptides were separated by chromatography with

446 the following parameters: Acclaim PepMap100 C18 pre-column (2 cm, 75 µm i.d., 3 µm, 100

447 Å), Pepmap-RSLC Proxeon C18 column (50 cm, 75 µm i.d., 2 µm, 100 Å), 300 nl/min flow

448 rate, a 98 min gradient from 95 % solvent A (water, 0.1 % formic acid) to 35 % solvent B (100

449 % acetonitrile, 0.1% formic acid). Peptides were analyzed in the Orbitrap cell, at a resolution

450 of 70,000, with a mass range of m/z 375-1500. Fragments were obtained by higher-energy

451 collisional dissociation (HCD) activation with a collisional energy of 28 %. MS/MS data were

452 acquired in the Orbitrap cell in a Top20 mode, at a resolution of 17,500. For the identification

453 step, all MS and MS/MS data were processed with the Proteome Discoverer software (Thermo

454 Scientific, version 2.2) and with the Mascot search engine (Matrix Science, version 5.1). The

455 mass tolerance was set to 6 ppm for precursor ions and 0.02 Da for fragments. The following

456 modifications were allowed: oxidation (M), phosphorylation (ST), acetylation (N-term of

457 protein), carbamidomethylation, (C). The SwissProt database (02/17) with the Homo sapiens

458 taxonomy and a database including all the viral proteins encoded by BTV were used in parallel.

459 Peptide identifications were validated using a 1% FDR (False Discovery Rate) threshold

460 calculated with the Percolator algorithm. Proteins with at least 2 unique peptides were

461 considered. Identified proteins were considered as potential partners of BTV-NS3 if no

462 identifications were reported in the control condition (mock infected- or empty pCI-neo-

463 3xFLAG transfected-cells).

464

21 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

465 Western blot analysis

466 Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel

467 electrophoresis (SDS-PAGE) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer

468 and transferred to a nitrocellulose membrane (Invitrogen). Proteins were detected using

469 standard immunoblotting techniques. 3×FLAG- and GST-tagged proteins were detected with a

470 mouse monoclonal HRP-conjugated anti-3×FLAG antibody (M2; Sigma-Aldrich) and a rabbit

471 polyclonal anti-GST antibody (Sigma-Aldrich), respectively. Specific antibodies (all from Cell

472 Signaling) were used to detect endogenous BRAF (clone D9T6S), phospho-ERK1/2 (clone-

473 E10), ERK1/2, phospho-eIF4E (Ser209), eIF4E. Secondary anti-mouse and anti-rabbit HRP-

474 conjugated antibodies were purchased from Invitrogen. Densitometric analysis of the gels was

475 performed using the ImageJ program.

476

477 Immunofluorescence assays

478 24-well plates (ibidi µ-plates, BioValley) were seeded with MDBK cells (1×105 cells

479 per well). One day later, cells were serum-starved and then infected with BTV (MOI=0.01). 18

480 h post-infection, cells were washed 3 times with PBS and then incubated with a 4% PFA

481 solution (Electron microscopy sciences) for 30 min at room temperature (RT), and then treated

482 with PBS-Glycine (0.1 M) and PBS-Triton (0.5%) for 5 min/each at RT to quench and

483 permeabilize the cells, respectively. Cells were washed 3 times with PBS and incubate for 1 h

484 with a PBS-BSA 1% blocking solution. Finally, cells were incubated with specific BRAF (Sc-

485 5284; Santa Cruz) and NS3 antibodies for 2 h at RT and then incubated for 1 h at RT in a PBS-

486 BSA 1% solution containing the dye Hoechst 33258 and secondary antibodies (anti-

487 rabbit/A11035 and anti-mouse/A11029; Thermofisher). Preparations were visualized using an

488 Axio observer Z1 fluorescence inverted microscope (Zeiss). Each experiment was repeated at

489 least 3 times.

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490

491 MAPK inhibitors and BRAF silencing

492 When specified, cells were treated with MEK1/2 specific inhibitor U0126 (20 µM final;

493 Promega). Transfections with siRNAs were performed using JetPRIME (Polyplus) according

494 to the manufacturer’s instructions. Control non-specific and BRAF specific siRNAs

495 (SMARTpool: ON-TARGETplus Human BRAF SiRNA) were purchased from Dharmacon

496 and were used at a final concentration of 50 nM.

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497 Acknowledgements

498

499 This work was supported by the Laboratoire d'Excellence “Integrative Biology of

500 Emerging Infectious Diseases” (grant no. ANR-10-LABX-62-IBEID). The LC-MS/MS

501 equipment was funded by the Region Ile-de-France (SESAME), the Paris-Diderot University

502 (ARS) and the CNRS. The funders had no role in study design, data collection and

503 interpretation, or the decision to submit the work for publication.

504 We would like to thank Dr. Frederick Arnaud, Dr. Marc Therrien, Dr. Pierre-Olivier

505 Vidalain, Dr. Maxime Ratinier, Dr. Virginie Doceul and all members of the UMR 1161

506 Virology for fruitful discussions. We thank Dr. Yves Jacob and Dr. Frederick Arnaud for

507 providing the pGal4-UAS-Luc plasmid and the NS3 antibody, respectively.

24 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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753

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754 Figure Legends 755

756 Figure 1. Activation of the MAPK/ERK pathway by BTV-NS3. HEK-293T cells were

757 transfected with pFA2-Elk1 to express Elk1 transcription factor fused to the DNA binding

758 domain of Gal4, pGal4-UAS-Luc that contains the firefly luciferase reporter gene downstream

759 of a promoter sequence containing Gal4 binding site, and pRL-CMV that drives Renilla

760 luciferase expression constitutively. In addition to these three plasmids, cells were co-

761 transfected with expression vectors encoding BTV ORFs in fusion with the 3xFLAG tag (B).

762 12 h after transfection, cells were serum-starved and 6 h later EGF was added at a final

763 concentration of 400 ng/ml (A, B). At the time of EGF stimulation, cells were also infected

764 with WT BTV at indicated MOIs or UV-inactivated BTV (MOI=0.05) (A). After 24 h, relative

765 luciferase activity was determined (A, B). All experiments were achieved in triplicate, and data

766 represent means ± SD. ND: not determined.

767

768 Figure 2. Only full-length BTV-NS3 interacts with BRAF and activates the MAPK/ERK

769 pathway. (A) Schematic representation of BTV-NS3 protein. Deletion fragments of BTV-NS3

770 were designed. Transmembrane (T) and extracellular domains (E) are indicated. (B) HEK-293T

771 cells were transfected with expression vectors encoding GST alone or fused to the full-length

772 NS3 protein (NS3FL) or its indicated fragments, and tested for the interaction with endogenous

773 BRAF. Total cell lysates were prepared 48 h post-transfection (cell lysate; middle panel), and

774 co-purifications of endogenous BRAF were assayed by pull-down using glutathione-sepharose

775 beads (pull-down; upper panel). GST-tagged NS3FL and fragments were detected by

776 immunoblotting using anti-GST antibody (pull-down; lower panel), while endogenous BRAF

777 was detected with a specific antibody. (C) As described in Figure 1, HEK-293T cells were

778 transfected with pFA2-Elk1, pGal4-UAS-Luc and pRL-CMV the expression vector encoding

779 3xFLAG-tagged NS3FL or fragments as indicated. 12 h after transfection, cells were serum-

37 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

780 starved and 6 h later EGF was added at a final concentration of 400 ng/ml. After 24 h, relative

781 luciferase activity was determined. All experiments were achieved in triplicate, and data

782 represent means ± SD. ND: not determined.

783

784 Figure 3. Stimulation of ERK1/2 and eIF4E phosphorylations by BTV infection or BTV-

785 NS3 expression. HEK-293T cells were transfected with an expression vector encoding

786 3xFLAG-tagged BTV-NS3 or the corresponding empty vector pCI-neo-3xFLAG (B). After 18

787 h, cells were serum-starved and, when indicated, infected with WT BTV (MOI=0.01) (A). 12

788 h later, cells were stimulated with 400 ng/ml of EGF. Phosphorylations of ERK1/2 and eIF4E

789 were measured at 10 min, 30 min, 2, 6 and 24 h after EGF stimulation (A, B). BTV infection

790 was confirmed by anti-NS3 immunoblotting (A, lower panel) and expression of 3xFLAG-

791 tagged BTV-NS3 in transfected cells was detected by anti-3xFLAG immunoblotting (B, lower

792 panel). Densitometric analysis of the gels were performed for p-ERK, ERK1/2, peIF4E, eIF4E

793 and the graphs represent the ratio phospho/total. Data presented are representative of at least

794 three independent experiments.

795

796 Figure 4. BRAF subcellular localization in the BTV infectious context. (A) MDBK cells

797 were serum-starved and infected with BTV at indicated MOIs for 24 h. Phosphorylated

798 ERK1/2, total ERK1/2 and BTV-NS3 were detected by western blot analysis. (B) MDBK cells

799 were serum-starved and infected with BTV (MOI=0.01). After 24 h, cells were fixed with 4%

800 PFA and labeled with the dye Hoechst 33258 to stain nuclei and with specific antibodies for

801 BRAF and BTV-NS3. Intracellular localization of Hoechst-stained nuclei (blue), endogenous

802 BRAF (green), and BTV-NS3 (red) were visualized by fluorescence microscopy (×63

803 magnification). Scale bars represent 20 µm.

804

38 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

805 Figure 5. Effects of U0126 inhibitor on BTV-induced MAPK/ERK pathway. (A) As

806 described in Figure 1, HEK-293T cells were transfected with pFA2-Elk1, pGal4-UAS-Luc and

807 pRL-CMV to determine the activation level of MAPK/ERK signaling pathway. In addition to

808 these three plasmids, cells were co-transfected with an expression vector encoding 3xFLAG-

809 tagged BTV-NS3 or the corresponding empty vector pCI-neo-3xFLAG. 12 h after transfection,

810 cells were serum-starved and 6 h later treated with 20 µM of MEK1/2 specific inhibitor U0126

811 when indicated. After 24 h, relative luciferase activity was determined. All experiments were

812 achieved in triplicate, and data represent means ± SD. (B) HEK-293T cells were serum-starved

813 and infected with BTV (MOI=0.01). At the time of infection, cells were also treated with U0126

814 inhibitor as indicated. After 24 h, phosphorylations of ERK1/2 and eIF4E were measured and

815 BTV infection was confirmed by anti-NS3 immunoblotting. (C) HEK-293T cells were serum-

816 starved and infected with BTV (MOI=0.01). After 24 h, supernatants were harvested and

817 titrated by TCID50/ml. The experiment was performed in triplicates, and data represent means

818 ± SD. *, p < 0.05.

819

820 Figure 6. BRAF silencing impairs activation of MAPK/ERK pathway by BTV. (A-D)

821 HEK-293T cells were transfected with non-specific or a BRAF-specific siRNA. One day later,

822 cells were either infected with BTV (MOI=0.01) (A, C) or transfected to express 3xFLAG-

823 tagged BTV-NS3 (B, D) as described in Figure 1. After 24 h, relative luciferase activity was

824 determined (A, B) and cell lysates were analyzed by immunoblotting with antibodies against

825 the indicated proteins (C, D). (A, B) All experiments were achieved in triplicate, and data

826 represent means ± SD. ***, p < 0.0005.

827

828 Figure 7. Comparative analysis of MAPK/ERK pathway for NS3 proteins from different

829 orbiviruses. (A, C) HEK-293T cells were transfected with expression vectors encoding GST

39 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

830 alone or fused to BTV8-NS3, BTV1-NS3, BTV27-NS3, EHDV-NS3, AHSV-NS3 or EEV-

831 NS3 as indicated, and tested for the interaction with endogenous BRAF. Total cell lysates were

832 prepared 48 h post-transfection (cell lysate; middle panel), and co-purifications of endogenous

833 BRAF were assayed by pull-down using glutathione-sepharose beads (pull-down; upper panel).

834 (B, D) As described in Figure 1, HEK-293T cells were transfected with pFA2-Elk1, pGal4-

835 UAS-Luc and pRL-CMV. In addition to these three plasmids, cells were co-transfected with an

836 expression vector encoding 3xFLAG-tagged BTV8-NS3, BTV1-NS3, BTV27-NS3, EHDV-

837 NS3, AHSV-NS3, EEV-NS3 or the corresponding empty vector pCI-neo-3xFLAG, as

838 indicated. 12 h after transfection, cells were serum-starved for 24 h and relative luciferase

839 activity was then determined. The experiment was performed in triplicate, and data represent

840 means ± SD. *** indicates that differences observed between BTV8-NS3, BTV1-NS3 or

841 BTV27-NS3 and the corresponding control vector pCI-neo-3xFLAG were statistically

842 significant p < 0.0005; ** indicates that differences observed between BTV-NS3 and the

843 corresponding control vector pCI-neo-3xFLAG were statistically significant p < 0.005; ns: non-

844 significant differences between EHDV-NS3, AHSV-NS3 or EEV-NS3 and the corresponding

845 control vector pCI-neo-3xFLAG.

846

847 Figure 8. Activation of MAPK/ERK pathway is not related to the inhibition of IFN-α/β

848 signaling by BTV-NS3. (A) HEK-293T cells were co-transfected with IFN-β-pGL3 plasmid

849 that contains the firefly luciferase reporter gene downstream of an IFN-β specific promoter

850 sequence, pRL-CMV reference plasmid, and pCI-neo-3xFLAG expression vectors encoding

851 for 3xFLAG alone or fused to NΔRIG-I (N-terminal CARDs of RIG-I) and BTV-NS3FL or

852 fragments. After 48 h, relative luciferase activity was determined. *** indicates that differences

853 observed between NS3FL, NS3118-229 or NS3D118-182 and the corresponding control vector pCI-

854 neo-3xFLAG were statistically significant p < 0.0005; ns: non-significant differences between

40 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

855 NS31-117 and the corresponding control vector pCI-neo-3xFLAG. (B) Same experiments as (A)

856 but 24 h after transfection, cells were serum-starved for 6 h and treated with 20 µM of U0126

857 as indicated. 24 h later, relative luciferase activity was determined. All experiments were

858 performed in triplicates, and data represent means ± SD. ND: not determined.

41 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B

90 v 8080 300 - EGF 300 - EGF 70 + EGF 250 + EGF 6060 50 200 40 150 luc(RLU) luc(RLU) 40 -1- -1- 30 100 Elk Elk 2020 50 10 ND ND ND 00 0 mocknon 0.MOI002 = MOI0.01 = MOI0.05 = 0.05BTV pCiNe o NS2 NS4 VP1 VP3 VP5 VP7 VP1 VP2 VP3 VP4 VP5 VP6 VP7 NS1 NS2 NS3 NS4 NS5

infecté 0,002 0,01 0,05 inactivé none WT BTV UV BTV bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B C 182 - 229 - 117 NS3 118 Δ 118 FL FL 1- 180 - EGFv N-ter T TE C-ter 160 + EGF NS3 NS3 none NS3 NS3 160 1 229 140 Pull-down BRAF 120120 NS31-117 100 N-ter v luc (RLU) 80 1 117 Cell lysate 80 BRAF -1- 60 Elk 4040 NS3118-229 20 T TE C-ter 118 229 0

Pull-down FL 117 182 229 - - one GST 1-

n NS3 -FL 118 118

NS3 NS3 Δ118-182 NS3 -1_ 117 Δ pCiNe o-vide NS3 NS3 -11 8_22 9

N-ter C-ter NS3

NS3 -delta TM1&2 NS3 1 117 182 229 bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. A B doi: P-eI P-eI 1/2ERK 1/2ERK P- P- 3 eI eI https://doi.org/10.1101/562421 xFlag Time Time ERK NS3 ERK F4E F4E F4E F4E

0 0 10 min 10 min mock 30 min none 30 min 2 h 2 h 6 h 6 h 24 h 24 h ;

0 0 this versionpostedFebruary27,2019. 10 min 10 min BTV 30 min NS3 30 min 2 h 2 h 6 h 6 h 24 h 24 h

P-eIF4E P-ERK P-eIF4E P-ERK (arbitrary units) (arbitrary units) (arbitrary units) (arbitrary units) 0,4 0,6 0,8 0,4 0,8 1,2 1,6 0,8 1,6 2,4 3,2 0,2 0,2 0,4 0,6 0,8 0 0 0 0 1 1 1 0 1 0 0 0 10min 30min 2 h 6 h 24 h 24 h 6 h 2 30min 10min 10min 30min 2 h 6 h 24 h 24 h 6 h 2 30min 10min 10min 30min 2 h 6 h 24 h 24 h 6 h 2 30min 10min 10min 30min 2 h 6 h 24 h 24 h 6 h 2 30min 10min The copyrightholderforthispreprint(which 3 3 3 3 5 5 5 5 Time Time Time Time BTV NS3 none mock BTV mock NS3 none bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B

mock BTV

BTV NS3 mock 0.002 0.01 0.05

P-ERK

BRAF

ERK1/2

merge NS3 + Hoechst bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A

300 300 DMSO 250 U0126 202000 150 luc (RLU) (RLU) luc

-1- 101000

Elk 50 00 pCiNeonone NS3NS3 B

mock BTV U0126 DMSO U0126 DMSO

P-ERK

ERK1/2

P-eIF4E

eIF4E

NS3

C

* 1,00E+044 DMSO U0126

1,00E+03/mL) 3 50

1,00E+022 (TCID

1,00E+0110 1 Log 1,00E+000 1 2 bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B

Si control Si BRAF Si control Si BRAF

90 *** v 78 *** 80 6 70 56 60 50 4

luc (RLU) 40 luc (RLU) 34 40 30 -1- -1- 2 2 20 Elk Elk 1 10 0 0 mock1 BTV2 mock3 BTV4 none1 NS32 none3 NS34

C D

Si control Si BRAF Si control Si BRAF none NS3 mock BTV none NS3 mock BTV

P-ERK P-ERK

ERK1/2 ERK1/2

BRAF BRAF

NS3 3xFlag bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B NS3 - NS3 NS3 - - BTV27 BTV1 none BTV8

Pull-down 20 *** BRAF 18 Cell lysate 16 BRAF 14 12 *** 12 ***

luc (RLU) 10

-1- 8 Pull-down Elk 6 GST 4 2 0 1 2 3 4 NS3 NS3 NS3 none - - - BTV8 BTV1 BTV27

C D NS3 NS3 - - NS3 NS3 - - EEV EHDV AHSV none BTV 1010 Pull-down ** 9 BRAF 8 Cell lysate 7 BRAF 6 5 luc (RLU) 4

-1- ns Pull-down 3

GST Elk 2 ns ns 1 0 pCineo-vide BTV_NS3 EHDV_NS3 AHSV_NS3 EEV_NS3 NS3 NS3 NS3 NS3 none - - - - BTV EEV AHSV EHDV bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A

+ NΔRIG-I 121200 ns 101000 80 *** 60 luc (RLU) (RLU) luc *** -β- 40

IFN 20 *** 00

pCiNeo pCiNeo NS3FL NS3 stop NS3 118- NS3 Δ118- 11 182 229 - - one one 1- neg pos 1177 229 182 n n 11 118 NS3 8 contrôle contrôle Δ NS3 NS3 NS3 B

+ NΔRIG-I 120120 DMSO 100100 U0126 80 60

luc (RLU) (RLU) luc 60

-β- 40

IFN 20 ND 00 none1 none2 NS33 FL bioRxiv preprint doi: https://doi.org/10.1101/562421; this version posted February 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1. List of the identified peptides corresponding to BRAF.

Sequence in BRAF Positions in BRAF Ions Score (by Search Engine): 3xFLAG-tagged NS3 Ions Score (by Search Engine): BTV infection QTAQGMDYLHAK [559-570] 6 31 LLFQGFR [254-260] 16 28 IGDFGLATVK [592-601] 56 38 SSSAPNVHINTIEPVNIDDLIR [363-384] 33 N/A DQIIFMVGR [663-671] 29 N/A LDALQQR [89-95] 39 N/A GYLSPDLSK [672-680] 17 N/A With a Percolator 1 % FDR threshold. N/A: not applicable.