Rayan ; Farhat

Karin Séron

Juliette Ferlin

Lucie Fénéant

Sandrine Belouzard

Lucie Goueslain

Catherine L Jackson

Jean Dubuisson

Yves Rouillé
email: yves.rouille@ibl.cnrs.fr

Goueslain

Identification of class II ADP-ribosylation factors as cellular 3 factors required for hepatitis C virus replication

Keywords: Infection, Membrane, Viruses (phages

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION 41

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the Flaviviridae 42 family. Like other positive RNA viruses, HCV genome is replicated in the cytoplasm of its 43 host cell. During HCV replication, internal membranes of the cell are rearranged and 44 these rearranged membranes likely are replication sites of the viral RNA genome. HCV-45 induced membrane rearrangements have been named membranous web (Egger et al., 46 2002). They include double membrane vesicles (DMV) and clusters of small single 47 membrane vesicles of ER origin [START_REF] Ferraris | Ultrastructural and biochemical 577 analyses of hepatitis C virus-associated host cell membranes[END_REF][START_REF] Romero-Brey | 632 Three-dimensional architecture and biogenesis of membrane structures associated with 633 hepatitis C virus replication[END_REF]. DMV 48 have also been observed in cells replicating RNA viruses of other families, including 49

Picornaviridae [START_REF] Limpens | The transformation of enterovirus replication 605 structures: a three-dimensional study of single-and double-membrane compartments[END_REF][START_REF] Belov | 523 Complex dynamic development of poliovirus membranous replication complexes[END_REF] and Coronaviridae (Knoops et al., 50 2008;[START_REF] Ulasli | Qualitative and 660 quantitative ultrastructural analysis of the membrane rearrangements induced by 661 coronavirus[END_REF], but not with other viruses of the Flaviviridae family (Welsch et 51 al., 2009;[START_REF] Gillespie | The endoplasmic 583 reticulum provides the membrane platform for biogenesis of the flavivirus replication 584 complex[END_REF][START_REF] Schmeiser | Morphogenesis of pestiviruses: 638 new insights from ultrastructural studies of strain Giraffe-1[END_REF]. 52 GBF1, a major regulator of membrane dynamics in the early secretory pathway, has 53 recently emerged as a host factor involved in the replication of several viruses of the 54 Picornaviridae [START_REF] Belov | A critical role of 517 a cellular membrane traffic protein in poliovirus RNA replication[END_REF][START_REF] Lanke | GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus 602 B3 RNA replication[END_REF], Coronaviridae (Verheije et al., 55 2008), and Flaviviridae [START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF][START_REF] Carpp | Quantitative proteomic 537 analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 538 (GBF1) in dengue infection[END_REF] families. GBF1 is a 56 brefeldin A (BFA)-sensitive guanine nucleotide exchange factor (GEF) for G-proteins of 57 the Arf family [START_REF] Claude | GBF1: 549 A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays 550 specificity for ADP-ribosylation factor 5[END_REF]. Arfs recruit and activate a number of effectors, which 58 function in vesicular transport, phospholipid metabolism, actin cytoskeleton regulation 59 and lipid droplet metabolism [START_REF] Donaldson | ARF family G proteins and their regulators: 564 roles in membrane transport, development and disease[END_REF][START_REF] Wright | Regulating the large Sec7 ARF guanine 685 nucleotide exchange factors: the when, where and how of activation[END_REF]. There 60 are 6 Arfs and 16 Arf-like proteins in mammalian cells. Arfs are divided into three 61 classes based on sequence homology. Class I contains Arf1-3, class II contains Arf4 and 62 Arf5, and Arf6 constitutes the sole member of class III [START_REF] Donaldson | ARF family G proteins and their regulators: 564 roles in membrane transport, development and disease[END_REF]63 Wright et al., 2014). As an ArfGEF, GBF1 shows selectivity for Arfs of classes I and II 64 [START_REF] Claude | GBF1: 549 A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays 550 specificity for ADP-ribosylation factor 5[END_REF][START_REF] Szul | 648 Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi 649 biogenesis and protein trafficking[END_REF]. GBF1 is a large protein of 206 kDa containing 6 65 [START_REF] Bui | Large Arf1 guanine nucleotide 534 exchange factors: evolution, domain structure, and roles in membrane trafficking and 535 human disease[END_REF]. The GEF activity is catalyzed by the Sec7 domain. 66

Our knowledge of the functions of the other conserved domains of GBF1 is still scarce 67 [START_REF] Bui | Large Arf1 guanine nucleotide 534 exchange factors: evolution, domain structure, and roles in membrane trafficking and 535 human disease[END_REF][START_REF] Belov | Poliovirus replication 520 requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1[END_REF][START_REF] Bouvet | Targeting 532 of the Arf-GEF GBF1 to lipid droplets and Golgi membranes[END_REF]. 68

The mechanism of action of GBF1 in viral infection is not yet understood. Several lines of 69 evidence indicate that GBF1 is required [START_REF] Verheije | Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated 670 ARF1 activation[END_REF][START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF]70 Carpp et al., 2014) and/or is recruited to viral replication complexes (Verheije et al., 71 2008;[START_REF] Richards | Generation 630 of unique poliovirus RNA replication organelles[END_REF] at the onset of viral replication, but not at later time points. 72

It is generally assumed to function as an ArfGEF activating Arf1, which in turn would 73 recruit the COP-I coatomer, a molecular machinery involved in intracellular transport, 74 which has also been reported to be required for the replication of several positive strand 75 RNA viruses, including HCV [START_REF] Gazina | Differential 580 requirements for COPI coats in formation of replication complexes among three genera 581 of Picornaviridae[END_REF][START_REF] Cherry | COPI 540 activity coupled with fatty acid biosynthesis is required for viral replication[END_REF][START_REF] Tai | A Functional Genomic Screen Identifies Cellular Cofactors of Hepatitis C Virus 652 Replication[END_REF]76 Wang et al., 2012). Accordingly, a GBF1-Arf1-COP-I pathway has been proposed to play a 77 role in the replication of HCV [START_REF] Matto | Role for ADP Ribosylation Factor 1 in the Regulation of Hepatitis C Virus 613 Replication[END_REF][START_REF] Zhang | ARF1 688 and GBF1 Generate a PI4P-Enriched Environment Supportive of Hepatitis C Virus 689 Replication[END_REF]Farhat et al., 2013) 78 and other viruses [START_REF] Wang | Class I ADP-Ribosylation Factors Are Involved in 675 Enterovirus 71 Replication[END_REF]. However, a direct functional link between GBF1 79 activation and COP-I function in HCV replication has not been experimentally 80 demonstrated. For instance, in poliovirus infection, the transient recruitment of GBF1 at 81 replication complexes is not coupled to COP-I recruitment [START_REF] Richards | Generation 630 of unique poliovirus RNA replication organelles[END_REF], 82 suggesting the existence of distinct functions for GBF1 at viral replication complexes and 83 in the secretory pathway of the cell. Indeed, GBF1 may primarily function in poliovirus 84 infection by activating or recruiting other cellular effectors essential for viral replication 85 in an Arf1-independent manner [START_REF] Belov | Poliovirus replication 520 requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1[END_REF]. During HCV replication, the 86 activation by GBF1 of an Arf1 effector different from COP-I, the phosphatidylinositol 4-87 kinase III β, has been proposed to be involved in HCV replication [START_REF] Zhang | ARF1 688 and GBF1 Generate a PI4P-Enriched Environment Supportive of Hepatitis C Virus 689 Replication[END_REF]. 88

However the involvement of this kinase during HCV replication is still controversial (Tai 89 et al., 2009;[START_REF] Vaillancourt | 663 Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA 664 replication[END_REF][START_REF] Berger | 526 Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C 527 virus replication[END_REF][START_REF] Borawski | Class III 529 phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of 530 hepatitis C virus replication[END_REF]Trotard et 90 For example, the mechanism of action of GBF1 during poliovirus replication has been 93

demonstrated not to depend on its catalytic Sec7 domain and therefore on its GEF 94 activity [START_REF] Belov | Poliovirus replication 520 requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1[END_REF]. In this study, we investigated the mechanism of action of 95 GBF1 in HCV replication. Our data support a model in which class II Arf proteins mediate 96 GBF1 function. 97

98

RESULTS

99

Role of the Sec7 domain of GBF1 in HCV infection. It has recently been reported that 100 GBF1 but not Arf activation is required for poliovirus replication [START_REF] Belov | Poliovirus replication 520 requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1[END_REF], 101

suggesting that GBF1 may have other functions than Arf activation during a viral 102 infection. More specifically, a catalytically inactive GBF1 truncation mutant lacking the 103 Sec7 domain has been reported to be sufficient for rescuing poliovirus replication from 104 BFA inhibition. To investigate if GBF1 could also function in a Sec7-independent manner 105 in HCV replication, we expressed a series of GBF1 truncation mutants (figure 1A) in 106 Huh-7 cells and infected them with HCV in the presence of BFA. We used a dose of 50 107 ng/ml BFA, which decreases HCV infection about 10 times and has minimal cytotoxic 108 effects in Huh-7 cells during the time scale of the HCV infection assay (Goueslain et al., 109 2010). We previously showed that BFA has no impact on HCV entry and inhibits the 110 replication step of the HCV life cycle [START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF]. GBF1 construct expression 111 and HCV infection were probed by immunoblotting at 30 hpi. At this time, only cells 112 initially infected are positive [START_REF] Afzal | 512 Regulation of core expression during the hepatitis C virus life cycle[END_REF], implying that the impact of BFA 113 inhibition of progeny virion release on HCV protein expression is negligible. 114 BFA treatment had no impact on the expression of GBF1 constructs (figure 1B). In the 115 absence of BFA, similar E1 expression levels were observed with all GBF1 constructs, 116 indicating that none of these constructs had any dominant-negative effect on HCV 117 infection. In the presence of BFA, E1 expression levels were reduced about 10-20 times 118 in untransfected cells or in cells expressing YFP. HCV infection was partially rescued in 119 cells expressing full-length wild-type or M832L BFA-resistant constructs, whereas the 120 E794K inactive mutant was unable to rescue HCV infection in the presence of BFA, as 121 previously reported [START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF]. Importantly, unlike for poliovirus, all the C-122 terminal deletion constructs including GBF1(1-710), which lacks a Sec7 domain, were 123 unable to rescue HCV infection in these complementation experiments (figure 1B andC). 124 Therefore, these results support a Sec7-dependent function of GBF1 in HCV infection. 125

As a control, we also quantified the impact of GBF1 constructs on the secretion of human 126 (Q71L). When overexpressed, these two constructs act as dominant negative mutants in 141 a protein transport assay and block the intracellular traffic at different steps of the early 142 secretory pathway [START_REF] Dascher | Dominant inhibitory mutants of ARF1 block 555 endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus[END_REF]. Arf mutants were expressed as EGFP or 143 mCherry fusion proteins in Huh-7 cells and the cells were infected with HCV. The 144 infection was monitored by immunofluorescence (figure 2A) and the percentage of 145 fluorescent cells infected was quantified. As expected, the expression of the T31N 146 mutant, which has a BFA-like effect, strongly inhibited HCV infection (figure 2B). The 147 Q71L mutant, which is locked in the active, GTP-bound form, also strongly inhibited HCV 148 infection, and the wild type Arf1 construct had a moderate impact compared to EGFP 149 and mCherry controls. On the other hand, Arf1 constructs did not have any effect on 150 adenovirus infection, indicating that the effects observed with HCV did not result from 151 any cytotoxic effects of Arf1 constructs. These results confirmed the involvement of an 152 Arf family member in HCV infection. 153 154 Role of Arf family members in HCV infection. The results with dominant negative 155 mutants of Arf1 suggest that Arf1 and/or other Arf family members are involved in HCV 156 infection. We used siRNA technology to determine which Arf proteins are specifically 157 involved in HCV infection. Given the BFA sensitivity of HCV infection, we focused our 158 study on class-I and class-II Arfs. Arf1, Arf3, Arf4 and Arf5 (human cells have no Arf2) 159 were targeted with pools of 4 siRNAs, except for Arf1, which was targeted by 2 siRNAs 160 only, because we found that the commercial Arf1 pool contains 2 individual siRNAs also 161 targeting Arf3 (supplementary figure 1). Surprisingly, the depletion of each Arf protein 162 resulted in a moderate (30-35%) inhibition of HCV infection (figure 3A). To assess the 163 extent of siRNA-mediated Arf depletion, we quantified mRNAs by quantitative RT-PCR. 164 The depletions were specific and reached 87% inhibition for Arf1 and about 95% 165 inhibition for the other Arfs (figure 3B). We also observed an up-regulation of Arf4 166 expression upon Arf1 and to a lesser extent Arf3 depletion, suggesting the existence of 167 compensatory mechanisms regulating the expression of Arf family members. 168

Arfs may have overlapping functions, and it has been reported that the depletion of 169 different pairs of Arfs results in specific phenotypes [START_REF] Volpicelli-Daley | Isoform-selective effects of 672 the depletion of ADP-ribosylation factors 1-5 on membrane traffic[END_REF]. 170

Therefore we considered that a specific pair of Arfs could be involved in HCV infection. 171

For this reason, we also depleted pairs of Arfs. Again, higher levels of Arf4 mRNA were 172 observed upon Arf1+Arf3 depletion or Arf1+Arf5 depletion, confirming the up-173 regulation of Arf4 in Arf1-depleted cells (figure 3B). Interestingly, HCV infection was 174 decreased down to 17±8% in cells depleted of both Arf4 and Arf5, whereas the 175 depletion of other pairs did not significantly decrease more HCV infection than single Arf 176 depletions (figure 3A). These results indicate a special importance of class-II Arfs in HCV 177 infection. 178 To rule out off-target effects, Arf4 or Arf5 were expressed in siRNA-treated cells. Arf 179 proteins of murine origin were expressed in Huh-7 cells simultaneously depleted of both 180

Arfs. Immunoblot analysis confirmed Arf5 expression (figure 3C). However, Arf4 181 expression could not be confirmed due to the lack of reactivity toward murine Arf4 of 182 the antibody. HCV infection was partially restored in Arf4-expressing cells and in Arf5-183 expressing cells (figure 3D). We also observed a slight decrease of HCV infection in cells 184 transfected with Arf4 in the control condition (figure 3D). The expression of Arf4 or Arf5 185 in double-depleted cells increased HCV infection up to levels similar to those observed 186 in cells depleted of a single Arf protein (figure 3A). In contrast, no rescue of HCV 187 infection was observed in cells transfected with siRNAs to PI4KA, as expected (figure 188 3D). 189 To further examine the impact of their depletion on the secretory pathway, we also 234 investigated the morphology of cellular compartments. We observed a more compact 235 Golgi morphology in cells simultaneously depleted of Arf4 and Arf5 than in control cells 236 using the cis-Golgi marker GM130 (figure 4B). In contrast, the depletion of Arf1 and Arf4 237 resulted in a fragmentation of the cis-Golgi, as previously reported for HeLa cells 238 [START_REF] Volpicelli-Daley | Isoform-selective effects of 672 the depletion of ADP-ribosylation factors 1-5 on membrane traffic[END_REF]. A similar scattered Golgi phenotype was observed upon 239 GBF1 depletion (figure 4B) or after a BFA treatment (data not shown). We did not 240 regulator of the early secretory pathway, GBF1 is also known to regulate lipid droplets 253 [START_REF] Guo | 589 Functional genomic screen reveals genes involved in lipid-droplet formation and 590 utilization[END_REF][START_REF] Beller | 515 COPI complex is a regulator of lipid homeostasis[END_REF][START_REF] Soni | Coatomer-dependent protein delivery to lipid droplets[END_REF], a cellular compartment playing a 254 critical role in the HCV life cycle. Given the lack of effect of class II Arfs on the secretory 255 pathway, we also investigated the morphology of lipid droplets after Arf depletion. Lipid 256 droplets were stained with BODIBY493/503 and the Golgi was also labeled with an 257 antibody to GM130 to verify the effect of Arf4 and Arf5 depletion. In cells simultaneously 258 depleted of Arf4 and Arf5, lipid droplets were larger than in control cells and were 259 packed together at the periphery, often in one extension of the cell, unlike in control 260 cells where smaller lipid droplets were usually scattered throughout the cytoplasm 261 (figure 5A). A similar effect was observed in cells treated for 24 hours with a low dose of 262 BFA (figure 5B), or in cells depleted of GBF1 (data not shown), suggesting that the action 263 of the pair of class II Arfs on lipid droplets is regulated by GBF1. Importantly, the 264 depletion of other Arf pairs did not result in a similar phenotype of accumulation of 265 In this study we investigated the mechanism of action of GBF1 in HCV infection. Our 273 results suggest that GBF1 functions in HCV infection by activating class II Arfs. This is 274

different from what has been reported for in poliovirus, for which its GEF activity and 275 Arf activation are not required [START_REF] Belov | Poliovirus replication 520 requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1[END_REF]. A series of GBF1 truncation mutants 276 inactive for regulating the secretory pathway of the cell, including a construct lacking a 277 Sec7 domain, were shown to complement BFA inhibition of poliovirus replication. Here 278 we showed that unlike poliovirus, the same series of GBF1 mutants do not support HCV 279 replication, clearly indicating a difference of action for GBF1 in poliovirus and HCV 280 replication. This requirement for GEF activity was confirmed with the use of Arf1 281 dominant negative mutants. 282

The function of GBF1 in HCV replication is also different from its function in membrane 283 traffic, as already suggested by our previous study of BFA resistant cell lines (Farhat et 284 al., 2013). Indeed our results suggest that GBF1 may act by activating different pairs of 285 Arf proteins for the control of HCV replication and of protein secretion. The pair Arf1-286 Arf4 mediates GBF1 control of the secretory pathway, as already reported (Volpicelli-287 Daley et al., 2005), whereas the pair Arf4-Arf5 mediates GBF1 function in HCV 288 replication. Recently, Arf4 and Arf5 have also been reported to be involved in dengue 289 virus infection, although for this virus, the function of this pair of Arfs was suggested to 290 [START_REF] Kudelko | Class 598 II ADP-ribosylation factors are required for efficient secretion of dengue viruses[END_REF]. Interestingly, the depletion of 291 Arf4 was reported to protect cells from BFA toxicity, and this effect also depends on 292 GBF1, Arf1 and Arf5 [START_REF] Reiling | A CREB3-ARF4 signalling pathway mediates the response to Golgi stress and 628 susceptibility to pathogens[END_REF]. 293

Based on siRNA experiments, previous studies, including our own, suggested an 294 involvement of Arf1 in HCV replication [START_REF] Matto | Role for ADP Ribosylation Factor 1 in the Regulation of Hepatitis C Virus 613 Replication[END_REF][START_REF] Zhang | ARF1 688 and GBF1 Generate a PI4P-Enriched Environment Supportive of Hepatitis C Virus 689 Replication[END_REF]Farhat et 295 al., 2013) that we did not observe in this study. However, in none of these previous 296 studies were the results of siRNA depletions confirmed by re-expressing Arf1, in order 297 to rule out off-target effects. Off-target effects are always a potential pitfall with siRNA 298 experiments, as exemplified by our finding that the commercial siRNA pool targeting 299 Arf1 also inhibits Arf3 expression. Reducing siRNA concentration may help to reduce 300 off-target effects. Using a more efficient transfection agent allowed us to decrease siRNA 301 concentration from 80 nM double transfections previously used down to 20 nM single 302 transfections in this study, with better depletion results on control proteins (data not 303 shown). With these new experimental conditions, Arf1 depletion resulted in a lower 304 inhibition of HCV replication (from about 50% in our previous study (Farhat et al., 305 2013) down to about 30% inhibition in this study) despite efficient reduction of Arf1 306 mRNA expression. All single Arf depletions similarly yielded low levels of inhibition of 307 HCV infection, in line with the reported lack of phenotype of single Arf depletion 308 [START_REF] Volpicelli-Daley | Isoform-selective effects of 672 the depletion of ADP-ribosylation factors 1-5 on membrane traffic[END_REF]. In contrast, we found a stronger inhibition of HCV 309 infection when cells were simultaneously depleted of both Arf4 and Arf5. This finding is 310 not inconsistent with the inhibition of HCV infection by Arf1 dominant negative mutants, 311 because trans dominant phenotypes often result from interactions with regulators (like 312

Arf-GEFs and Arf-GAPs in this case). This suggests that Arf1, Arf4 and Arf5 share 313 common regulators, as also indicated by their common inhibition by BFA. 314 al., 2005). In our study, the depletion of Arf4 and Arf5 displayed a BFA-like effect on 316 lipid droplets morphology and on HCV infection that the pair Arf1-Arf4 did not. On the 317 other hand, the depletion of the Arf1-Arf4 pair resulted in a BFA-like inhibition of HSA 318 and ApoE secretion, whereas the depletion of the Arf4-Arf5 pair did not. Cells depleted 319 of Arf1 and Arf4 also displayed a scattered Golgi very similar to what is observed in 320 BFA-treated cells or in cells depleted of GBF1, whereas cells depleted of Arf4 and Arf5 321 had a more compact Golgi morphology than control cells. This effect is not mimicked by 322 GBF1 inhibition, but is rather reminiscent of cells treated with latrunculin B, an inhibitor 323 of actin polymerization [START_REF] Valderrama | 666 Actin microfilaments facilitate the retrograde transport from the Golgi complex to the 667 endoplasmic reticulum in mammalian cells[END_REF][START_REF] Dippold | GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch 562 and shape the Golgi to promote budding[END_REF]. Therefore, the 324 Arf4-Arf5 pair could potentially activate an effector involved in the regulation of the 325 actin cytoskeleton. Alternatively, we can also speculate that this effect could result from 326 an alteration of the metabolism of phosphoinositides, because actin fibers are linked to 327 Golgi membranes by PI4P-interacting protein GOLPH3 [START_REF] Dippold | GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch 562 and shape the Golgi to promote budding[END_REF]. Taken 328 together these results suggest that GBF1 fulfills its different cellular functions by 329 activating different pairs of Arfs, which in turn control different aspects of the cellular 330 metabolism. 331 An intriguing question is how each pair of Arfs controls specific pathways. This appears 332 to be a common feature of G-proteins of the Arf family [START_REF] Panic | The ARF-like GTPases Arl1p and Arl3p act 624 in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus[END_REF]Setty et al., 333 2003;[START_REF] Volpicelli-Daley | Isoform-selective effects of 672 the depletion of ADP-ribosylation factors 1-5 on membrane traffic[END_REF][START_REF] Cohen | Active 552 Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains[END_REF]. A redundancy of action is the 334 simplest explanation, although it is quite difficult to match this model with the diversity 335 of phenotypes observed with the depletion of different pairs of BFA-sensitive Arfs 336 [START_REF] Volpicelli-Daley | Isoform-selective effects of 672 the depletion of ADP-ribosylation factors 1-5 on membrane traffic[END_REF]. Another model for Arf pair specificity could be the 337 formation of Arf dimers, at the site of interaction with effectors. However, such a kind of 338 interactions has not yet been reported for any Arf effector to our knowledge. It is also 339 of the other one. Different models compatible with this possibility have been proposed 341 [START_REF] Cohen | Active 552 Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains[END_REF][START_REF] Chun | 545 Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound 546 class II Arfs associate with the ER-Golgi intermediate compartment independently of 547 23 GBF1[END_REF]. A cascade of recruitment has been reported for 342 other Arf family members [START_REF] Panic | The ARF-like GTPases Arl1p and Arl3p act 624 in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus[END_REF][START_REF] Setty | Golgi recruitment 640 of GRIP domain proteins by Arf-like GTPase 1 is regulated by Arf-like GTPase 3[END_REF][START_REF] Cohen | Active 552 Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains[END_REF]343 Christis and Munro, 2012). This hypothesis would be more compatible if similar 344 phenotypes were observed in cells depleted of either protein and in double depleted 345 cells. However, if the selectivity of the system were not stringent, then it could be 346 compatible with an inhibition only visible in double depleted cells. It would be 347 interesting to determine if such a mode of action actually occurs for Arf1-Arf4 and Arf4-348 Arf5 pairs. 349

In conclusion, our results indicate that the role of GBF1 in HCV replication is mediated 350 by its ArfGEF activity and is potentially related to a function of regulation of lipid 351 metabolism, rather than of regulation of the protein secretory pathway. Interestingly, 352 two members of the Arf family, class II Arf4 and Arf5, appear to be of special importance 353 for mediating GBF1 function in HCV replication. Although very little is known about the 354 specific functions of class II Arfs, data from our study and from another group 355 [START_REF] Takashima | GBF1-654 Arf-COPI-ArfGAP-mediated Golgi-to-ER transport involved in regulation of lipid 655 homeostasis[END_REF] suggest their involvement in the control of lipid metabolism, as 356 evidenced by the abnormal morphology of lipid droplets in cells depleted of both Arf4 357 and Arf5. However, their mechanism of action is unknown. A role for the drosophila Arf1 358 homologue Arf79F in controlling the localization of enzymes of the triglyceride 359 metabolism to oleic acid-induced lipid droplets was recently described (Wilfling et al., 360 2014). It is not yet clear how this might relate to our results with class II Arfs-depleted 361 cells, because the depletion of Arf1 reduced the size of lipid droplets in drosophila cells, 362 instead of increasing it as we observed for simultaneous depletion of both Arf4 and Arf5 363 in Huh-7 cells. Further studies will be needed to determine the nature of the enzymes or 364 Antibodies. Mouse anti-E1 mAb A4 [START_REF] Dubuisson | 567 Formation and intracellular localization of hepatitis C virus envelope glycoprotein 568 complexes expressed by recombinant vaccinia and Sindbis viruses[END_REF] was produced in vitro by 375 using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. Mouse 376 anti-NS5A mAb 9E10 [START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF] DNA constructs. GBF1 deletion mutants were described previously [START_REF] Niu | Dynamics of 618 GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi[END_REF]387 Belov et al., 2010) HCVcc. The virus JFH1-CSN6A4 used in this study was based on JFH1, and contained cell 407 culture adaptive mutations [START_REF] Delgrange | 558 Robust production of infectious viral particles in Huh-7 cells by introducing mutations in 559 hepatitis C virus structural proteins[END_REF] and a reconstituted A4 epitope in E1, 408 as previously described [START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF]. The plasmid pJFH1-CSN6A4 was 409 linearized with XbaI and treated with the Mung Bean Nuclease (New England Biolabs). 410

In vitro transcripts were generated using the Megascript kit according to the 411 manufacturer's protocol (Ambion). Ten micrograms of in vitro transcribed RNA were 412 delivered into Huh-7 cells by electroporation as described [START_REF] Kato | Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon[END_REF]. For virus 413 production, electroporated cells were passaged 3 days after electroporation and grown 414 19 siRNA was used. Then, the diluted transfection reagent was added to the siRNA and the 440 mixture was incubated for 30 min at room temperature. At the end of this incubation, 441 2.5 10 5 freshly trypsinized cells in a volume of 2 ml of culture medium were added to the 442 transfection mix and the cells were returned to 37°C. Cells were trypsinized 24 h later 443 and seeded on glass cover slips, and analyzed by immunofluorescence 3 or 4 days after 444 siRNA transfection. For quantifying HCV infection, siRNA-treated cells were infected 48 445 h after trypsinization. Just before infection, extra wells of cells treated with each siRNA 446 were used to extract RNA for quantifying the depletion efficiency. Infected cells were 447 stained with anti-E1 mAb A4 and DAPI at 30 hpi and HCV-infected were counted. At 448 least 5000 cells were counted per experiment for each condition. 449

Immunoblotting. Cells were rinsed 3 times with cold PBS, and lysed at 4°C for 20 min 450 in a buffer containing 50 mM TrisCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% Triton-X, 451 0.1% SDS, 1 mM PMSF, and a mix of protease inhibitors (Complete). Insoluble material 452 was removed by centrifugation at 4°C. The protein content was determined by the 453 bicinchoninic acid method as recommended by the manufacturer (Sigma), using bovine 454 serum albumin as the standard. The proteins were then resolved by SDS-PAGE and 455 transferred onto nitrocellulose membranes (Hybond-ECL; Amersham) using a Trans-456 Blot apparatus (Bio-Rad). Proteins of interest were revealed with specific primary 457 antibodies, followed by species-specific secondary antibodies conjugated to peroxidase. 458 Proteins were visualized using enhanced chemiluminescence (ECL Plus, GE healthcare). 459

The signals were recorded using a LAS 3000 apparatus (Fujifilm). Quantification of 460 unsaturated signals was carried out using the gel quantification function of ImageJ. 461

Immunofluorescence microscopy.

Indirect immunofluorescence labeling was 462 performed as previously described [START_REF] Rouillé | 635 Subcellular localization of hepatitis C virus structural proteins in a cell culture system 636 that efficiently replicates the virus[END_REF]. Lipid droplets were stained 463 with BODIBY 493/503 (0.5 µg/ml; Invitrogen) for 10 min at room temperature. Nuclei 464 carried out with an LSM780 confocal microscope (Zeiss) using a 63X oil immersion 466 objective with a 1.4 numerical aperture. Signals were sequentially collected using single 467 fluorescence excitation and acquisition settings to avoid crossover. Images were 468 processed using Adobe Photoshop software CS4. 469 BFA rescue experiments. Cells were transfected with GBF1 mutants and then infected 470 with JFH1-CSN6A4 48 h post transfection in the presence of 50 ng/ml of BFA, or the 471 corresponding volume of ethanol (BFA solvent). Cells were lysed at 30 hpi and the 472 expression levels of HCV E1 (A4) were measured by Western blot. The expression level 473 of each mutant with ethanol was set to 100%. The relative expression of HCV E1 of the 474 BFA treated samples was normalized to the ethanol control. 475 Secretion assays. Sub-confluent cell cultures grown in 12-well plates were incubated 476 for 24 h in 1 ml of complete culture medium. Culture media were collected and 477 centrifuged to remove cells debris. Cells were rinsed with PBS, and lysed for 20 min on 478 ice. The HSA concentration in the supernatants and lysates was determined by ELISA, 479 using human serum albumin (HSA) as a standard, as described [START_REF] Snooks | Vectorial entry and release of hepatitis A virus in polarized human hepatocytes[END_REF]. 480 Apolipoprotein E was quantified using a commercial ELISA kit from Mabtech. The 481 percentage of secretion was calculated as the percentage of HSA/apoE in the medium 482 divided by the total amount of HSA/apoE in the medium and the lysate. 483

Replication assay. The construct used for the replication assay (HCVcc-Rluc/∆E1E2) 484 was as previously described [START_REF] Goueslain | Identification of GBF1 as a cellular factor required for hepatitis C virus RNA 587 replication[END_REF]. Huh-7 cells were electroporated 485 with HCVcc-Rluc/∆E1E2 in vitro transcribed RNA and seeded in 24-well plates. The 486 luciferase activity was measured 4 h, 24 h, 48 h and 72 h post-electroporation using the 487 Renilla luciferase assay system kit from Promega. 488 analysis was performed using the Taqman® pre-designed gene expression assay 493 approach (Applied Biosystems), using 1 µl of cDNA and premade probes designed by the 494 manufacturer. The ratio of the mRNA level of each gene to that of large ribosomal 495 protein P0 (RPLP0) endogenous control mRNA was calculated by the ∆∆Ct method 496 [START_REF] Livak | Analysis of relative gene expression data using 610 real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF], and a value of 100 was assigned to control siRNA-497 transfected cells. Each experiment was performed in triplicate and repeated three times. 498

HCVpp. The luciferase-based HCV-pseudotyped retroviral particles (HCVpp) infection 499 assay was performed as previously described [START_REF] Op De Beeck | 621 Characterization of functional hepatitis C virus envelope glycoproteins[END_REF]. 500

ACKNOWLEDGMENTS 502

This work was supported by grants from the "Agence Nationale de la Recherche sur le 503 SIDA et les hépatites virales" (ANRS) and "Agence Nationale de la Recherche" (ANR) 504 through ERA-NET Infect-ERA program (ANR-13-IFEC-0002-01). 505

We thank B. Hoflack, C.M. Rice and T. Wakita for providing us with reagents. We are 506 grateful to Sophana Ung for his assistance in the illustrations. Some data were generated 507 with the help of the Imaging Core Facility of the campus Calmette (BICeL). 508

The authors have no conflict of interest to declare. 509

  2009). Other possibilities for GBF1 function during viral replication include the 91 activation of other members of the Arf family or mechanisms unrelated to Arf activation. 92

  serum albumin (HSA) by BFA-treated Huh-7 cells. A well-established function of GBF1 is 127 the control of membrane dynamics in the early secretory pathway, and this function 128 relies on Arf activation. Therefore, HSA secretion is a marker of GBF1-mediated Arf 129 activation in these experiments. The treatment of Huh-7 cells with 50 ng/ml BFA 130 decreased albumin secretion in control mock-, or YFP-transfected cells (figure1D). As 131 expected, wild type and M832L GBF1 constructs partially restored albumin secretion, 132 but none of the other constructs did. These results indicate a similar requirement of 133 GBF1 domains for HCV infection and for Arf activation and are consistent with a 134 requirement of ArfGEF activity to promote HCV infection. 135 136 Arf1 dominant negative mutants inhibit HCV infection. Our results with GBF1 137 truncation mutants suggested that the activation of at least one BFA-sensitive Arf family 138 member is required for HCV infection. We further investigated this question using two 139 Arf1 affecting either GDP exchange (T31N) or GTP hydrolysis 140

  the replication step of the HCV life cycle is affected by the simultaneous 190 depletion of class-II Arfs, siRNA-transfected Huh-7 cells were electroporated with an in 191 vitro-transcribed recombinant ∆E1E2 JFH1 genomic RNA expressing a Renilla luciferase 192 reporter. Measuring luciferase activity over a 72-h time course assessed replication. 193 Luciferase activity was not inhibited at 4h post electroporation, and was reduced about 194 9 times in cells depleted of Arf4 and Arf5 at 48 and 72 h post electroporation (figure 3E), 195 indicating an inhibition of HCV replication with no impact on the initial translation level 196 of HCV RNA. 197 We also investigated HCV entry in Huh-7 cells simultaneously depleted of both class II 198 Arfs using HCVpp. As controls, we used VSV-Gpp, which rely on endocytosis and pH-199 dependent fusion for entry, like HCVpp, and RD114pp, which enter by a pH-independent 200 mechanism. All pseudoparticles were similarly inhibited by the double depletion of Arf4 201 and Arf5 (supplementary figure 2), indicating a post-fusion inhibition of pseudoparticle 202 entry. Therefore, we cannot conclude on the function of class II Arfs during the entry 203 step of the HCV life cycle. 204 205 Class II Arfs are not recruited in HCV replication complexes. The intracellular 206 localization of Arf4 and Arf5 in Huh-7 cells was analyzed by immunofluorescence 207 confocal microscopy. Cells were transfected with expression vectors for Arf4-GFP or 208 Arf5-GFP. Both Arf4-GFP and Arf5-GFP were observed in Golgi-like perinuclear 209 structures together with a diffuse staining of the cytosol and the nucleus 210(supplementary figure3), which is consistent with the dual localization of Arf proteins 211 as membrane-associated and soluble proteins. Arf4-and Arf5-positive perinuclear 212 structures were also labeled with an antibody to GM130 (supplementary figure3A), 213 indicating that they are localized in the cis-Golgi. Interestingly, Arf4 and Arf5 214 perinuclear structures also colocalized with GBF1 (supplementary figure3B), the localization of Arf4-GFP and Arf5-GFP was compared to that of NS3 216 and NS5A, two markers of HCV replication complexes. We did not observe any major 217 change of Arf4 and Arf5 patterns (supplementary figure4), except in Arf4-GFP 218 expressing cells, where the Golgi appears less structured. This tendency toward a less 219 compact Golgi was also observed in non-infected cells (supplementary figure3). Very 220 few levels of colocalization of Arfs and NS3 (supplementary figure 4A) or NS5A 221 (supplementary figure 4B) were observed, indicating that class II Arfs are not 222 permanently recruited to HCV replication complexes. 223 224 Impact of Arfs depletion on the secretory pathway. To get insight into a mechanism of 225 action of the pair of class-II Arfs, we analyzed their involvement in the regulation of 226 secretion. Following depletion of different pairs of Arfs, we monitored the secretion of 227 serum albumin and VLDL-associated apolipoprotein E (apoE), two proteins expressed 228 and constitutively secreted by Huh-7 cells. Albumin and apoE secretion were inhibited 229 in cells with reduced levels of both Arf1 and Arf4, but were not affected by the depletion 230 of any other pair of Arfs, including Arf4 and Arf 5 (figure 4A). These results indicate that 231 the simultaneous depletion of class II Arfs has no functional impact on the secretory 232 pathway. 233

  change in cells depleted of other pairs of Arfs 241 (supplementary figure5). A compaction of the Golgi complex was also observed in class 242 II Arfs-depleted cells with other markers. In these cells, GBF1 and ERGIC53, two proteins 243 located both in the cis-Golgi and the ERGIC, and TGN46, a marker of the trans-Golgi 244 network, all showed a more compact localization (figure4B), indicating that the effect is 245 not restricted to the cis-Golgi. Again, the depletion of Arf1 and Arf4 or of GBF1 resulted 246 in a scattered pattern of ERGIC53, GBF1 and TGN46 (figure4B). No other Arf pair 247 produced similar phenotypes of scattered or compacted patterns with any of the 248 markers (data not shown). These results indicate that the pair of class II Arfs does not 249 contribute to the Golgi scattering effect of GBF1 inhibition. 250 251 Impact of class II Arfs depletion on the lipid droplets. In addition to its role as a 252

  droplets. Lipid droplets were very similar to controls for all depletions 266 except for the pair Arf1 and Arf4, and to some extent the pair Arf3 and Arf4, the 267 depletion of which resulted in a reduced number of enlarged lipid droplets 268 (supplementary figure5). This result suggests that the pair of class II Arfs could 269 participate in GBF1-mediated regulation of lipid metabolism. 270 271 DISCUSSION 272

  the assembly step

  already reported for different pairs of Arfs (Volpicelli-Daley et 315

  recruitment or the activation of one Arf would depend on the activation 340

  lipid metabolism that are effectors of Arf4 and Arf5. These 365 proteins may constitute host factors critical for the replication of HCV and potentially Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline 370 (PBS), goat and fetal calf sera (FCS), BODIBY 493/503 and 4',6-diamidino-2-371 phenylindole (DAPI) were purchased from Life Technologies. Mowiol 4-88 was from 372 Calbiochem. Protease inhibitors mix (Complete) was from Roche. Other chemicals were 373 from Sigma. 374

  was kindly provided by C. M. Rice (The 377 Rockefeller University). Mouse anti-NS3 mAb 1848 was from Virostat. Rabbit anti-378 human Arf4 mAb (ab171746) was from Abcam. Mouse anti-Arf5 mAb 1B4 was from 379 Abnova. Sheep anti-TGN46 was from Serotec. Mouse anti-GBF1 and anti-GM130 mAbs 380 were from Transduction Laboratories. Mouse anti-β-tubulin mAb (TUB 2.1) was from 381 Sigma. Mouse anti-HSA (ZMHSA1) was from Invitrogen. Goat anti-HSA (507313) was 382 from Calbiochem. Mouse anti-GFP mAb was from Roche. Alexa 555-conjugated donkey 383 anti-sheep IgG antibody was from Life Technologies. Peroxidase-conjugated goat anti-384 mouse, and anti-sheep IgG, and cyanine 3-conjugated goat anti-mouse IgG were from 385 Jackson Immunoresearch. 386

  The culture medium was collected every day, titrated, aliquoted and 415 stored at -80°C. For infection assays, sub-confluent naïve Huh-7 cells grown in a P24 416 well were incubated with 50 µl of this virus preparation diluted to 200 µl of medium for 417 2 hours (M.O.I. ~0.5), and the inoculate was replaced with fresh culture medium. In 418 order to reach near 100% infected cells in experiments with Arf1 mutants, cells were 419 infected with 200 µl of undiluted HCVcc stock with a higher titre, (M.O.I. ~5). In all 420 experiments, infections were scored at 30 hpi. 421Adenovirus. A recombinant defective adenovirus expressing a green fluorescent 422 protein (EGFP) was as previously described[START_REF] Farhat | Hepatitis C virus replication and Golgi function in brefeldin a-resistant 575 hepatoma-derived cells[END_REF]. Cells were infected for 423 1 hour at 37°C, and fixed for 20 minutes with PFA 3% at 16 hours post-infection. 424 DNA transfection. Twenty-four hours before transfection, cells were seeded in 24-well 425 clusters to reach ~70% confluence the next day. Cells were transfected with 0.5 μg of 426 plasmid DNA mixed with Trans-IT LT1 reagent following the instructions of the 427 manufacturer (Mirus). Cells transfected with pCEP4-based constructs were co-428 transfected with pPUR (clonetech) at a 1:20 ratio, and selected for with 5 µg/ml 429 puromycin for 3 days, and cultured with no puromycin for 4 days before siRNA 430 transfection. 431 RNA interference. RNA interference experiments were carried out with pools of four 432 different synthetic double-stranded siRNAs to the same target (on-target plus smart 433 pool reagents from Dharmacon). Due to an off-target effect with the pool against Arf1 we 434 used a mix of 2 individual siRNAs (J-011580-05-0005 & J-011580-08-0005). The control 435 used in this study was the on-target plus non-targeting siRNA #1 (D-001810-01-20). 436For siRNA transfection, 3 μl of lipofectamine RNAi MAX (Life Technologies) were added 437 to 0.5 ml of D-PBS and incubated for 3 min. In a 6-well plate, 2.5 μl of siRNA at 20μM 438 were spotted in the center of a well. In case of double siRNA transfection, 1.25 µl of each 439

  DAPI. For colocalization experiments, confocal microscopy was 465

  PCR. Total RNA was extracted from siRNA-transfected and control 489 cells using Nucleospin RNA II extraction kit (Macherey-Nagel), which includes a DNAse I 490 treatment. cDNA was obtained from RNA using the High Capacity cDNA Reverse 491 transcription kit (Life Technologies) in a final volume of 20 µl. Quantitative RT-PCR 492

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure S2 .Figure S3 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345S2S312345 FIGURES LEGENDS 693

Cellular Microbiology

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