Sonia Assil

Séverin Coléon

Congcong Dong

Elodie Décembre

Lee Sherry

Omran Allatif

Brian Webster

Marlène Dreux
email: marlene.dreux@ens-lyon.fr

Plasmacytoid Dendritic Cells and Infected Cells form an Interferogenic Synapse Required for Antiviral Responses

Type I interferon (IFN-I) is critical for antiviral defense, and plasmacytoid dendritic cells (pDCs) are a predominant source of IFN-I during virus infection. pDC-mediated antiviral responses are stimulated upon physical contact with infected cells, during which immunostimulatory viral RNA is transferred to pDCs, leading to IFN production via the nucleic acid sensor TLR7. Using dengue, hepatitis C, and Zika viruses, we demonstrate that the contact site of pDCs with infected cells is a specialized platform, we term the interferogenic synapse, which enables viral RNA transfer and antiviral responses. This synapse is formed via αLβ2 integrin/ICAM-1 adhesion complexes and the recruitment of the actin network and endocytic machinery. TLR7 signaling in pDCs promotes interferogenic synapse establishment and provides feed-forward regulation sustaining pDC contacts with infected cells. This interferogenic synapse may allow pDCs to scan infected cells and locally secrete IFN-I, thereby confining a potentially deleterious response.

Introduction

Type I interferon (i.e., IFNα/β, referred to as IFN-I) response is pivotal for protection against viral infections. It is initiated by the recognition of pathogen-associated molecular patterns (PAMPs) including viral nucleic acids by cellular pathogen recognition receptors (PRRs) such as toll-like receptors (TLRs). This leads to the secretion of IFN-I, type III IFNs, inflammatory cytokines and to the expression of an array of IFN-stimulated genes (ISGs) [START_REF] Hoffmann | Interferons and viruses: an evolutionary arms race of molecular interactions[END_REF]. This first line of host response suppresses viral spread and jump-starts the adaptive immune response.

All cells possess signaling pathways designed to trigger antiviral responses against invading viruses. Nonetheless, virtually all viruses have evolved mechanisms to inhibit these hostsensing pathways within cells that they infect [START_REF] Garcia-Sastre | Ten Strategies of Interferon Evasion by Viruses[END_REF]. For example, hepatitis C (HCV), dengue (DENV) and Zika (ZIKV) viruses encode viral proteases, which inactivate the adaptors of host antiviral sensing [START_REF] Garcia-Sastre | Ten Strategies of Interferon Evasion by Viruses[END_REF]. Despite viral countermeasures, cytokines and ISGs are detected in infected humans and play pivotal roles in infection clearance and pathogenicity, suggesting the existence of alternative pathogen-sensing mechanisms [START_REF] Martina | Dengue virus pathogenesis: an integrated view[END_REF][START_REF] Snell | Type I Interferon in Chronic Virus Infection and Cancer[END_REF][START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF]. Along this line, we and others uncovered that plasmacytoid dendritic cells (pDCs) produce robust levels of IFN-I in response to physical contact with infected cells [START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF]. This response is unopposed by viral products as pDCs are not productively infected. pDCs function as sentinels of viral infections and are a major early source of IFN-I in vivo during viral infections [START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF]. This response is predominantly triggered via recognition of viral RNA and DNA species by endolysosome-localized TLR7 and TLR9, respectively. pDC response is pivotal for control of viral infection as recently demonstrated e.g., for DENV [START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF][START_REF] Webster | Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses[END_REF]. IFN-I production by pDCs also contributes to several autoimmune or inflammatory diseases, and pDCs are pivotal in cancers [START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF]. Despite this large spectrum of regulatory functions of pDCs, the molecular bases controlling their activation are still largely enigmatic.

We previously demonstrated that immunostimulatory viral RNA can be transferred by noncanonical/non-infectious carriers from infected cells to pDCs, leading to IFN production via the TLR7 sensor. These PAMP-carriers include exosomes and immature viral particles in the context of HCV and DENV, respectively [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. The activation of pDCs by exosomal transfer of viral RNA has been also shown for the sensing of genetically distant viruses [START_REF] Feng | Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells[END_REF][START_REF] Wieland | Human plasmacytoid dendritic cells sense lymphocytic choriomeningitis virus-infected cells in vitro[END_REF]. Importantly, while various types of carrier can transfer PAMPs, these pDC activations require physical contact with infected cells [START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF]. For example, HCV infected cells secrete exosomes into the culture supernatant at concentrations that are below an excitatory threshold that could be reached in the intercellular space during cell contacts [START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. In the context of DENV, viral components (i.e. envelope proteins) accumulate at the cell contact between infected cells and pDCs [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF]. Albeit not universal, the cell contact is increasingly recognized as a requirement for pDC-mediated antiviral state triggered by many genetically distant RNA viruses (e.g., Flaviviridae, Picornaviridae, Arenaviridae, Retroviridae, Togaviridae) and in different species [START_REF] Bruni | Viral entry route determines how human plasmacytoid dendritic cells produce type I interferons[END_REF][START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF][START_REF] Feng | Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells[END_REF][START_REF] Feng | Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells[END_REF][START_REF] Lepelley | Innate sensing of HIV-infected cells[END_REF][START_REF] Takahashi | Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection[END_REF][START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF][START_REF] Webster | Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses[END_REF][START_REF] Wieland | Human plasmacytoid dendritic cells sense lymphocytic choriomeningitis virus-infected cells in vitro[END_REF], nonetheless how the cell contact mediates pDC response is still largely unknown.

Here, we explored the molecular mechanisms underlying the establishment of contacts between pDCs and infected cells, its modulation and impact on the activation of an antiviral state using DENV, HCV and ZIKV as viral models. Our results uncovered that pDCs established cell adhesion with infected cells via αLβ2 integrin complex and ICAM-1. We showed that regulators of actin network polarized together with components of the endocytosis machinery at contact sites. These actin-mediated reorganizations define a remodeled territory acting as a platform for the transfer of PAMP-carriers to the endolysosomal TLR7, leading to pDC-mediated antiviral response. We further demonstrated that the molecular rearrangements at contact sites were potentiated by the TLR7-mediated response, which thus acts as a positive feed-forward regulation of pDC activation. The formation of interferogenic synapse is thus a pDC attribute critical for their robust IFN response.

Results

The αLβ2 integrin/ICAM-1 adhesion molecules are involved in pDC activation by contacts with DENV infected cells

The pDC response to many viruses requires contact, so we sought to define the molecular basis for establishment of physical contacts between pDCs and infected cells. We tested whether cell adhesion complexes can establish pDC/infected cell contact leading to pDC activation. We analyzed the surface expression of selected representatives of cell adhesion/homing complexes, known to be key for pDC functions, e.g., migration and antigen presentation [START_REF] Sozzani | Trafficking properties of plasmacytoid dendritic cells in health and disease[END_REF][START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF]. L-selectin (CD62L), αL integrin (ITGAL, CD11a) and intercellular adhesion molecule (ICAM)-1 (CD54) were all expressed at the pDC surface (Figure S1A). ICAM-1 was also present at the surface of the donor cells (i.e., infected and uninfected cells), while E-cadherin was present only on donor cell surface (Figure S1A). Next we tested the role of cell adhesion molecules in pDC IFNα production induced by contact with DENV infected cells in antibody-mediated blocking assays. The blockade of L-selectin and E-cadherin had no significant impact on pDC IFNα production induced by DENV infected cells (Figure 1A andS1B). In sharp contrast, inhibition of both αL integrin and its ligand ICAM-1 [START_REF] Rothlein | A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1[END_REF] prevented the pDC IFNα response in a dose-dependent manner, along with production of IFNβ and type III IFNs (IL-29/28A/28B) (Figure 1A-B andS1B-C). Viral infectivity and RNA levels were maintained upon similar treatment of infected cells in absence of pDCs, thus ruling out nonspecific effects on DENV replication (Figure 1A, S1B-C). The regulation of the establishment of pDC-infected cell contacts by the αL integrin and its ligand ICAM-1 was determined by imaging flow cytometry technology (Figure S1D). We showed that blocking of both αL integrin and ICAM-1 significantly prevented contacts of pDCs with DENV infected cells (Figure 1C).

Next, to define how the pDC response to contact with DENV infected cells inhibits viral propagation, we set-up an assay consisting of pDCs cocultured with GFP -/DENV infected cells and GFP + /uninfected cells. The results demonstrated that pDC response readily prevented viral spread from GFP -/DENV infected cells to the cocultured GFP + /uninfected cells, and consistently diminished the replication in GFP -cells, i.e., infected prior to the coculture (Figure 1D-E). The inhibition of pDC/infected contact via blockage of the αL integrin restored the viral propagation to levels comparable to those in the absence of pDC (Figure 1D-E). Consistently, the analysis of infectious virus production by DENV infected cells demonstrated similar inhibition by cocultured pDCs (compared to the absence of pDCs) and the restoration of the viral production upon inhibition of αL integrin (Figure 1F). This indicates that the establishment of cell-contact via adhesion molecules is required for pDCmediated antiviral response.

Further, we assessed the impact of silencing ICAM-1 expression in infected cells. pDC IFNα production was strongly reduced when ICAM-1 expression was silenced in DENV infected cells (Figure 1G-H). In absence of pDC, reduced ICAM-1 expression has no impact on virus replication, yet slightly diminished the release of DENV (Figure 1H). Although we cannot formally exclude that decreased viral production could contribute to lower pDC activation, pDC IFNα production was disproportionally reduced when ICAM-1 expression was silenced in cocultured infected cells (Figure 1H). Of note, this is in accordance with the results obtained with ICAM-1 blocking antibody suggesting a regulation by ICAM-1 independent of viral production (Figure 1A andS1B-C). Together, our results identified αL integrin/ICAM-1 as a key cell adhesion complex for the sensing of DENV infected cells by pDC leading to a potent antiviral response.

Cell polarity of the adhesion molecules at the pDC/DENV infected cells contact

Next we demonstrated that αL integrin specifically accumulated at the contact site of pDC/DENV infected cell (i.e., over 90% of the analyzed contacts), as opposed to marginal accumulation at contact of CD123, a surface molecule not involved in cell adhesion (Figure 2A-C andS2A-B). The αL integrin forms complex with β2 integrin, which is known to undergo conformational rearrangement to an open form compatible with high-affinity interaction with its ligands, including ICAM-1 [START_REF] Shimaoka | Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation[END_REF]. The open/high-affinity conformation of αLβ2 integrin complex polarized at the contact site of pDC/infected cell on the pDC side (Figure 2D-F andS2C-D). Of note, cell polarity of αL integrin and the high-affinity conformation of αLβ2 integrin complex was absent when pDCs were not in contact with infected cells, shown by the quantification of αL integrin accumulation versus diffuse localization around the pDCs (Figure 2G-I andS2E-F), and thus validating the specificity of the polarization upon cell contact. Further, in agreement with the engagement of the αLβ2 integrin complex with its ligand, ICAM-1, the latter was detected at contact on the surface of infected cells apposed to polarized αL integrin on pDC side (Figure 2J-L). Our results demonstrate that the open/high affinity conformation αLβ2 integrin complex accumulates at contact, facing its partner ICAM-1.

The αLβ2 integrin/ICAM-1 adhesion molecules are required for pDC activation by distinct viruses

The activation of pDCs by HCV infected cells also requires physical contact, yet this involves a distinct PAMP-carrier i.e., exosome (versus immature viral particles for DENV) [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. We thus tested whether αLβ2 integrin/ICAM-1 adhesion complexes were also required for pDC activation by HCV infected cells. Similar to DENV, ICAM-1 is present at the surface of HCV infected cells (Figure S3D) and blocking of both αL integrin and ICAM-1 diminished the pDC IFNα response to HCV infected cells, in doseresponse experiments, without any non-specific effect on HCV viral production -in absence of pDC (Figure 3A andS3A). Consistently, blocking both αL integrin and ICAM-1 significantly prevented contacts of pDCs with HCV infected cells (Figure 3B). Although the presence of ICAM-1 on PAMP-carriers could additionally contribute to their tethering to the surface of the pDCs, altogether these results demonstrated that the αLβ2 integrin/ICAM-1 interaction regulates the formation of pDC-infected cell conjugate and are required for pDC activation, independently of the nature of PAMP-carrier. In sharp contrast, anti-αL integrin and anti-ICAM-1 did not prevent pDC IFNα production triggered by cell-free virus i.e., influenza virus (Figure 3C), suggesting that αLβ2 integrin/ICAM-1 interaction regulates cell contact mediated-pDC activation.

Next we extended our demonstration to ZIKV, which also represents an important health concern [START_REF] Miner | Zika Virus Pathogenesis and Tissue Tropism[END_REF]. Using epidemic ZIKV strains (i.e., isolated from patients in Brazil and French Polynesia; H/PF), we demonstrated that robust pDC IFNα productions were induced when pDCs were in physical contact with ZIKV infected cells (Figure 3D; coculture set-up). On the contrary, no IFNα response was detected when pDCs were exposed to ZIKV infectious supernatants (SN) or upon physical separation of pDCs from ZIKV infected cells by a semi-permeable membrane in transwell (TW) experiments (Figure 3D). In accordance with TLR7 localized in endolysosome, and like DENV/HCV, pDC activation by ZIKV infected cells involved endocytosis (Figure S3B). Importantly, we showed that, similar to what is observed for DENV/HCV, pDC response to ZIKV infected cells is mediated by αL integrin/ICAM-1 adhesion molecules as shown by the reduced production of IFNα, IFNβ and type III IFNs by antibody blocking (Figure 3F-G, S3C andS3E).

Next, we demonstrated that, similar to the observation with DENV, pDC response to contact with ZIKV infected cells prevented ZIKV transmission to uninfected cells (GFP + ), replication in the cells infected prior to coculture (GFP -) and viral production (Figure 3H-I andS3F). Of note, the inhibition of αL integrin in infected cell/pDC cocultures greatly restored viral propagation to levels similar to those in the absence of pDCs (Figure 3H-I andS3F), indicated that the establishment of cell-contact is required for pDC-mediated antiviral response against ZIKV. Together, our results demonstrate that the establishment of pDC/infected cell interactions via cell adhesion complex is a prerequisite for the antiviral function of pDC in response to cells infected by distinct viruses.

Polarization at contact site of actin network and regulators contribute to pDC response

The engagement of integrins with their ligand can induce the local recruitment of the actin network, notably via Arp2/3 complex, which mediate actin nucleation by branching of the actin filaments within network [START_REF] Demali | Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion[END_REF][START_REF] Demali | Force transmission at cell-cell and cell-matrix adhesions[END_REF][START_REF] Rotty | New insights into the regulation and cellular functions of the ARP2/3 complex[END_REF][START_REF] Vicente-Manzanares | Integrins in cell migration-the actin connection[END_REF]. This is in line with our preceding observations for the presence of actin at pDC/DENV infected cell contact [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF]. Here, we showed that actin network accumulated at cell contacts, using both HCV infected cells and the subgenomic replicon (SGR) model for HCV replication (Figure S4A-D). HCV SGR cells do not produce virus but trigger pDC IFN response, as previously reported [START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF][START_REF] Takahashi | Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection[END_REF] and thus demonstrating that cell polarity at contact is independent of viral production (Figure S4A-B). Non-specific polarized immunodetection was ruled out by the simultaneous analysis of another intracellular component, the protein disulfide-isomerase (PDI), an ER marker and by the expected decrease of actin clustering upon treatment with inhibitor of actin polymerization i.e., Latrunculin B (Figure S4B,D).

We thus sought to define the subcellular localization of key regulators of actin network scaffolding at the plasma membrane. We first selected the phosphatidylinositol 4,5 biphosphate (PI(4,5)P2) because it is a plasma membrane-localized modified lipid known to recruit actin-adaptor proteins and to regulate the clustering of integrins as well as clathrinmediated endocytosis [START_REF] Jost | Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation[END_REF][START_REF] Martel | Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides[END_REF][START_REF] Posor | Phosphoinositides in endocytosis[END_REF][START_REF] Vicente-Manzanares | Integrins in cell migration-the actin connection[END_REF]. The type I phosphatidylinositol-4-phosphate-5-kinase α (referred to as PIP5KIα) generates PI(4,5)P2 from its PI4P precursor [START_REF] Doughman | Phosphatidylinositol phosphate kinases put PI4,5P(2) in its place[END_REF]. We showed that PI(4,5)P2 and PIP5KIα co-clustered at the contact sites of pDCs with cells infected either by DENV or HCV, or with HCV replicating (rep) cells (Figure 4A-L). The 3Dreconstruction of consecutive Z-stacks further revealed that the co-clusters were present on the side of pDCs (Figure 4C,G,K). The quantification of PI(4,5)P2/PIP5KIα co-clustering revealed that intact actin network was required for the polarization at contact, as demonstrated by the decrease in phenotype when using inhibitors of Arp2/3 complex and actin polymerization (Figure 4D,H,L). Of note, the frequency of PI(4,5)P2/PIP5KIα co-clustering at pDC/infected cell contacts was lower compared to the polarization of the αL integrin (Figure 2C and2I), likely reflecting the transient nature and/or differential kinetics of these recruitments at contact as compared to the adhesion complexes.

Next, we tested the functional role of actin network regulators, including Arp2/3 complex, and the Rho GTPase CDC42, which also regulates actin polymerization, and along with PI(4,5)P2, is known to induce actin nucleation via Arp2/3 [START_REF] Higgs | Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex[END_REF][START_REF] Rohatgi | The interaction between N-WASP and the Arp2/3 complex links Cdc42dependent signals to actin assembly[END_REF][START_REF] Rozelle | Phosphatidylinositol 4,5bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3[END_REF]. The pharmacological inhibition of either Arp2/3 complex, CDC42 or actin polymerization prevented pDC IFNα production induced by DENV or HCV infected cells, without non-specific effect on the viral replication of DENV and HCV in absence of pDC (Figure 4M-O, S4E-G andS4I-L). Of note, similar pharmacological inhibition of Arp2/3 did not prevent pDC IFNα production triggered by cell-free influenza virus (Figure 4P), implying that Arp2/3-mediated actin nucleation is pivotal for pDC response induced by contact with infected cells. This assumption was validated by the reduced frequency of pDC/infected cell contacts upon inhibition of Arp2/3 and actin polymerization (Figure 4Q-R andS4H). Our results underscore that integrin-mediated adhesion and rearrangement at contact of actin network via notably Arp2/3, are pivotal for the establishment and/or reinforcement of the cell contacts with infected cells and subsequent pDC activation, presumably by a concerted structuring of the contact site.

Components of the endocytosis machinery polarize at contacts

The immunostimulatory viral RNAs are transferred by vesicles (i.e., exosome or immature virion) from infected cells to pDCs and induce IFN-I response via clathrin-mediated endocytosis by pDCs and recognition through endolysosome-localized TLR7 (Figure S3B) [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF][START_REF] Takahashi | Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection[END_REF][START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF]. Of note, PI(4,5)P2, which clustered at contact (Figure 4A-L) is known to mark the plasma membrane and, together with cargo proteins, is pivotal for the initiation of clathrin-coated pit formation and consistently, PIP5KIα via PI(4,5)P2 synthesis control the rate and growth of clathrin-coated pits [START_REF] Jost | Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation[END_REF][START_REF] Posor | Phosphoinositides in endocytosis[END_REF]. We thus tested whether the components of the endocytosis machinery could polarize at contact and thereby facilitate the internalization of PAMP-carriers by pDCs. We observed co-clusters of PI(4,5)P2 and Early Endosome Antigen 1 (EEA1) at the contact sites of pDCs with DENV infected cells, and similarly with HCV replicating cells (Figure 5A-F, 5J-K andS5A-B). The 3Dreconstruction of consecutive Z-stack imaging suggested that the PI(4,5)P2/EEA1 coclustering was present on the pDC side of the contact (Figure 5C and5F). Similarly, clathrin co-clustered with actin on the pDC side, as revealed by the 3D-reconstruction of contact sites (Figure 5G-I, 5L andS5C-F). The cell polarity of both these markers at contact required an intact actin network, as demonstrated by using inhibitors of Ap2/3 complex and actin polymerization (Figure 5J-L). Together our results suggest that components of the endocytosis machinery polarize at the contact between pDCs and infected cells, thus representing a functional remodeling to favor a localized endocytosis of PAMP-carriers leading to pDC activation via the endolysosomal TLR7.

TLR7-induced pDCs establish sustained contact with virally infected cells

The rearrangement of the contact site into a specialized territory on the side of pDCs suggests the existence of a dynamic process within pDCs when in contact and sensing infected cells.

We hypothesized that the sensing by pDCs of PAMP-carriers released by infected cells, and thereby the activated state of the pDCs could control the contact establishment and/or the polarization of pDC cellular machineries. The frequency of pDCs conjugated with infected cells was significantly higher as compared to uninfected cells (Figure 6A). Similar results were obtained for DENV and HCV infected cells, thus showing that the potentiation of contacts by sensing of virally infected cells is not virus-restricted.

To further demonstrate that pDC contact with infected cells are more stable compared to uninfected cells, we developed a live-imaging approach (using spinning-disk confocal microscopy) to quantify the duration of pDC contact with infected cells as compared to uninfected cells (Figure S6A). Distinct categories of contact duration were observed, including short-lived contacts (<100 min), intermediate duration contacts of low frequencies and sustained contact (>350 min) (Figure 6B-I). Importantly, the frequency of long-lasting contacts was higher when pDCs were cocultured with DENV infected cells as compared to contact with uninfected cells (Figure 6B and6F). In accordance, as highlighted in tracking motion analysis pDC motility, and consistently the number of contact per pDCs, were reduced when cocultured with infected as compared to uninfected cells (Figure S6B-C andS6K).

Similar results were obtained in the context of pDC cocultured with HCV replicating cells (Figure 6C and 6G), suggesting this regulation is not virus-restricted and independent of the type of PAMP-carriers. Live-imaging experiments with blocking anti-αL integrin revealed that the long-lasting contacts with infected cells involved αL integrin (Figure 6D, 6H andS6E).

Furthermore, the blocking anti-αL integrin decreased the frequency of conjugates even when added later in course of pDC/infected cell coculture and leading to reduced IFNα production by pDCs (Figure S6G-I).

Our results demonstrated that pDCs established sustained contact with infected cells, thus implying that a molecular regulation of the contact by either sensing of the infected status of cells and/or the activating state of pDCs promotes their retention at the contact of infected cells. We thus tested the regulation of the pDC/infected cell contact by TLR7-induced signaling. To address this question, we used a specific TLR7 inhibitor (i.e., IRS661), which diminished the pDC IFNα production induced by DENV infected cells, as previously reported [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF]. We found that the inhibition of TLR7 signaling diminished the frequency of the long-lived contacts of pDCs with infected cells (Figure 6B, 6F and S6D).

Inhibition of TLR7 signaling thus reverted the duration of pDC contacts to those observed with uninfected cells. Accordingly, TLR7 agonist increased the frequency of long-lasting contact of pDCs with non-infected cells, without a marked impact on pDC velocity (Figure 6E, 6I, S6F, S6J and S6L-M). Altogether our results demonstrate that TLR7-signaling induced by recognition of infected cells exerts a feed-forward regulation leading to sustained contact of pDCs with infected cells.

Recognition of viral infection favors polarization of cellular components at contacts via TLR7-induced signaling

Next, we tested whether the sustained contact observed with infected cells was associated with increased polarization of the identified cell adhesion molecules at the contact sites. The total surface expression of ICAM-1 did not markedly change when comparing uninfected versus cells infected by the different viral models (Figure S1A and S3D), neither did the level of αL integrin on pDC cell surface when cocultured with either infected or uninfected cells (Figure S7A-B). Importantly, the co-accumulation at contact of αLintegrin/ICAM-1 significantly augmented when pDCs were in contact with DENV or HCV infected cells as compared to uninfected cells (Figure 7A-B). In accordance with the known interplay between αLintegrin/ICAM-1 and actin network/regulators [START_REF] Demali | Force transmission at cell-cell and cell-matrix adhesions[END_REF], we showed that the polarization of actin network regulators including PI(4,5)P2/PIP5KIα increased upon contact with DENV infected cells and similarly for HCV infected and HCV replicating cells (Figure

7C, 7F, 7I).

We demonstrated that the inhibition of TLR7-induced signaling prevented the establishment of long-lived contact of pDCs with infected cells (Figure 6B and6F). By analogy to insideout signaling defined in the context of other cell contact including neurological and immunological synapses [START_REF] Hogg | The insider's guide to leukocyte integrin signalling and function[END_REF][START_REF] Park | Integrins in synapse regulation[END_REF], we hypothesized that TLR7-induced signaling might promote the cell polarity. We showed that the inhibition of TLR7 significantly reduced coclustering of PI(4,5)P2/PIP5KIα at contact, along with endocytosis components, including the coclustering of PI(4,5)P2/EEA1 and clathrin/actin (Figure 7C-D, 7F-G, 7I-L). The coclusters of αL integrin and the high-affinity conformation of αLβ2 integrin complex together with the endocytosis components (i.e., clathrin and EEA1, respectively) at the contact site of pDCs were also subjected to regulation by TLR7-mediated pDC activation (Figure 7E, 7H andS7C-D). This further implies a connection between components of the cell adhesion, actin network and the endocytosis machinery modulated by TLR7 signaling. Similar results were obtained using DENV, HCV and ZIKV infected cells (Figure 7 and S7), highlighting that the control by TLR7-mediated signaling of pDC polarity is neither restricted to specific virus nor PAMP-carrier. Our results demonstrate that the sensing of infected cells via TLR7 sensor induces cell polarity of regulators of adhesion, actin network and endocytosis machinery at contact on the pDC side, thus acting as a positive feedback regulation for the efficient transfer of PAMP-carriers and pDC response.

Discussion

pDCs are important mediators of IFN-I response. We demonstrate that pDC activation is mediated by the establishment of sustained contact with infected cells via cell adhesion molecules, αLβ2 integrin complex and ICAM-1. This is associated with the polarization of the actin network and its regulators as well as components of the endocytosis machinery, at the contact site within pDCs, requiring and facilitating sensing of viral RNA by endolysosomelocalized-TLR7. Furthermore, we provide compelling evidence that TLR7-induced signaling in pDCs regulates the rearrangement of a polarized platform at contact site and prolongs cell contacts, thus acting as a feed-forward regulation for the transfer of PAMP-carrier to the pDCs. Cell polarity is thus a previously unappreciated primary attribute of pDCs necessary to sense virally infected cells and respond by a robust antiviral response, which prevents viral spread. We propose to call this functional structuring the interferogenic synapse, by analogy to previously described synapses, while functionally driving a distinct cellular response.

PAMP transfer via physical contact and cell polarity: the interferogenic synapse

Different types of synapses have been defined including neurological, immunological and virological synapses, which differ in composition and signal transferred but are all specialized cell junctions to convey information between cells. Across all synapses, this informationsharing capacity is supported by adhesion complexes, thereby focusing signaling, secretion and internalization at the point of cell contact [START_REF] Dustin | Signaling and Polarized Communication Across the T Cell Immunological Synapse[END_REF][START_REF] Hogg | The insider's guide to leukocyte integrin signalling and function[END_REF][START_REF] Park | Integrins in synapse regulation[END_REF]. Likewise, we showed that adhesion molecules accumulate at the contact site of pDC/infected cell, and are required for pDC IFN response. Moreover, identical to the immunological synapse, the αLβ2 integrin complex is present on the signal recipient cells (pDCs vs T cells) and ICAM-1 on the signal delivering cells (infected cells vs antigen presenting cells) [START_REF] Dustin | Signaling and Polarized Communication Across the T Cell Immunological Synapse[END_REF]. Again like all synapses, the actin network (and regulators) polarizes at contact and acts as a functional structuring platform. Analogous to the neurological synapse, the mechanism of pDC activation involved a secreted activating signal. In accordance with the transfer of PAMPs via vesicles and the endolysosomal localization of the sensor (i.e., TLR7), endocytosis components polarize at contacts. Cell polarity of vesicular trafficking is a hallmark of other synapses, primarily defined for secretory pathways, e.g., local secretion of synaptic vesicles or lytic granules at the neurological and immunological "lytic/NK" synapses, respectively. Additionally, the endocytosis machinery also polarizes at the level of the immunological synapse and regulates T cell activation via the internalization and recycling of the T cell receptor (TCR) [START_REF] Compeer | A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation[END_REF][START_REF] Das | Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes[END_REF]. Importantly, the feed-forward regulation by TLR7-induced signaling is reminiscent of the stabilization of the cell polarity and contact described for immunological and neurological synapses, referred to as inside-out signaling and induced by the response/activation in recipient cells [START_REF] Dustin | Signaling and Polarized Communication Across the T Cell Immunological Synapse[END_REF][START_REF] Hogg | The insider's guide to leukocyte integrin signalling and function[END_REF]. For example, T-cell receptor engagement at the immunological synapse reinforces cell adhesion by the activation of integrin ectodomain [START_REF] Dustin | Signaling and Polarized Communication Across the T Cell Immunological Synapse[END_REF][START_REF] Hogg | The insider's guide to leukocyte integrin signalling and function[END_REF].

Therefore, the interferogenic synapse presents hallmarks of other synapses, while being deployed to achieve a distinct cellular response, the IFN response, thus representing a previously unappreciated type of synapse.

Scanning function of pDCs regulated by a TLR7-centered feed-forward signaling.

We propose that the immune surveillance by pDCs involves a scanning function by physical cell contacts (Figure S8). In this 'scanning' model, a pDC encounters a cell and initially establishes a transient contact. When the cell is not infected (i.e., in the absence of transfer of PAMPs), the non-activated pDC resumes its motion. By contrast, when a pDC encounters an infected cell, PAMP transfer during the initial contact activates TLR7 in the pDC. In turn, TLR7-mediated signaling potentiates the structural reorganization at the contact site. These molecular rearrangements include polarized endocytosis components, which can facilitate the subsequent transfer of PAMPs to pDCs and further strengthen TLR7 activation. TLR7induced signaling primes the establishment of sustained pDC/infected cell contact, which in turn further promotes TLR7 activation, leading to a feed-forward regulatory loop culminating in sustained contacts as well as robust and localized IFN production. As such, the sensing mechanism by pDCs can be seen as a "relay switch": a relatively small initial 'PAMP current' leads to a 'high amperage' flow of PAMPs, via a functional platform for efficient PAMP transfer at the contact sites leading to a potent pDC-mediated antiviral response. TLR7-induced signaling is expected to induce a bifurcated signaling: the transcriptional activators IRF7 and nuclear factor-κB (NF-kB), respectively leading to type I/III IFNs and other inflammatory cytokine response. One might hypothesize that the autocrine/paracrine action of IFNs (and/or subsequently induced-ISGs or other cytokines) could potentiate the interferogenic synapse. Alternatively, the signaling downstream of TLR7, including the phosphorylation cascade might directly reinforce cell contact/polarity [START_REF] Olayioye | Multi-level control of actin dynamics by protein kinase D[END_REF][START_REF] Park | Protein kinase D1 is essential for MyD88dependent TLR signaling pathway[END_REF][START_REF] Saitoh | TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells[END_REF].

Local response to viral infection by pDCs.

Systemic and massive productions of IFNs and other inflammatory cytokines are known to be detrimental to the host, as they correlate with systemic homeostatic dysfunction, including tissue damage e.g., increased vascular permeability in the context of DENV [START_REF] Costa | Inflammatory and Innate Immune Responses in Dengue Infection: Protection versus Disease Induction[END_REF]. Here we uncovered that the organization of a functional territory in pDCs at the contact site with infected cells is required for IFN-I production, thus implying that this antiviral response is confined to the proximity of infected cells. This is reminiscent of previous reports showing a pivotal in vivo antiviral regulation by early pDC response, despite undetectable or very transient/limited pDC-derived IFN-I at systemic levels [START_REF] Swiecki | Plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral NK and CD8(+) T cell accrual[END_REF][START_REF] Webster | Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses[END_REF], thus positioning pDC IFN-I secretion as a response likely confined to the infected site. Therefore, the cell contact-dependent robust pDC response could have evolved in favor of the host fitness to locally respond at the infected sites, and thereby to thwart the otherwise harmful systemic IFN and inflammatory responses.

Albeit not universal, the requirement for cell contact with infected cells is increasingly recognized for pDC activation by a large spectrum of genetically divergent viruses [START_REF] Webster | Cell-Cell Sensing of Viral Infection by Plasmacytoid Dendritic Cells[END_REF]. It is therefore likely that the interferogenic synapse that we describe here for pDC response to DENV, ZIKV and HCV via distinct PAMP-carriers, is also at play for pDC activation by other viruses. Additionally, pDCs can participate in the priming of both immunogenic and tolerogenic adaptive immunity, being also increasingly recognized as pivotal mediators in pathogenesis of autoimmune diseases and cancers [START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF]. It is possible that the interferogenic synapse is of importance for pDC-mediated sensing in these other contexts. were 800x10 6 and 2x10 6 cells, respectively, with a typical purity of >95% pDCs. Isolated pDCs were maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS, 10 mM HEPES, 100 units/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, non-essential amino acids and 1 mM sodium pyruvate at 37°C/5% CO2.

The down regulation of ICAM-1 in huh7.5.1 cells was achieved using lentivirus-based vector expressing shRNA against ICAM-1 (TRCN0000372477, target sequence:

GCCCGAGCTCAAGTGTCTAAA, Sigma-Aldrich), produced using 293T cells (female), as we previously described [START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. Cells were transduced 4 days prior to infection by DENV for 48 hours and followed by coculture with pDCs.

Preparation of viral stocks and infections.

Viral stocks of HCV JFH1 strain [START_REF] Zhong | The efficiency of RNA extraction and reverse transcription-real-time quantitative PCR (RT-qPCR) was controlled by the addition of carrier RNAs encoding Xef1α (xenopus transcription factor 1α) in vitro transcripts in supernatants diluted in GTC buffer. DENV, HCV and ZIKV RNA, Xef1α and intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were determined by RT-qPCR using cDNA synthesis and qPCR kits (Life Technologies) and analyzed using StepOnePlus Real-Time PCR system (Life Technologies), using previously described primers[END_REF] and the prototypic DENV-2 strain New Guinea C (NGC) (AF038403) [START_REF] Kroschewski | Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain[END_REF] were produced as previously described [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. The clinical isolate of epidemic strains of ZIKV including from Brazil (PE243_KX197192) [START_REF] Donald | Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil[END_REF] The inhibitors used were Latrunculin B (Sigma-Aldrich); Arp2/3 complex inhibitor I (CK-666, Merck Millipore); CDC42 inhibitor (ML141, Sigma-Aldrich); chlorpromazine (CPZ, Sigma-Aldrich), dynasore (Sigma-Aldrich); TLR7 inhibitor IRS661 (5'-TGCTTGCAAGCTTGCAA GCA-3') synthesized on a phosphorothioate backbone [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF].

The other reagents were TLR7 agonist (Imiquimod; IMQ; Invivogen); Ficoll-Hypaque (GE that the polarization of endocytic components is slightly higher at this time point. After 4%

PFA fixation and three PBS washes, immunostainings were performed without additional permeabilization step, as previously [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Meertens | The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry[END_REF]. After blocking step (PBS 3% BSA), primary antibody antibodies were diluted in 3% BSA-PBS and added to the cell for one hour incubation at room temperature. Primary antibodies used in this study include mouse anti-actin (20 µg/mL); rabbit anti-PDI (2 µg/mL); mouse anti-PI(4,5)P2

(20 µg/mL); goat anti-PIPK1α (4 µg/mL); rabbit anti-clathrin (2 µg/mL); rabbit anti-EEA1 (2 µg/mL); mouse anti-ICAM-1 (10 µg/mL); mouse anti-αL integrin subunit (clone 38 LSBio at 5 µg/mL); mouse anti-integrin β2 I-like domain, i.e., open/high-affinity conformation of αLβ2 integrin complex [START_REF] Nordenfelt | Coordinated integrin activation by actin-dependent force during T-cell migration[END_REF][START_REF] Salas | Transition from rolling to firm adhesion can be mimicked by extension of integrin alphaLbeta2 in an intermediate affinity state[END_REF][START_REF] Sen | Ligand and cationinduced structural alterations of the leukocyte integrin LFA-1[END_REF] (clone m24 at 5 µg/mL); APC-conjugated mouse anti-CD123 (clone AC145 at 5 µg/mL). After three washes with PBS, cells were incubated with the appropriate anti-mouse, rabbit or goat antibody

[conjugated with Alexa 488-, 555-or 647-fluorochrome] or Alexa-conjugated antibody against specific mouse isotypes [i.e., anti-IgG1 for detection of m24 epitope of Lβ2 integrin complex and anti-IgG2a for detection of the integrin αL subunit] at 2 µg/mL in 3% BSA-PBS and add to the cells along with Hoechst diluted at 1:1000 (Molecular Probes) for 1 hour incubation at room temperature. After three gentle washes with PBS, cells were observed with a Zeiss LSM 710 laser scanning confocal microscope. The quantification of the phenotypes defined to as clusters or co-clusters at the contact, outside contact or no cluster/co-clusters were performed using Image J software package (http://rsb.info.nih.gov/ij); Results were validated in a "double-blind" set-up. IMARIS (Bitplane Inc.) software package with coloc tools was used for the 3D-reconstructions of the consecutive Z-stacks.

using Zeiss Axiovert 135 or Olympus CKX53 microscopes.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data are presented as the mean values ± standard deviation (SD). The figure legends report the number of independent experiments.

Statistical analysis was performed using R software environment for statistical computing and graphics, version 3.3.2. For levels of IFNα productions, viral RNA levels, viral titers and cell surface expression, the values were considered relative to untreated references for each independent experiment and analysis using an one-way ANOVA on ranks (Kruskal-Wallis test). When the test was significant, we used the Nemenyi post hoc test for multiple comparisons of mean rank sums to determine which contrast(s) between individual experimental condition pairs was significant. For the statistical analysis of polarization of structure/component at contact (assessed by confocal microscopy analysis), the distribution of the analyzed pDCs in contact with infected cells that presents co-clusters at the contact (without or with additional cluster outside contact) versus no co-cluster at contact (without or with cluster outside contact) were compared using Fisher's exact test for count data. Similar results were obtained when using the test for equality of proportions. Statistical analysis of viral transmission from GFP -infected cells to GFP + uninfected cells was performed using one-way ANOVA, followed by Tukey multiple comparisons of means. The results of imaging flow cytometry (using Image Stream X technology) were analyzed using the Wilcoxon Unpaired test. For statistical analysis of the duration of contact (assessed by live-imaging using spinning-disk confocal microscopy analysis), a test for pairwise equality of proportions was applied to determine whether the duration of contact depends on the experimental conditions by comparing the frequency of the indicated time lengths. Data considered significant demonstrated adjusted p-value by False Discovery Rate (FDR) less than 0.05.

Figure 1 .

 1 Figure 1. Identification of adhesion molecules involved for pDC-mediated antiviral response against DENV. (A) Quantification of IFNα in supernatants of pDCs cocultured with DENV infected Huh-7.5.1 cells that were treated or not with blocking antibodies against L-selectin, E-cadherin, αL integrin or ICAM-1 at 10 μg/ml. The intracellular and extracellular viral RNA levels and extracellular infectious virus production by DENV infected cells treated with blocking antibodies were analyzed in absence of pDC, but with treatment conditions similar to the analysis of IFNαproduction in cocultures (i.e., incubation time and concentration). Results are expressed as percentages relative to untreated cells; mean±SD; 4-7 independent experiments. The statistically significances reflected by p-values are indicated in the table; not significant (NS; p ≥ 0.05). (B) Quantification of IFNβ and type III IFNs (IL-29/28A/28B) in supernatants of DENV infected cell/pDC cocultures treated, or not, with blocking anti-αL integrin as in A. Results expressed as pg/ml; mean±SD. (C) Quantification of conjugates between pDCs and DENV infected cells by Image Stream X technology, as exemplified in Figure S1D. Celltracer violet stained-pDCs were cocultured with GFP +

Figure 4 .Figure 6 .

 46 Figure 4. Actin network regulators polarize at the contact and an intact actin network is required for pDC response. (A-L) Confocal microscopy imaging of immunostained PI(4,5)P2 and PIP5KIα in 4-5 hour cocultures of pDCs (DiI stained) and DENV (A-C), HCV infected cells (E-G) and HCV replicating (rep) Huh7.5.1c2 cells (I-K), nuclei (Hoechst). Imaging of PI(4,5)P2 and PIP5KIα projected on the phase contrast (A, E, I), confocal stacks of magnification of yellow-boxed pDC (B, F, J) and 3D-reconstruction of consecutive Zstacks with colocalization of PI(4,5)P2/PIP5KIα in white (C, G, K). (D, H, L) Quantification of the phenotype of PI(4,5)P2/PIP5KIα co-clustering as indicated (i.e., co-cluster at the contacts, outside the contact or no co-cluster) in pDCs cocultured with infected cells and treated with inhibitors of Arp 2/3 complex (CK-666; 60 μM) or actin polymerization (Latrunculin B, Lat B; 1 μM). Results of 3 independent experiments (D; n>53, H; n>40, L; n>21 analyzed contacts per condition) are expressed as the percentage of total number of analyzed pDCs in contact; mean±SD; p-value as indicated. (M-P). Quantification of IFNα in supernatants of pDCs cocultured with DENV infected (M), HCV infected (N) or HCV rep Huh7.5.1c2 cells (O) treated or not with inhibitor of Arp 2/3 complex (CK-666). The levels of

Figure 7 .

 7 Figure 7. TLR7-mediated pDC activation potentiates cell polarity at the contact. (A-B) Quantification of the αL integrin accumulation and αL integrin/ICAM-1 co-accumulation at the contact between pDCs and DENV (A) or HCV (B) infected cells versus uninfected (cont) cells, analyzed as in Figure 2J-L. Results are expressed as the percentage of indicated phenotype relative to the total number of analyzed contacts; 4 (A; n>62 analyzed contacts per condition) and 3 (B; n>46) independent experiments; mean±SD. (C-L) Quantification of the co-clustering of PI(4,5)P2/PIP5KIα (C, F, I), PI(4,5)P2/EEA1 (D, G), clathrin/actin (J-L), αL integrin/clathrin (E) and high-affinity conformation αLβ2 integrin complex/EEA1 (H), as

Acknowledgements

We thank A. D. Davidson (University of Bristol, UK) for the molecular clone NGC, A. Kohn (MRC-University of Glasgow Centre for Virus Research, UK) and R. Freitas de Oliveira Franca (FIOCRUZ, Recife, Brazil) for clinical isolate ZIKV Brazil (PE243_KX197192), the European Virus Archive for H/PF, C. Rice (Rockeffeler University, New York, NY) for the anti-HCV NS5A, P. Despres (PIMIT, Université de La Réunion-INSERM France) for providing the anti-DENV E, F.V. Chisari (Scripps Research Institute, La Jolla, CA) for the Huh7.5.1 cells and V. Lotteau (CIRI, Lyon, France) for Influenza A Virus. We are grateful to Y. Jaillais, H Paidassi, T. Walzer, S. Munoz-Gonzalez for critical reading of the manuscript, and to our colleagues for encouragement and help. The PLATIM platform of SFR Biosciences Lyon-Gerland (UMS3444/US8) for imaging analysis and technical assistance. We acknowledge the contribution of the EFS Confluence-Lyon. This work was supported by grants from the 'Agence Nationale pour la Recherche' (ANR-JCJC-EXAMIN), the 'Agence Nationale pour la Recherche contre le SIDA et les Hépatites Virales' (ANRS-AO 2017-01), the 'Fondation pour le recherche médicale' (DBI20141231313 DREUX Bioinfo 2014) and LabEx Ecofect Grant ANR-11-LABX-0048. SA's PhD fellowship was sponsored by French ministry and Ligue contre le cancer. The post-doc fellowship for BW and DC were sponsored by EMBO and ANRS, respectively.

The authors declare no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: SA, SC, DC, LS, BW, MD. Performed the experiments: SA, SC, DC, ED, LS. Analyzed the data: SA, SC, DC, ED, LS, OA, BW, MD.

Wrote the paper: SA, SC, MD.

Declaration of Interests

STAR Methods

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Marlène Dreux (marlene.dreux@ens-lyon.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lines and primary cell cultures.

Huh7.5.1, Huh-7.5.1c2 cells and Huh-7.5.1c2 cells (male) that harbor the JFH-1 subgenomic replicon (HCV rep cells) [START_REF] Kato | Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon[END_REF] were maintained in Dulbecco's modified Eagle medium (DMEM) (Life Technologies) supplemented with 10% FBS, 100 units (U)/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and non-essential amino acids (Life Technologies) at 37°C/5% CO2 [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF].

The pDCs were isolated from blood or cytapheresis units from healthy adult human volunteers which was obtained from the 'Etablissement Français du Sang' (EFS; Auvergne Rhône Alpes, France) under the convention EFS 16-2066 and according to procedures approved by the EFS committee. Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Information on sex and age was not available for all subjects, however was not relevant for the purpose of the study, since we previously showed that pDC responses are within the range for all donors [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF].

PBMCs were isolated using Ficoll-Hypaque density centrifugation and pDCs were positively selected from PBMCs using BDCA-4-magnetic beads (MACS Miltenyi Biotec), as previously described [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF][START_REF] Dreux | Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity[END_REF]. The typical yields of PBMCs and pDCs (Corning); Vybrant cell-labeling solution (CM-DiI, Life Technologies); Celltrace Violet cell Proliferation kit (Life Technologies); Hoescht and Fc receptor blocking reagent (MACS Miltenyi Biotec); cDNA synthesis and qPCR kits (Life Technologies); poly-L-lysin (P6282, Sigma-Aldrich).

METHOD DETAILS

Immunostaining and FACS analysis.

Surface immunostainings were performed on freshly isolated pDCs, DENV infected and uninfected Huh-7.5.1 cells and cocultured cells that were harvested and resuspended using 0.48 mM EDTA-PBS solution. Surface adhesion molecules were detected by a 30 minuteincubation at 4°C with 5 µg/mL of mouse anti-αL integrin (clone 38); mouse anti-ICAM-1 (Clone LB-2); mouse anti-E-cadherin (clone SHE78-7); Vioblue-conjugated mouse anti-Lselectin/CD62L (clone 145/15) diluted in staining buffer (PBS -1% FBS), followed by PBS washes. For αL integrin, ICAM-1 and E-cadherin detection, cells were incubated for 20 minutes with APC-conjugated-anti-mouse antibody diluted in staining buffer (2 µg/mL) and cells were fixed in PFA4% (20 min, 4°C). Flow cytometric analysis was performed using a BD FACS Canto II, and the data were analyzed with Flow Jo software (Tree Star).

Co-culture experiments.

Unless otherwise indicated, 10 4 pDCs were co-cultured with 2x10 4 infected cells or uninfected parental cells in a 200 µl final volume in 96-well round-bottom plates incubated at 37 °C/5% CO2. Eighteen to twenty hours later, cell-culture supernatants were collected and the levels of IFNα and, when indicated, IFNβ and type III IFNs, were measured using a commercially available specific ELISA kit (PBL Interferon Source) following the manufacturer's instructions. To optimize the pDC response to HCV replicating (SGR) cells, 2x10 4 pDCs were co-cultured with 10 5 cells for 22-to-24 hours.

Viral spread assay.

5x10e4 GFP+ uninfected cells and 5x10e4 GFP-cells, that were infected by DENV or ZIKV, as indicated, for 48 hours prior to coculture, were cocultured with 2x10e4 pDCs, 48 hourincubation. When indicated, the cocultures were treated by anti-αL integrin at 10 μg/mL. The levels of the viral spread from infected cells (GFP-) to uninfected cells (GFP+) during coculture were determined by FACS analysis of the frequency of infected cells in the GFP+ cell population. Similar detection in GFP-populations demonstrated the inhibition of viral replication during coculture. The infected cells were detected by immunostaining of dsRNA, a marker of viral replication. Briefly, after 48 hours of coculture, cells were fixed with 4% PFA, followed by PBS washes and a permeabilization step with 90% methanol/10% PBS for 1 hour-incubation at 20°C. The mouse anti-dsRNA (clone J2; Scicons) was diluted at 1 μg/mL staining buffer (PBS -1% FBS) and added to the cells for an hour incubation at 4°C. After PBS wash, cells were incubated with anti-mouse IgG2a conjugated with Alexa 647fluorochrome and diluted staining buffer with 30 minute incubation at 4°C. Flow cytometric analysis was performed using a BD FACS Canto II, and the data were analyzed with Flow Jo software (Tree Star).

Imaging combined with flow cytometry analysis.

Huh 7.5.1c2 cells were transduced with lentiviral based vector pseudotyped with VSV glycoprotein to stably express GFP, as previously described [START_REF] Decembre | Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells[END_REF]Dreux et al., 2009). Forty-eight hours prior co-culture with pDCs, GFP-expressing Huh 7.5.1c2 cells were infected by HCV or DENV as above-described. After immuno-isolation, pDCs were stained by using Celltrace Violet Cell Proliferation kit (Life Technologies) by 20 minute-incubation at 37°C in the dark. Labeled pDCs were then spinned down and resuspended into pDC coculture medium. 10 5 GFP-expressing HCV/DENV infected or uninfected cells were co-cultured with 4 x 10 4 pDCs in low-adherence micro-plate designed for cell harvesting by temperature reduction (Nunc UpCell 96F Microwell Plate from Thermo Scientific) for 4-5 hours at 37°C in presence or not of inhibitor, as indicated. The co-cultured cells were detached and harvested by 20 minute-incubation at room temperature. After 2% PFA fixation, cells are washed twice with staining buffer (PBS 2% FBS). Co-cultured cells were analyzed by Image Stream X technology (Amnis) at magnification x40 using IDEAS software. The cell population defined as pDC/HCV-infected or uninfected cell conjugates comprises conjugates of at least one Celltrace Violet (CTV) positive cell and at least one GFP positive cell among the total of CTV positive cells, GFP positive cells and conjugates. The cell populations were sorted by using masks (IDEAS software) to eliminate cells not in focus and/or with saturating fluorescent signal, conjugate population was further selected by with Area dilate IDEAS mask (i.e., double fluorescent signal without overlay) and then sorted based of cell size of the positive cells (i.e., fluorescent signal area). Post-cell sorting, the accuracy of the gated cell populations in regards to the defined criteria was controlled by a visual inspection of the individual pictures in the gated cells population (Figure S1D).

Immunostaining for confocal analysis.

After immuno-isolation, 4 x 10 4 pDCs were stained by using either 0.5 µM Vybrant celllabeling solution (CM-DiI, Life Technologies) by successive incubations for 10 and 15 minutes at 37°C and 4°C, respectively or Celltrace Violet cell Proliferation kit (Life Technologies) as above-described. Labeled pDCs were washed twice with PBS and then cocultured with 2 x 10 4 DENV infected Huh7.5.1 cells or HCV infected Huh7.5.1c2 cells at 37°C for 4-5 hours, in a 96-Well Optical-Bottom Plate pre-coated with 8 µg/mL poly-L-lysin Live imaging of coculture.

Huh 7.5.1c2 cells were transduced with lentiviral based vector to stably express GFP and infected, or not, with HCV or DENV 48 hours before co-culture, as above described. GFPexpressing DENV infected cells or HCV replicating cells and corresponding uninfected control (cont) cells were seeded (2 x 10 4 cells per well) in a 96-Well Optical-Bottom Plate pre-coated with poly-L-lysin (30 minutes incubation at 37°C with 8 µg/mL poly-L-lysin).

Isolated pDCs were stained with Vybrant cell-labeling solution (CM-DiI, Life Technologies) as above described. When pDCs were added to the seeded infected (or uninfected) cells, the cocultures were imaged every 5 minutes for 10 hours at magnification x10 with a BSL3-based spinning-disc confocal microscope (AxioObserver Z1, Zeiss). The cocultures were maintained at 37°C, 5% CO2 in an incubation chamber throughout the course of the experiments. The analyses of pDC motion (as illustrated in Figure S6) were performed using projection of Z-stacks with maximal intensity (i.e., about 3-to-5 selected Z-stacks per fields out of 15 Z-stacks in total). The quantification of the duration of contacts between pDCs and infected cells versus uninfected cells were performed using Image J software package (http://rsb.info.nih.gov/ij). The calculations of cell positions for the motion graphs were performed using Trackmate plug-in of Image J software. The representation with violin plots were obtained using R software environment for statistical computing and graphics, version