Malene Ringkjøbing Jensen

Filip Yabukarski

Guillaume Communie

Eric Condamine

Caroline Mas

Valentina Volchkova

Nicolas Tarbouriech

Jean-Marie Bourhis

Viktor Volchkov

Martin Blackledge

Marc Jamin
email: marc.jamin@ibs.fr

STRUCTURAL DESCRIPTION OF THE NIPAH VIRUS PHOSPHOPROTEIN AND ITS INTERACTION WITH STAT1

Keywords: Nipah virus, phosphoprotein, Mononegavirales, intrinsically disordered protein, small-angle X-ray scattering, nuclear magnetic resonance, X-ray crystallography

The structural characterization of modular proteins containing long intrinsically disordered regions (IDRs) intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches.

Nipah virus (NiV) phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoviruses. Here, we combine multiple biophysical methods, including X-ray crystallography, NMR spectroscopy and small-angle X-ray scattering (SAXS), to characterize the structure of this protein and provide an atomistic representation of the fulllength protein in the form of a conformational ensemble. We show that full-length NiV phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain.

Using NMR spectroscopy and SAXS, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, while others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the phosphoprotein, comprised between residues 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1, and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new insights into how the phosphoprotein and the non-structural V and W proteins of NiV perform their multiple functions.

2

STATEMENT OF SIGNIFICANCE

The intrinsically disordered phosphoprotein of Nipah virus orchestrates viral genome transcription, replication and encapsidation and shares a long N-terminal disordered region with the non-structural V and W proteins involved in blocking the interferon responses. By combining multiple biophysical methods, including X-ray crystallography, NMR spectroscopy and small-angle X-ray scattering, we characterized the structure and flexibility of this protein, identified several putative short linear motifs of interaction with viral and cellular partners and investigated the interaction with one of them. The protein appears as a nanometer-size octopus comprising a central tetrameric core and displaying binding sites on its flexible arms to recruit multiple viral and cellular partners, thus providing new insights into how this protein fulfills its multiple functions.

INTRODUCTION

Nipah virus (NiV) is one of the most pathogenic and deadly viruses infecting humans [START_REF] Chua | Nipah virus: a recently emergent deadly paramyxovirus[END_REF]. It is an emergent zoonotic virus that naturally infects fruit bats in South-East Asia, but that can also infect other hosts including pigs, dogs, cats, horses and humans, where it becomes highly pathogenic. Since its discovery in 1998 in Malaysia, NiV has caused regular outbreaks of severe human encephalitis and respiratory illness with a fatality rate ranging from 40% to 90% mainly in Bangladesh and more recently in Philippines and India [START_REF] Chua | Nipah virus: a recently emergent deadly paramyxovirus[END_REF][START_REF] Arunkumar | Outbreak investigation of nipah virus disease in Kerala, India, 2018[END_REF]. With its ability to transmit from man to man and the lack of vaccine or efficient treatment, NiV has the potential to cause a pandemic and to be used in biological warfare [START_REF] Luby | The pandemic potential of Nipah virus[END_REF]. It is consequently classified as a biosafety level-4 (BSL-4) pathogen.

NiV is an enveloped virus with a nonsegmented negative-sense RNA genome, which belongs to the order Mononegavirales and the family Paramyxoviridae, although, several features in its genome organization and in its protein sequences led to its classification into a new genus, Henipavirus, with the highly pathogenic Hendra virus and the non-pathogenic Cedar virus [START_REF] Wang | The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae[END_REF].

The multiplication of NiV in a suitable host cell, like that of all members of the Mononegavirales, requires both (i) transcription of the single-stranded RNA genome into monocistronic, capped and polyadenylated messenger RNAs (positive-sense RNA) that can be used by the cellular ribosomes to generate new viral proteins and (ii) replication of the negative-sense RNA genome into positive-sense antigenome copies, which are then used to generate a progeny of negative-sense genomes. The enzymatic machinery that carries out these activities is conserved among all members of the Mononegavirales and is unique in biology and thus represents an attractive target for the development of much-needed antiviral compounds. It consists of a megadalton-sized multi-molecular complex located in the core of the viral particle and timely released in the cytoplasm of the host cell [START_REF] Halpin | Nipah virus conforms to the rule of six in a minigenome replication assay[END_REF][START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF]. This transcription/replication complex is composed of the genomic RNA and of three viral proteins: the nucleoprotein (N), the phosphoprotein (P) and the large RNA-dependent RNA polymerase (L). With the help of the phosphoprotein acting as a chaperone, the nucleoprotein assembles into linear homopolymers that coats both negative-sense genomic and positivesense antigenomic RNAs, forming long, helical nucleocapsids [START_REF] Gutsche | Near-atomic cryo-EM structure of the helical measles virus nucleocapsid[END_REF]. These nucleocapsids constitute the biologically active templates used by the polymerase complex, which consists of the L polymerase and the phosphoprotein acting as a non-catalytic cofactor. Structural and dynamical studies of the unassembled nucleoprotein (N 0 ) in complex with the phosphoprotein [START_REF] Leyrat | Structure of the vesicular stomatitis virus N-P complex[END_REF][START_REF] Yabukarski | Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone[END_REF][START_REF] Milles | An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function[END_REF][START_REF] Aggarwal | Structure of the paramyxovirus PIV5 nucleoprotein in complex with an amino-terminal peptide of the phosphoprotein[END_REF] and of nucleocapsids [START_REF] Gutsche | Near-atomic cryo-EM structure of the helical measles virus nucleocapsid[END_REF][START_REF] Milles | Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence[END_REF] have started to unravel the mechanisms of assembly and remodeling of the nucleocapsids during the transcription and replication processes [START_REF] Jamin | Non-segmented negative-sense RNA virusesstructural data brings new insights into nucleocapsid assembly[END_REF]. The multidomain L polymerase catalyzes RNA synthesis, but also the capping, methylation and polyadenylation of the viral messenger RNAs [START_REF] Jordan | Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase[END_REF]. The underlying mechanisms of the functioning of this polymerase remain largely to be deciphered [START_REF] Ogino | RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus[END_REF], but its structure in complex with its cofactor the phosphoprotein is being revealed by recent cryo-electron microscopic reconstructions of the complex from various Mononegavirales [START_REF] Gilman | Structure of the Respiratory Syncytial Virus Polymerase Complex[END_REF][START_REF] Pan | Structure of the human metapneumovirus polymerase phosphoprotein complex[END_REF][START_REF] Jenni | Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor[END_REF][START_REF] Horwitz | Structure of a rabies virus polymerase complex from electron cryo-microscopy[END_REF], including a first low-resolution reconstruction of the NiV polymerase [START_REF] Jordan | Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase[END_REF]. The third essential component, the phosphoprotein, is thus a multitasking protein that orchestrates the interplay between the different components of the transcription/replication complex. The phosphoprotein takes its name from early observations showing that it is the most heavily phosphorylated viral protein in cells infected by different members of the Mononegavirales [START_REF]Fields Virology[END_REF]. In NiV phosphoprotein, residues Ser224 and Thr239 are the major phosphorylation sites [START_REF] Shiell | Sites of phosphorylation of P and V proteins from Hendra and Nipah viruses: Newly emerged members of Paramyxoviridae[END_REF] and a binding site for Polo-like kinase 1 is located near residue 130 [START_REF] Ludlow | Henipavirus V Protein Association with Polo-Like Kinase Reveals Functional Overlap with STAT1 Binding and Interferon Evasion[END_REF], but as for other Mononegavirales, the role of these phosphorylations in the viral replication cycle remains elusive [START_REF] Fuentes | Phosphorylation of paramyxovirus phosphoprotein and its role in viral gene expression Sandra[END_REF].

All phosphoproteins of the Mononegavirales share the properties of binding the L polymerase, the unassembled nucleoprotein and the nucleocapsid through independent structural modules [START_REF] Jamin | Non-segmented negative-sense RNA virusesstructural data brings new insights into nucleocapsid assembly[END_REF]. In this way, the phosphoprotein promotes the initiation of RNA synthesis, confers processivity to the polymerase, maintains the unassembled nucleoprotein in an RNA-free and soluble form and delivers the latter to the site of RNA synthesis in order to encapsulate the nascent RNAs [START_REF] Yabukarski | Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone[END_REF][START_REF] Milles | An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function[END_REF][START_REF] Milles | Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence[END_REF][START_REF] Morin | Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase[END_REF][START_REF] Ranadheera | The interaction between the Nipah virus nucleocapsid protein and phosphoprotein regulates virus replication[END_REF][START_REF] Bloyet | Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength[END_REF][START_REF] Bloyet | Regulation of measles virus gene expression by P protein coiledcoil properties[END_REF][START_REF] Cox | The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient[END_REF]. Despite common functions, similarities in the phosphoprotein sequence are hardly found beyond the level of the family and in some cases of the genus [START_REF] Karlin | Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins[END_REF][START_REF] Mccarthy | Reassessing conflicting evolutionary histories of the Paramyxoviridae and the origins of respiroviruses with Bayesian multigene phylogenies[END_REF]. However, all phosphoproteins of the Mononegavirales are multimeric and share a common architecture with a long N-terminal intrinsically disordered region (PNTR) and a C-terminal region (PCTR) that includes the multimerization domain (PMD), a linker region (PCT-LINK) and most often a folded domain at the C-terminal end (PXD) [START_REF] Karlin | Structural disorder and modular organization in Paramyxovirinae N and P[END_REF][START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF] (Figure 1B). They also share a rather common localization of functional modules [START_REF] Jordan | Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase[END_REF][START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF][START_REF] Chan | Mapping of domains responsible for nucleocapsid protein-phosphoprotein interaction of henipaviruses[END_REF][START_REF] Bruhn | A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function[END_REF], and thus, NiV phosphoprotein exhibits a structural architecture and an organization of the functional modules involved in binding the nucleoprotein and the polymerase that are typical of the family Paramyxoviridae, although the protein of henipaviruses is appreciably longer than that of the other genera of Paramyxoviridae [START_REF] Ranadheera | The interaction between the Nipah virus nucleocapsid protein and phosphoprotein regulates virus replication[END_REF][START_REF] Chan | Mapping of domains responsible for nucleocapsid protein-phosphoprotein interaction of henipaviruses[END_REF][START_REF] Omi-Furutani | Novel phosphoprotein-interacting region in Nipah virus nucleocapsid protein and its involvement in viral replication[END_REF] (Figure 1B): (i) The N-terminal disordered region of more than 400 aa contains a short region (aa 1-38) at the N-terminal end that acts as a molecular recognition element or MoRE (i.e. a region that is partially disordered in isolation but that adopts a stable conformation upon binding to a partner [START_REF] Fuxreiter | Preformed structural elements feature in partner recognition by intrinsically unstructured proteins[END_REF]) for recruiting and chaperoning the RNA-free unassembled nucleoprotein (N 0 ) required for the assembly of new nucleocapsids during genome replication [START_REF] Yabukarski | Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone[END_REF]. (ii) The tetramerization domain of NiV (aa 475-578) [START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF] is involved in the interaction with and in the function of the L polymerase [START_REF] Bloyet | Regulation of measles virus gene expression by P protein coiledcoil properties[END_REF][START_REF] Bruhn | A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function[END_REF][START_REF] Tarbouriech | Tetrameric coiled coil domain of Sendai virus phosphoprotein[END_REF]. (iii) A folded domain at the C-terminal end of the protein, named the X domain (PXD) (aa 655-709) that binds to a MoRE in the C-terminal disordered tail of the nucleoprotein (NTAIL) [START_REF] Habchi | Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus[END_REF]. (iv) A third region of weak interaction with the nucleoprotein that has recently been identified in measles virus phosphoprotein, appears to be conserved in NiV phosphoprotein (aa 330-350) [START_REF] Milles | An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function[END_REF]. Despite all this knowledge, we are still lacking a structural characterization of the full-length NiV phosphoprotein.

In addition, besides its roles in the transcription and replication of the viral genome, the phosphoprotein of many Mononegavirales also plays roles in hijacking cellular machineries and in counteracting the host immune responses [START_REF] Audsley | Paramyxovirus evasion of innate immunity: Diverse strategies for common targets[END_REF]. As in other Paramyxoviridae, the NiV P gene codes for multiple proteins through the use of alternative open reading frames, and these additional proteins act as virulence factors, in particular by directly targeting the Signal Transducer and Activator of Transcription (STAT) proteins and blocking the interferon signaling pathway [START_REF] Audsley | Paramyxovirus evasion of innate immunity: Diverse strategies for common targets[END_REF]. The NiV P gene encodes three proteins, the phosphoprotein and the non-structural V and W proteins. The gene contains an editing site in the part coding for the N-terminal region of the phosphoprotein, where the polymerase stutters during transcription and incorporates one, two or more pseudo-templated Gs resulting in shifts of the reading frame [START_REF] Kulkarni | Nipah virus edits its P gene at high frequency to express the V and W proteins[END_REF]. The different messenger RNAs generated through this process lead to the expression of either the phosphoprotein or of one of the non-structural V (+1 ORF) or W (+2 ORF) proteins, which share their 405 N-terminal residues with the phosphoprotein but have specific C-terminal moieties of 51 and 44 amino acids, respectively (Figure 1B) [START_REF] Kulkarni | Nipah virus edits its P gene at high frequency to express the V and W proteins[END_REF]. In addition, a second gene coding for the C protein is overprinted on the initial region of the P gene in the same reading frame as the V protein (+1 ORF) (Figure 1B) and is expressed from the three different mRNAs by a leaky ribosome scanning mechanism [START_REF] Lo | Evolution and Structural Organization of the C Proteins of Paramyxovirinae[END_REF]. However, in contrast to some other Paramyxoviridae, NiV phosphoprotein, V and W proteins block interferon signaling by preventing the phosphorylation of STAT1 and by sequestering STAT1 and STAT2 into non-functional complexes [START_REF] Rodriguez | Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation[END_REF]. The phosphoprotein and the V protein retain STAT proteins in the cytoplasm, whereas the W protein sequesters them in the nucleus. The N-terminal part comprised between residues 100 and 300, shared by the three proteins, is critical for interferon evasion. The binding site for STAT1 was first localized between residues 100 and 160 [START_REF] Rodriguez | Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion[END_REF][START_REF] Shaw | Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively[END_REF] and then further mapped down to residues 114 to 140 [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF].

Mutation of different residues in this region abrogate interferon inhibition [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF][START_REF] Hagmaier | A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species[END_REF]. The interaction with STAT2 requires the entire100-300 region, in particular a motif between residues 230 and 237 and the prior binding of STAT1 [START_REF] Rodriguez | Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion[END_REF]. Furthermore, the region between residues 174 and 192 contains a CRM1-dependent nuclear export signal (NES), which allows the V protein to shuttle between the nucleus and cytoplasm and remove STAT1 out of the nucleus [START_REF] Rodriguez | Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation[END_REF][START_REF] Rodriguez | Hendra Virus V Protein Inhibits Interferon Signaling by Preventing STAT1 and STAT2 Nuclear Accumulation[END_REF][START_REF] Smith | Structural basis for importin alpha 3 specificity of W proteins in Hendra and Nipah viruses[END_REF][START_REF] Atkinson | Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus v[END_REF], whereas the W protein maintains phosphorylated STAT1 in the nucleus through the presence of a nuclear localization signal (NLS) in its unique C-terminal moiety [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF][START_REF] Shaw | Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively[END_REF]. Finally, the region around residue 130, overlapping with the STAT1 binding site interacts with polo-like kinase 1, and this interaction results in the phosphorylation of the V protein [START_REF] Ludlow | Henipavirus V Protein Association with Polo-Like Kinase Reveals Functional Overlap with STAT1 Binding and Interferon Evasion[END_REF].

Here, we investigate the structure of full-length NiV phosphoprotein (PFL). We determine its oligomeric state by biophysical methods and demonstrate that the multimerization domain is the only region of the phosphoprotein involved in its tetramerization. We solve the crystal structure of the tetramerization domain and use NMR spectroscopy to investigate the structural properties of the long N-terminal region, of the C-terminal linker predicted to be intrinsically disordered and of the C-terminal X domain. We demonstrate the independence of the different N-terminal chains in the tetrameric protein and identify several regions with transiently populated secondary structure elements, which could be molecular recognition elements (MoRE) for different viral or cellular partners. We generate an ensemble of conformers of full-length phosphoprotein that incorporates the crystal structure of the tetramerization domain and a homology model of the X domain and that simultaneously accounts for NMR and small-angle X-ray scattering data from three different constructs of the protein. Finally, we investigate the interaction between STAT1 and a fragment of 100 amino acids derived from the N-terminal region of the phosphoprotein. NMR spectroscopy is used to validate the binding site in the phosphoprotein, while isothermal titration calorimetry and cellbased assays are combined with site-directed mutagenesis to map important residues for the interaction.

MATERIALS AND METHODS

Disorder meta-prediction

The location of intrinsically disordered regions (IDRs) in NiV phosphoprotein was predicted by submitting its amino acid sequence to 16 different algorithms accessible through web servers and by calculating a consensus prediction as described previously [START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF][START_REF] Ribeiro | Solution Structure of the C-Terminal Nucleoprotein-RNA Binding Domain of the Vesicular Stomatitis Virus Phosphoprotein[END_REF]. The calculated disorder score (D-score) varies between 0 and 1, and generally, regions with a Dscore lower than 0.5 are intrinsically disordered, whereas regions with a score higher than 0.5 are either folded domains [START_REF] Ribeiro | Solution Structure of the C-Terminal Nucleoprotein-RNA Binding Domain of the Vesicular Stomatitis Virus Phosphoprotein[END_REF][START_REF] Ivanov | Structure of the Dimerization Domain of the Rabies Virus Phosphoprotein[END_REF][START_REF] Martinez | Structure of the C-terminal domain of lettuce necrotic yellows virus phosphoprotein[END_REF] or potential molecular recognition elements for partner proteins [START_REF] Leyrat | The N0-binding region of the vesicular stomatitis virus phosphoprotein is globally disordered but contains transient α-helices[END_REF].

Protein expression and purification

On the basis of our predictions, different fragments of the phosphoprotein (Uniprot: Q9IK91) were cloned between the NcoI and BamHI restriction sites of a pET28 vector, such that each construct included a C-terminal His6-tag with a Leu-Glu linker. For the constructs lacking the N-terminal end of the protein, with the exception of the P655-709 construct which starts at Met655, a Met residue or a Met-Ala dipeptide (construct P173-240) was introduced at the Nterminal end of the fragment. Three single mutants of P92-190: Y116A, G120E, G125E and a triple mutant: H117A/Q118A/H1119A, were generated by reverse PCR using the QuickChange mutagenesis protocol (Agilent). All constructs were verified by DNA sequencing.

The proteins were expressed in E. coli BL21-RIL. Cells were grown in Luria Bertoni medium at 37°C until the optical density (O.D.) at 600 nm reached a value of 0.6, and protein expression was then induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were allowed to grow for 3 hours. For producing the selenomethionine-substituted form of the P471-580 construct, the bacterial cells were grown in minimal medium and SeMet was added before induction of the heterologous protein expression. In each case, the cells were harvested by centrifugation, and the pellet was resuspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl (buffer A) and EDTA-free protease inhibitor cocktail (Roche). The cells were broken by sonication, and the soluble fraction was loaded onto a Ni 2+ column equilibrated in buffer A. After washing the column with buffer A containing 20 mM imidazole, the protein was eluted with buffer A containing 300 mM imidazole. The protein was further purified by size exclusion chromatography on a Superdex S75 (GE Healthcare) equilibrated in buffer A or in 20 mM Bis-Tris buffer at pH 6.0 containing 150 mM NaCl, 50 mM arginine, 50 mM glutamate and 0.5 mM TCEP (buffer B) for NMR experiments or in 20 mM sodium phosphate buffer at pH 7.5 containing 150 mM NaCl (buffer C) for ITC experiments. Each protein preparation was checked by SDS-PAGE and SEC-MALLS (see below). A fragment of the gene coding for residues 132 to 683 of human STAT1 was cloned in pET28, was expressed in E. coli and was purified as previously described [START_REF] Vinkemeier | DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites[END_REF]. Briefly, after ammonium sulfate precipitation, the protein was solubilized in a 20 mM Tris-HCl buffer at pH 8.8 containing 150 mM NaCl, and the solution was loaded onto a SP sepharose column (GE healthcare) equilibrated in the same buffer. The protein was not retained on the column and eluted in the flow-through. After concentration, the protein was further purified by loading the solution containing STAT1132-683 onto a Superdex S200 (GE Healthcare) size exclusion column equilibrated in buffer C.

SEC-MALLS experiments

Size exclusion chromatography (SEC) combined with on-line detection by multi-angle laser light scattering (MALLS) and refractometry (RI) is a method for measuring the absolute molecular mass (MM) of a particle in solution that is independent of its dimensions and shape [START_REF] Wyatt | Submicrometer Particle Sizing by Multiangle Light Scattering following Fractionation[END_REF]. SEC was performed with TSK G4000 (Tosohbioscience) or S200 Superdex column (GE Healthcare) equilibrated with 20 mM Tris-HCl buffer containing 150 mM NaCl, and the columns were calibrated with globular standard proteins of known hydrodynamic radius (Rh) [START_REF] Uversky | Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule[END_REF]. Chromatographic separations were performed at 20°C with a flow rate of 0.5 or 0.8 mL.min -1 , with on-line MALLS detection with a DAWN-HELEOS II detector (Wyatt Technology Corp.) using a laser emitting at 690 nm, and protein concentration was measured on-line by the use of differential refractive index measurements using an Optilab T-rEX detector (Wyatt Technology Corp.) and a refractive index increment, dn/dc, of 0.185 mL.g -1 .

The weight-averaged molecular mass was calculated using the ASTRA software (Wyatt Technology Corp.).

Small angle X-ray scattering experiments

Small angle X-ray scattering (SAXS) data were collected at the BioSAXS beamline (ID14-3) of the ESRF [START_REF] Pernot | New beamline dedicated to solution scattering from biological macromolecules at the ESRF[END_REF]. The scattering from the buffer alone was measured before and after each sample measurement and used for background subtraction. All data analyses were performed using the program PRIMUS from the ATSAS 3.0.0 package [START_REF] Konarev | ATSAS 2.1, a program package for small-angle scattering data analysis[END_REF].

NMR spectroscopy

The spectral assignment of the different constructs of NiV P protein (P1-100, P92-190, P173-240, P223-319, P300-401, P387-479, P588-650 and PXD (residues 655-709)) at 25°C in buffer B using BEST-type triple resonance experiments [START_REF] Lescop | A set of BEST triple-resonance experiments for time-optimized protein resonance assignment[END_REF]. The NMR experiments were acquired at a 1 H frequency of 600 or 800 MHz (Agilent spectrometers). Six experiments were acquired for each construct: HNCO, intra-residue HN(CA)CO, HN(CO)CA and intra-residue HNCA, HN(COCA)CB and intra-residue HN(CA)CB. All spectra were processed in NMRPipe [START_REF] Delaglio | NMRPipe: a multidimensional spectral processing system based on UNIX pipes[END_REF], analyzed in Sparky (61) and automatic assignment of spin systems was done using MARS [START_REF] Jung | Mars -robust automatic backbone assignment of proteins[END_REF] followed by manual verification. The assigned chemical shifts have been deposited in the BMRB database under accession numbers 50098, 50099, 50100, 50101, 50102, 50103, 50105. The completeness of the assignments was above 97% for all constructs with the unassigned residues mostly belonging to the N-termini that undergo fast solvent exchange.

Only the construct P387-479 had a lower assignment completeness (92%) with residues S399-I402 being unassigned. Secondary structure propensities (SSPs) were calculated from the experimental Cα and Cβ chemical shifts according to Marsh et al. [START_REF] Marsh | Sensitivity of secondary structure propensities to sequence differences between alpha-and gamma-synuclein: implications for fibrillation[END_REF].

X-ray crystallography

Initial crystallization conditions for the tetramerization domain (aa 471-580) were identified at the High Throughput Crystallization platform of the PSB, (https://htxlab.embl.fr). The crystals used for data collection were obtained in 7% PEG 3350, 1 M LiCl, 0.1 M sodium citrate, 0.1 M arginine, 4% hexanediol, 0.001% NaN3 at pH 5.0, and were frozen with 15% glycerol as cryo-protectant. X-ray diffraction data were collected at the ID29 beamline of the ESRF at a wavelength of 0.9793 Å and at Proxima1 beamline of SOLEIL at a wavelength of 0.97895 Å and at a temperature of 100 K and were processed with the XDS package [START_REF] Kabsch | Xds[END_REF]. Initial phases were obtained using the anomalous scattering from selenium atoms by the SAD method with the program HKL2MAP [START_REF] Pape | HKL2MAP: a graphical user interface for phasing with SHELX programs[END_REF]. A model was initially constructed with the Autobuild program (66) from the phenix suite (67) and subsequently refined with the phenix.refine program [START_REF] Afonine | Towards automated crystallographic structure refinement with phenix.refine[END_REF] and Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]. The geometry of the final model was checked with MolProbity (70). In the model, 98.04% of residues had backbone dihedral angles in the favored region of the Ramachandran plot, 1.83% fall in the allowed regions and 0.13% were outliers. Figures were generated with PyMol (71) and Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF].

Modeling of NiV phosphoprotein as an ensemble of conformers

An ensemble of 10,000 full-length NiV phosphoprotein conformers was generated with the program Flexible-Meccano [START_REF] Bernado | A structural model for unfolded proteins from residual dipolar couplings and small-angle x-ray scattering[END_REF]. These included the high-resolution three-dimensional structures of the tetramerization domain (PMD) and an homology model of the C-terminal X domain based on the known HeV PXD structure (74) by using Modeller [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF]. To accurately model the disordered N-terminal region, the ASTEROIDS selection algorithm [START_REF] Jensen | Defining conformational ensembles of intrinsically disordered and partially folded proteins directly from chemical shifts[END_REF] was first applied to each of the sub-constructs of the phosphoprotein in order to obtain an ensemble of conformers of these regions in agreement with experimental 1 H N , 15 N, 13 Cα, 13 Cβ, and 13 C' chemical shifts. Briefly, in successive rounds of selection, 5 × 200 pairs of (ϕ, ψ) dihedral angles were selected for each residue from a pool of 10,000 conformers assembled by Flexible-Meccano such that the difference between ensemble-averaged 1 H, 15 N and 13 C chemical shifts predicted by the program SPARTA [START_REF] Shen | Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology[END_REF] and corresponding experimental values was minimized. The selected dihedral angles were then used in generation of a new set of 10,000 conformers for the next round of selection. The (ϕ, ψ) databases for each subconstruct of the phosphoprotein thus obtained after four rounds of selection were combined and used by Flexible-Meccano to build conformations of full-length protein. Residues in the linker region between the tetramerization domain and the X domain were stochastically assigned amino-acid specific backbone dihedral angles in agreement with random coil statistics. In the disordered regions, side chains were constructed using the program SCCOMP [START_REF] Eyal | Importance of solvent accessibility and contact surfaces in modeling side-chain conformations in proteins[END_REF]. Scattering curves were calculated with CRYSOL (ATSAS 3.0.0) (79) and ensemble selections were performed using ASTEROIDS (80) by combining both the experimental NMR and SAXS data. In order to account for conformational heterogeneity within different regions of the full-length protein, the ASTEROIDS target function combined SAXS data from three constructs: PFL, P1-580 and P471-709, representing the full-length tetramer, and the tetramerization domain with the N-terminal and C-terminal parts of the protein, respectively.

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) experiments for characterizing the interaction between the five fragments P92-190 (WT, Y116A, G120E, G125E, H117A/Q118A/H1119A) and STAT1132-683 were performed with a MicroCal iTC200 system (North Hampton, MA). The STAT1 solution at a concentration of 100 μM in buffer C was loaded in the calorimetric cell and titrated at 25°C, typically, by performing 16 injections of 2.5 μl of the wild-type (WT) P92-190 solution or of one of the variants (at concentrations ranging between 1.5 and 2.5 mM) in the same buffer. Protein samples and buffers were degassed before data collection. The heat generated for each injection was obtained by integrating the calorimetric signal. The heat resulting from the binding reaction between the two proteins was obtained as the difference between the heat of the reaction and the corresponding heat of dilution. The data were processed using Origin 7.0 (Malvern Panalytical, Malvern, UK) and fitted to a single-site binding model.

STAT1 localization in NiV infected cells

Generation of recombinant NiV was described elsewhere [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF]. NiV-G121E and NiV-G135E contain a single amino acid change in the P protein at position 121 or 135. To monitor the effect of NiV infection on endogenous STAT1, Vero E6 cells were grown on polymer coverslips of the µ-Slide VI 0.4 and were mock infected or infected with recombinant NiVs (WT, G121E or G135E) at a multiplicity of infection (m.o.i.) of 0.5. At 1 h post-infection, the virus inoculum was replaced with DMEM containing 2.5% fetal bovine serum and the cells were incubated at 37°C for 28 h. The cells were washed with serum-free medium and then treated with 1,000 U/mL of human interferon-β (PBL) in serum-free DMEM for 40 min at 37°C. For untreated controls, infected cells were incubated with IFN-free medium. Cells were washed twice with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde-PBS solution for 20 min at room temperature, and subsequently treated with 0.1 M glycine for 10 min. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and washed with a blocking solution (2% BSA in PBS). Immunofluorescence staining was performed with mouse anti-phospho-STAT1 (pY701) (BD Biosciences) (1:250 dilution) antibody or rabbit anti-NiV matrix protein (1:500) antibody.

RESULTS

NiV phosphoprotein has a modular architecture

The presence of intrinsically disordered regions (IDR) in a protein can be predicted from its amino acid sequence by different computational approaches [START_REF] Ferron | A practical overview of protein disorder prediction methods[END_REF][START_REF] Meng | Comprehensive review of methods for prediction of intrinsic disorder and its molecular functions[END_REF]. Previously, we devised a meta-prediction method that calculates a consensus disordered score (D-score) between 16 independent disorder predictors by a simple voting procedure [START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF] and found that by applying a threshold at 0.5 to distinguish between ordered and disordered regions we were able to precisely predicts boundaries between folded domains and IDRs [START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF][START_REF] Ribeiro | Solution Structure of the C-Terminal Nucleoprotein-RNA Binding Domain of the Vesicular Stomatitis Virus Phosphoprotein[END_REF][START_REF] Martinez | Structure of the C-terminal domain of lettuce necrotic yellows virus phosphoprotein[END_REF][START_REF] Ivanov | Structure of the dimerization domain of the rabies virus phosphoprotein[END_REF] as well as the existence of MoREs [START_REF] Fuxreiter | Preformed structural elements feature in partner recognition by intrinsically unstructured proteins[END_REF].

The D-score profile calculated from NiV phosphoprotein sequence reveals three disordered regions of D-score < 0.5 (named IDR1-3) and several regions of D-score > 0.5 predictive of structural order (Figures 1A and1B) in agreement with the Hydrophobic Cluster Analysis plot (Supplementary Figure S1) [START_REF] Callebaut | Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives[END_REF]. Two of these structured regions of more than 100 aa between residues 475 and 598 (averaged D-score of 0.82) and between residues 658 and 709 (averaged D-score of 0.69) coincide with the known localization of the folded tetramerization (PMD) and X domains (PXD), respectively [START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF][START_REF] Habchi | Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus[END_REF]. A shorter region of D-score > 0.5 at the Nterminal end (aa 1-29) corresponds to the α-MoRE known to bind the unassembled nucleoprotein N 0 (9). However, the D-score also identifies other putatively ordered regions. A region of D-score > 0.5, also spanning more than 100 aa between residues 102 and 219, encompasses the putative binding sites for STAT1, STAT2 and Polo-like kinase 1 as well as the CRM1-dependent NES (Figure 1B). A shorter region of D-score > 0.5 is localized within the C-terminal linker (aa 607-658). Finally, borderline regions appear between residues 310 and 450 in the form of clearly visible peaks in the D-score even if the score remains under the threshold of 0.5. One of these peak encompasses the conserved ultra-weak nucleoprotein binding motif recently discovered in measles virus phosphoprotein [START_REF] Milles | An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function[END_REF], while two other ones overlap with the regions encoding the specific moiety of the V and W proteins (Figure 1B).

Protein production and quality control

We expressed the full-length NiV phosphoprotein and its different fragments as recombinant proteins in Escherichia coli (Figure 1C). The full-length protein and the different protein fragments were soluble and eluted as single peaks from the SEC column. The molecular mass determined by on-line multi-angle laser light scattering (MALLS) detection was constant throughout the chromatographic peak (polydispersity index, Mw/Mn lower than 1.01) indicating that each sample was monodisperse (Table 1).

NiV full-length phosphoprotein is a tetramer of large size

The molecular mass of full-length NiV phosphoprotein (PFL) measured by on-line MALLS is 306 ± 30 kDa, in agreement with the molecular mass calculated for the tetramer (4 x 79.367 = 317.468 kDa) (Figure 2 and Table 1). Its hydrodynamic radius (Rh) measured by SEC is 9.3 ± 0.2 nm, a value that is intermediate between those expected for a compact globular protein (Rh,theo = 6.0 nm) or an unfolded chain (Rh,theo = 16.8 nm) of the same molecular mass (Table 1). Analyses of the different protein fragments by SEC-MALLS indicate that the central region is solely responsible for the tetramerization of the protein in accordance with crosslinking experiments [START_REF] Salvamani | Oligomerization state of the multimerization domain of Nipah virus phosphoprotein[END_REF] and the recent crystal structure of the NiV phosphoprotein multimerization domain [START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF]. The fragments P1-580 and P471-709, which both encompass the tetramerization domain, and the fragment P471-580, which closely corresponds to this domain, have molecular masses determined by SEC-MALLS of 250 ± 25 kDa, 113 ± 10 kDa and 58 ± 6 kDa (Figure 2), respectively, in agreement with the theoretical molecular masses expected for their tetrameric forms (Table 1). Similarly to the full-length protein, the hydrodynamic radius of these fragments is intermediate between those expected for compact globular proteins and for unfolded chains of the same molecular mass (Table 1). By contrast, all other fragments are monomeric and those from the N-terminal region have a hydrodynamic radius close to that of an unfolded chain (Table 1 and see below). SAXS curves were recorded at three different protein concentrations for the full-length phosphoprotein (Supplementary Figure S2) and for both tetrameric constructs: P1-580, (Supplementary Figure S3) and P471-709 (Supplementary Figure S4). For each protein, the scattering profile shows an absence of concentration dependence and the Guinier plots at low Q values are linear, indicating the absence of aggregation or of significant interchain interactions (Supplementary Figures S2, S3 andS4). We note, however, an unexplained overestimation of more than 30% in the molecular mass of full-length phosphoprotein and of P471-709 calculated by using the calibration with bovine serum albumin (BSA) (Table 2). The dimensionless Kratky plot and the pair-distance distribution function revealed ambivalent features for the three proteins, supporting the presence of both structured and disordered parts (Supplementary Figures S2,S3 and S4 and Table 2) [START_REF] Bizien | A Brief Survey of State-of-the-Art BioSAXS[END_REF]. A peak slightly above 1.1 is visible in the Kratky plot at Q.Rg 2 likely reflecting the presence of the folded tetramerization and X domains. For full-length phosphoprotein and the fragment P1-580, the value evolves towards a plateau at higher Q.Rg indicating the presence of a large fraction of disordered regions in agreement with the asymmetric bell-shape curve in the P(r) plots. For the fragment P471-709, a maximum is also present at Q.Rg 5 in the Kratky plot, and two maxima at r 4 nm and r 8.5 nm, likely reflecting on the long-range contacts between the tetramerization domain and the four X domains. The radius of gyration determined from the Guinier plot and the longest pair distance Dmax were almost identical for the full-length protein and the N-terminal fragment P1-580 indicating the overall size of these molecules is mainly determined by the N-terminal disordered region (Table 1). The corresponding values for the C-terminal fragment P471-709 was clearly smaller in agreement with the largest fraction of residues involved in structured domains (∼65% of folded residues) (Table 1).

Crystal structure of the tetramerization domain

The fragment P471-580 crystallized in different conditions and different space groups. We solved the structure by the SAD method using selenomethionine labeled protein crystallized in space group P1211 (see Table 3 for details of data collection and statistics) The asymmetric unit contains two copies of a tetramer (Supplementary Figures S5A andS5B), in which each monomer is composed of two short helices (helices α1 and α2) and a long C-terminal helix (helix α3 -aa 508-570) (Figures 3A and3B). The long helix of each monomer is composed of eight complete heptad repeats and a stammer at position 540-542 resulting in a kink near residue Pro544 (Figure 3C). Within one tetramer, the four long helices fold into a parallel coiled-coil forming a central shaft, while the short helices form a crown around the Nterminal end of this shaft (Figures 3A and3B). The global net charge of the tetrameric domain is negative at neutral pH, and the calculated electrostatic surface potential shows an asymmetrical distribution, with a negative head and a patch of negative charges at the Cterminal end of the coiled coil shaft, whereas the central part of the coiled-coil shaft is rather neutral (Supplementary Figure S5C).

The N-terminal part of the tetrameric domain exhibits four-fold symmetry, but in the coiledcoil shaft, the four-fold symmetry is progressively replaced by a two-fold symmetry when going from the N-terminal end to the C-terminal end (Figures 3D and3E). The monomers within a tetramer superimpose by pairs with Cα r.m.s.d. values around 0.6 (two-fold symmetrical mate) and 1.0 Å (other two chains). Each protomer Ni contacts the three other protomers. The long helix α3 of Ni is packed between helix α3 of the Ni-1 and Ni+1 protomers, while helix α2 contacts the Ni+1 protomer and helix α1 contacts the Ni+2 protomer (Figure 3B).

In the corresponding tetrameric domain of Sendai virus phosphoprotein, each protomer only interacts with two other adjacent protomers [START_REF] Tarbouriech | Tetrameric coiled coil domain of Sendai virus phosphoprotein[END_REF].

The tetrameric structure of NiV tetramerization domain is stabilized by multiple interchain interactions, each protomer forming on average 19 H-bonds and 11 salt bridges with the others protomers and burying a surface area of 2,200 Å 2 between each pair of adjacent protomers (for a total of 8,800 Å 2 of accessible surface area buried in the tetrameric structure). The interior of the coiled-coil shaft is composed of 19 sidechain layers, mainly hydrophobic but with four layers of polar residues (C512, S515, G519 and A522) at the Nterminal end and one near the C-terminal end (T560) (Figure 3C).

The structure is very similar to the crystal structure of NiV tetramerization domain published by Saphire and coworkers [START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF] with, however, two noticeable differences: (1) Whereas the constructs are similar in length, the coiled-coil region in our structure is six residues shorter than in the previous structure (62 aa instead of 68 aa), with a length intermediate between that of Sendai virus (64 aa) [START_REF] Tarbouriech | Tetrameric coiled coil domain of Sendai virus phosphoprotein[END_REF] and that of measles virus (60 aa) [START_REF] Communie | Structure of the tetramerization domain of measles virus phosphoprotein[END_REF], suggesting crystal contacts play a role in the stabilization of this part of the structure. (2) The C-terminal part of the coiled coil exhibits a slightly different superhelical frequency (-3.39° versus -3.63°) resulting in a different superhelical pitch (158 Å versus 148 Å) (Supplementary Figure S5D). The mapping of crystallographic B-factors onto the structure confirms that the C-terminal end of the coiled-coil shaft is more flexible than the remaining part of the molecule (Supplementary Figure S5E) as shown previously [START_REF] Bruhn | A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function[END_REF][START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF].

The N-terminal region and the C-terminal linker are globally disordered

Several fragments of about 100 amino acids covering the entire N-terminal region but also the linker in the C-terminal part (PCT-LINK) that is predicted to be at least partially disordered (IDR3 in Figure 1A) and the X domain were produced as 13 C, 15 N-labeled proteins and purified (Figure 1C). Each fragment from the N-terminal region has a hydrodynamic radius (Rh) significantly larger than that expected for a globular protein of the same molecular mass and slightly smaller than that expected for a fully unfolded protein in a good solvent (Table 1). SAXS data recorded for these fragments revealed no concentration dependence. For the different N-terminal fragments and for the fragment corresponding to the C-terminal linker (P588-650), the experimental radius of gyration (Rg) values determined using the Guinier approximation were close to the Rg values calculated for a random coil according to Flory's power-law dependence on chain length using parameters for chemically unfolded proteins [START_REF] Fitzkee | Reassessing random-coil statistics in unfolded proteins[END_REF] or for intrinsically disordered proteins (89) (Table 1), in line with the proposal that these regions are disordered. By contrast, the radius of gyration for the X domain is significantly smaller than that of a disordered chain of the same length, as expected for a folded X domain similar to that of Hendra virus [START_REF] Communie | Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus[END_REF].

To characterize the structure of these fragments in solution, we carried out assignment of the backbone NMR resonances and calculated their secondary structure propensities (SSP) from the experimental Cα and Cβ chemical shifts. The 1 H-15 N two-dimensional heteronuclear single quantum coherence (HSQC) spectra of the different N-terminal fragments exhibit poor chemical shift dispersion of the amide 1 H resonances as is typical for a disordered protein [START_REF] Zhang | Structural characterization of folded and unfolded states of an SH3 domain in equilibrium in aqueous buffer[END_REF] (Supplementary Figure S6). The combined SSP score for the entire N-terminal region (PNTR) reveals the presence of several transient α-helices (Figure 4A). In particular, we observe two transiently populated helical elements at the very N-terminus that make up the binding region for N 0 (9). In the N 0 -P complex, these helical elements (aa 1-29) bind to the C-terminal domain of the nucleoprotein mainly through hydrophobic interactions, forming a stable helix, kinked at residue Q21 [START_REF] Yabukarski | Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone[END_REF]. In addition, a third helical element with a population of around 40% is observed at residues 340-355, which coincides with the putative additional weak binding region for the nucleoprotein recently discovered in measles virus [START_REF] Milles | An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function[END_REF]. The 1 H- 15 N HSQC spectrum of the fragment P588-650 corresponding to the C-terminal linker (PCT-LINK) is also typical of a disordered protein (Supplementary Figure S6) and the combined SSP score also revealed the presence of low populated α-helices (aa 600-625) (Figure 4B). By contrast, the 1 H- 15 N HSQC spectrum of the isolated X domain, (PXD, aa 655-709) confirms that this domain is folded in solution, and the chemical shift assignments clearly show that a three-helix bundle structure is predominant in solution (Figure 4C) as also observed in the corresponding domains of measles, mumps, Sendai, and Hendra viruses [START_REF] Communie | Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus[END_REF][START_REF] Johansson | Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein[END_REF][START_REF] Cox | Structural and functional characterization of the mumps virus phosphoprotein[END_REF][START_REF] Blanchard | Structure and dynamics of the nucleocapsid-binding domain of the Sendai virus phosphoprotein in solution[END_REF] . The HSQC spectrum of the tetrameric full-length phosphoprotein (a tetrameric protein complex of 2,868 aa with our construct) contains resonances with limited chemical shift dispersion that superposed well with the resonances from the residues of the N-terminal disordered part of the protein (Figure 4D and Supplementary Figure S6). This clearly indicates that the N-terminal part is flexible with similar conformational behavior in the tetramer as in the isolated peptides and negligible interchain contacts. No resonances for the tetrameric domain, C-terminal linker or the X domain could be identified in the spectrum of full-length phosphoprotein, possibly reflecting slower rotational tumbling caused by a hydrodynamic drag due to the connection to the tetrameric domain and the presence of the X domain at the C-terminus, although we cannot rule out the possibility of interactions between the four C-terminal regions that may also give rise to broadening.

Overall, these results show that (i) the N-terminal region of NiV phosphoprotein is intrinsically disordered and exchanges between multiple conformers on a fast timescale, (ii) this region contains transient α-helical elements, (iii) there are no significant interactions between the N-terminal region of the four protomers within the tetrameric phosphoprotein and (iv) the C-terminal linker also seem to be disordered and to contain transient α-helical elements.

Ensemble description of full-length NiV phosphoprotein tetramer

To study the conformational diversity of NiV phosphoprotein, we generated ensembles of all-atom conformations of the full-length protein and of both tetrameric fragments: P1-580 and P471-709. We used our crystal structure of the tetrameric domain (Figure 3), a homology model of the X domain based on the known structure of the X domain of Hendra virus [START_REF] Communie | Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus[END_REF], heteronuclear backbone chemical shifts (Cα, Cβ, C' and 15 N) for the N-terminal fragments (Figure 4) and SAXS data for the three proteins (Supplementary Figures S2,S3 and S4) as experimental constraints to delineate the contours of conformational space sampled by the full-length protein. We used a combine fit of multiple curves to select conformational subensembles of 10 conformers for which the three average theoretical scattering curves reproduced the three experimental curves. The average SAXS curve calculated for this ensemble is comparable to the experimental SAXS curve measured for the full-length phosphoprotein, while the average curves calculated with the properly truncated forms of the same 10 conformers are comparable to the experimental SAXS curves obtained for P1-580 and P471-709 (Figure 5A). A global χ 2 value of 1.53 and the plot of reduced residuals indicate the quality of the fit (Figure 5A). It should be noted that the independent fits performed for each of the three proteins yielded ensembles of conformers with averaged Rg values in agreement with the experimental values (full-length P: 13 ± 3 nm; P471-709: 6.3 ± 0.5 nm; P1-508: 12 ± 3 nm), but did not improve the overall quality of the fits (Supplementary figure S7). Some discrepancies between the experimental and fitted curves are visible. The downward deviation observed at low Q values for full-length phosphoprotein and to a lesser extent for the P1-580 fragment can be due to intermolecular interactions, whereas the discrepancy observed between the experimental and calculated curves for the fragment P471-709 at Q values > 1.0 nm - 1 (Figure 5A) might result from non-optimal buffer subtraction as already observed for proteins such as this fragment, which comprises both folded and unfolded domains that may have distinct electron densities [START_REF] Bernadó | Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering[END_REF][START_REF] Brennich | How to analyze and present SAS data for publication[END_REF]. Alternatively, it can also result from a bias in the construction of the initial ensemble of the full-length protein. As the initial ensemble of conformers was built for full-length protein and was then truncated to generate the initial ensembles for the fragments P1-580 and P471-709, it is possible that the large N-terminal disordered region has constrained the X domains to position at one pole of the tetramerization domain, whereas such constrain is absent in the fragment P471-709. Finally, additional structural organization or inter-chain interactions might be present in the C-terminal part of the phosphoprotein, which has not been taken into account in our models. It is thus remarkable that the same set of conformers is able to represent the curves obtained for the three constructions. The Rg values of the initial ensemble for the full-length protein exhibited broad Gaussian distribution centered around 12 nm, and the Rg values of the selected ensembles of 10 conformers exhibit a similar broad distribution (Figure 5B), suggesting the absence of constraints on the overall size of the protein. Figure 5C shows a typical model of the fulllength phosphoprotein and Figure 5D shows the overlays of the 10 conformers of each construct selected in the same fitting process for reproducing the data shown panel A.

The N-terminal region of NiV phosphoprotein binds human STAT1 with low affinity

The region of NiV phosphoprotein that binds STAT1 was previously mapped between residues 100 and 160 [START_REF] Rodriguez | Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation[END_REF][START_REF] Rodriguez | Hendra Virus V Protein Inhibits Interferon Signaling by Preventing STAT1 and STAT2 Nuclear Accumulation[END_REF], which contains several highly conserved residues among henipaviruses (Figure 6A). Point mutations within residues 81 to 120 of the phosphoprotein affected the expression of a minireplicon suggesting that this region is involved in the functioning of the polymerase machine, while mutations within residues 111 and 140 prevented the inhibition of interferon signaling [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF].

To investigate the interaction between NiV phosphoprotein and STAT1, we used the fragment P92-190 that contains the entire putative binding site for STAT1, and we expressed and purified a fragment of human STAT1 that comprises residues 132 to 683 encompassing the coiled-coil domain, the DNA binding domain, the linker domain and the SH2 domain (96) (Figure 6B).

The missing 131 N-terminal residues and 38 C-terminal residues from the STAT1α isoform are not required for interaction with the phosphoprotein, V or W proteins of NiV [START_REF] Rodriguez | Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion[END_REF]. The protein STAT1132-683 eluted from the SEC column as a single peak and its molecular mass determined by SEC-MALLS of 64 ± 6 kDa is in good agreement with the theoretical mass of 64.1 kDa (Figure 6C). The fragment P92-190 alone also eluted as a single peak and its molecular mass of 11 ± 1 kDa is in agreement with the theoretical mass of the monomer. In a first approach, we mixed STAT1132-683 (~54 µM) with P92-190 (~540 µM) and loaded the mixture on a SEC column after 2 hour incubation at 20°C. Both proteins eluted independently at elution volumes and with molecular masses corresponding to isolated STAT1132-683 and P92-190 (Figure 6C). In a second approach, we used NMR spectroscopy and recorded 1 H-15 N HSQC spectra of 15 N labeled P92-190 at a final concentration of ~22 µM in the absence or presence of unlabeled STAT1132-683 (~44 µM final concentration) (Figure 6D). In a complex of the size expected for the heterodimer STAT1132-683-P92-190 (~75 kDa), NMR signals are strongly broadened in protonated samples, thus precluding their detection. By comparing the HSQC spectra, we find a partial reduction of the intensity in the presence of STAT1132-683 for resonances corresponding to residues 100 to 150 (Figure 6E). The most affected resonance intensities are within the region of residues 110 to 140, which contains several residues of crucial importance for blocking STAT1 signaling in cell-based assays [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF]. We interpret this result as evidence that, under these experimental conditions, only a fraction of P92-190 molecules bind STAT1132-683, and we estimate the dissociation constant for STAT1132-683 in the range of 10 to 100 µM. We note that conformational exchange contributions from the exchange between free and STAT1-bound P92-190 could also be the source of some of the observed line broadening in the interaction experiments.

We used isothermal titration calorimetry (ITC) and site-directed mutagenesis to further characterize the interaction between the two protein fragments. We used STAT1132-683 as receptor at a concentration of 100 µM and performed titration with P92-190 as ligand at an initial concentration between 1.5 and 2.5 mM. Despite that complete saturation was not achieved, the binding isotherm can be fitted to a single binding site model, yielding a dissociation constant (Kd) of 100 ± 15 µM (∆ = + = -5.4 kcal.mol -1 ) (from five independent measurements) (Figure 7A), in good agreement with the NMR interaction experiment (Figure 6E). The favorable enthalpy of binding (∆ ) of -9.5 kcal.mol -1 is partially balanced by an unfavorable entropic contribution of 4.1 kcal.mol -1 (∆ = -14 cal.mol -1 .K -1 ) (Table 4).

Point mutations Y116A and G125E as well as a triple alanine mutation H117A/D118A/H119A ( 117 AAA 119 ) in full-length NiV phosphoprotein were previously shown to block interferon signaling and to disrupt the interaction with STAT1 [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF][START_REF] Hagmaier | A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species[END_REF]. By contrast, the single amino-acid mutation G120E had no effect [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF]. We expressed and purified

NiV phosphoprotein fragments encompassing residues 92 to 190 (P92-190) and harboring these different mutations (Y116A, G120E, G125E and H117A/Q118A/H1119A). Each protein eluted as a single peak from the size exclusion chromatography column with a molecular mass calculated from MALLS in agreement with the expected molecular mass for a monomeric protein. We performed calorimetric titrations of STAT1132-683 with these different variants, but we were unable to detect any interaction with the variants Y116A and H117A/Q118A/H1119A. With the G120E variant, the affinity was comparable to that of the WT protein (Figure 7B-F and Table 4). With the G125E variant, the affinity is slightly lower than that of WT protein, with a Kd value of 210 ± 90 µM, although the difference in ∆ of 0.4 kcal/mol is not significant (Table 4). These results confirm that the interaction between the N-terminal region of NiV phosphoprotein and STAT1 is direct but weak.

In order to further highlight the importance of the STAT1-binding domain (amino acids 110 to 140) that is present in the phosphoprotein but also in the V and W proteins, we also used recombinant WT or mutant viruses, which were generated by reverse genetics ( 6) and possess either intact or a defective P gene. Two mutant viruses containing substitutions at positions 121 and 135 of the phosphoprotein, V and W proteins (NiV-G121E and NiV-G135E) were compared with NiV WT for their ability to block translocation into the nucleus of endogenous tyrosine-phosphorylated STAT1 activated by interferon (Figure 7G). NiV matrix protein was used as a marker of infection and a specific anti tyrosine-phosphorylated STAT1 antibody was used to monitor the localization of NiV P gene specific proteins (Figure 7G). Little or no tyrosine-phosphorylated STAT1 was detectable in the nucleus of cells infected with the WT NiV, whereas adjacent, uninfected cells exhibited clear nuclear phospho-STAT1 staining. By contrast, a loss of STAT1 binding ability in NiV G121E resulted in strong and apparent tyrosine-phosphorylated STAT1 signal present in the nuclei (Figure 7G). Interestingly, NiV-G135E mirrored NiV WT and showed a similar pattern of disruption of normal STAT1

trafficking. Earlier it had been shown that the V protein harboring the same substitution failed to detectably bind STAT1 in immunoprecipitation studies but inhibited ISG54-driven reporter induction, albeit less efficiently [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF]. Our data suggest that this mutant retains sufficient STAT1-binding activity but that the interaction is too weak to be seen in simple co-expression co-immunoprecipitation assays. The mechanism for such behavior will need to be further investigated, in particular with respect to the implication of STAT2.

DISCUSSION

The highly disordered, tetrameric NiV phosphoprotein is an integral component of the transcription/replication machinery of this dangerous virus and of all members of the Mononegavirales and a hitherto largely unexplored target for viral inhibition. Due to the size and flexible nature of the phosphoprotein its conformational behavior has remained unexplored.

Here, we present the first atomistic representation of full length NiV phosphoprotein in solution. We show that, under our experimental conditions, full-length NiV phosphoprotein as well as two other long fragments containing the tetramerization domain also form tetramers in solution (Figure 2), and we have obtained a second crystal structure of this tetramerization domain [START_REF] Bruhn | Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain[END_REF][START_REF] Salvamani | Oligomerization state of the multimerization domain of Nipah virus phosphoprotein[END_REF]. The phosphoprotein of other paramyxoviruses also contains a tetrameric domain [START_REF] Tarbouriech | Tetrameric coiled coil domain of Sendai virus phosphoprotein[END_REF][START_REF] Cox | Structural and functional characterization of the mumps virus phosphoprotein[END_REF][START_REF] Communie | Structure of the tetramerization domain of measles virus phosphoprotein[END_REF][START_REF] Blocquel | Coiled-coil deformations in crystal structures: The measles virus phosphoprotein multimerization domain as an illustrative example[END_REF], although the existence of a trimeric form in solution has been described [START_REF] Blocquel | Coiled-coil deformations in crystal structures: The measles virus phosphoprotein multimerization domain as an illustrative example[END_REF][START_REF] Beltrandi | Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies[END_REF]. NiV tetramerization domain has dimensions and crystal structure similar to those of Sendai virus tetramerization domain with a parallel tetrameric coiled-coil and a crown of helices at its N-terminal end [START_REF] Tarbouriech | Tetrameric coiled coil domain of Sendai virus phosphoprotein[END_REF]. By contrast, the tetramerization domains of measles and mumps viruses lack the crown of helices, and while the structure of measles domain is also a parallel tetrameric coiled-coil, that of mumps domain is composed of two pairs of parallel helices packed in opposite orientation [START_REF] Cox | Structural and functional characterization of the mumps virus phosphoprotein[END_REF][START_REF] Communie | Structure of the tetramerization domain of measles virus phosphoprotein[END_REF]. Phylogenetic trees based on the phosphoprotein sequences but also on the whole genome sequences place the genus

Henipavirus in an intermediate position between the genera Morbillivirus (measles virus) and

Respirovirus (Sendai virus), while the genus Rubulavirus (mumps virus) is more distant (100, 101). The crown of helices may thus be an ancestral feature lost by some viruses or a modern one acquired by others, that stabilizes the central coiled coil shaft or provides additional function. Up to now, no function has been specifically associated with the N-terminal part of the tetramerization domain, and mutations that perturb the crystallographic water molecules inside the coiled-coil shaft at the crown level had no effect on phosphoprotein function in a minireplicon system [START_REF] Bruhn | A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function[END_REF]. Mapping the crystallographic B-factors on the domain structure shows that the N-terminal part of the central coiled-coil, upstream of the kink, is more rigid than the downstream part (Supplementary Figure S5E). Although the crystallographic packing can influence B factors, the conservation of this dynamical feature in the tetramerization domain of the related measles virus and Sendai virus argues for functional relevance. The presence of inter-helical networks of h-bonds and salt-bridges in the crown and in the upper part of the coiled-coil preceding the kink region could explain this additional rigidity of the structure. By contrast, a conserved basic patch in the C-terminal part of the shaft is known to interact with the polymerase and mutations in this part affect polymerase activity (34) (Figures 3C and3F). Multiple sequence alignment of Henipavirus phosphoprotein shows that the crown is not the most conserved part of the tetramerization domain, while it reveals a highly conserved region following the basic patch and straddling the C-terminal end of the coiled coil, and the N-terminal part of the PCT-LINK (aa 565-600) (Figures 3E and3F). This region includes the less rigid part of the structure (Supplementary Figure S5E), and in related Mononegavirales, it interacts directly with L polymerase [START_REF] Gilman | Structure of the Respiratory Syncytial Virus Polymerase Complex[END_REF][START_REF] Pan | Structure of the human metapneumovirus polymerase phosphoprotein complex[END_REF]. Because the polymerase changes conformation while it operates and moves along its RNA template, the interaction between the phosphoprotein and the polymerase is submitted to tensional, shear and/or rotational stresses and thus likely requires some flexibility in the protein interfaces to maintain the attachment.

Previous sequence analyses predicted that the N-terminal part of the phosphoprotein encompassing the region common to the V and W proteins and extending up to the tetramerization domain, is an intrinsically disordered region except for the N-terminal end [START_REF] Karlin | Structural disorder and modular organization in Paramyxovirinae N and P[END_REF]102), which consists of a MoRE that binds and chaperones unassembled N 0 (9). The Dscore calculated as a consensus of multiple disorder predictions reveals a more complex picture with intervening putatively ordered regions and undefined regions (Figure 1A).

Experimentally, we confirm the overall disorder of the N-terminal region of the phosphoprotein. The hydrodynamic radius determined by SEC and the radius of gyration determined by SAXS indicate that each fragment in the N-terminal region (Table 1) is rather extended, in accordance with the behavior of intrinsically disordered polypeptide chains in water [START_REF] Bernadó | A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering[END_REF]. The poor chemical shift dispersion of the NMR 1 H- 15 N HSQC spectra also supports the high flexibility of the different fragments, but the assignment of the spectra revealed the presence of transient secondary structure elements (Figure 4D and Supplementary Figure S6), and an overlay of the SSP parameter (Figure 4A) and D-score where the N-terminal region and the C-terminal linker are highly flexible and adopt random coil conformation, is in agreement with the SAXS and NMR data (Figure 5).

The ensemble model of full-length phosphoprotein provides new insights about the conformation of this protein in solution (Figure 5). Overall, the molecule appears elongated in the direction of the tetramerization domain shaft with the four X domains clustering at the C-terminal end (Figure 5D). This spatial arrangement certainly provides an ideal positioning of the four X domains for interacting with the polymerase or the nucleocapsid, and for "walking" along the latter, driven by the polymerase. The four N-terminal chains mainly occupy the other end and the sides of the tetramerization domain, likely creating the steric hindrance that pushes the X domains towards one end. When multiple decoys are overlaid with respect to the tetramerization domain, thus mimicking the time average spatial occupancy of the four flexible N-terminal chains and four C-terminal linkers (Figure 5D), the central tetramerization domain including the basic site of putative interaction with the L polymerase is masked. The recent structure of the polymerase complexes of two related viruses revealed that one of the chain of the phosphoprotein is mainly involved in the interaction with the L polymerase, but the three other chains are also binding [START_REF] Gilman | Structure of the Respiratory Syncytial Virus Polymerase Complex[END_REF][START_REF] Pan | Structure of the human metapneumovirus polymerase phosphoprotein complex[END_REF]. These multiple interactions could be required to move the N-terminal chains of the phosphoprotein away from the multimerization domain and to allow access of the polymerase to the central tetrameric shaft.

The tetrameric NiV phosphoprotein is thus a large molecule, with a hydrodynamic radius of 9.3 ± 0.3 nm and a radius of gyration of 10.9 ± 0.2 nm. The Rg/Rh ratio is a sensitive indicator of chain compactness; experimental values for this ratio lie between limits of about 0.8 for globular proteins (103) and of about 1.5 or higher for random coils (104). The value of 1.18

found here for NiV full-length phosphoprotein is in agreement with an elongated molecule and/or the presence of long intrinsically disordered regions and with the values of 1.0 and 1.14 that we previously reported for the full-length phosphoproteins of vesicular stomatitis virus and rabies virus (105), respectively. Despite that these phosphoproteins of the family Rhabdoviridae are dimeric and less than half its size, they share a similar architecture with NiV phosphoprotein [START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF]. Assuming a partial specific volume of 0.73 cm 3 .gr -1 , the intrinsic molecular volume of a protein the size of tetrameric NiV phosphoprotein should be about 390 nm 3 , whereas the volume of the equivalent sphere calculated by using the experimental Rh value is 3,400 nm 3 , almost ten-fold larger. Multiple copies of this protein are packaged in the

Figure S8

). A comparison with measles virus phosphoprotein shows that most functional and structural regions are conserved but that NiV phosphoprotein contains an additional region in the N-terminal region between residues 150 and 300 that is common to the V and W proteins, and is required for binding STAT2 (Supplementary Figure S9). The binding site of STAT1 appears to be conserved between NiV and measles virus, being localized between residues 110 and 140, but in measles virus the binding of STAT2 is mediated by the specific Cterminal region of the V protein (107). We speculate that NiV phosphoprotein, V and W proteins have thus evolved by acquiring an additional extension within their common Nterminal region that recruits STAT2. The first 405 N-terminal amino acids of N-terminal region are common to NiV phosphoprotein, V and W proteins (Figure 1B), and these three proteins block interferon signaling by preventing the phosphorylation of STAT1 and by sequestering both STAT1 and STAT2 proteins in the cytoplasm and the nucleus [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF][START_REF] Rodriguez | Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation[END_REF][START_REF] Rodriguez | Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion[END_REF][START_REF] Shaw | Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively[END_REF].

The primary binding site of STAT1 was localized between residues 110 and 140 of the Nterminal region by co-immunoprecipitation [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF] and this localization was confirmed here by a direct interaction assay. The short section of only 30 amino acids forming this binding site in the NiV phosphoprotein suggests that it is a typical example of a short linear motif that folds upon binding to recruit a protein partner (108) as used by numerous viruses to hijack cellular machineries (109) by mimicking cellular proteins, such as STAT1 itself, which uses a short linear motif within its transactivation domain (TAD) to interact with the TAZ1 domain of the CBP/p300 transcriptional coactivator protein (110).

The Y116A mutant and the H117A/D118A/H119A triple mutant lost their ability to bind STAT1. Similarly, the mutant G121E prevents the accumulation of phosphorylated STAT1 in the nucleus of infected cells. Surprisingly, the G125E mutant, which was unable to inhibit IFN signaling and failed to co-precipitate STAT1 and STAT2 [START_REF] Ciancanelli | Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism[END_REF][START_REF] Hagmaier | A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species[END_REF], shows similar binding affinity for STAT1 as the WT protein by ITC measurement. By contrast, the G135E mutant conserves the ability to block the accumulation of phosphorylated STAT1 in the nucleus, while it also fails to co-precipitate STAT1. Because the affinity between the phosphoprotein and STAT1 is weak, it is possible that the co-precipitation with STAT1 also requires the presence of STAT2 as found for measles virus (107), and that the mutations of Gly125 and Gly135 perturb the co-interaction between STAT1 and STAT2 rather than the direct interaction with STAT1. It is also possible that the difference in affinity, although small, is sufficient to produce an effect in vivo and that interactions with WT and the G125E variant are influenced differently by intracellular conditions such as molecular crowding.

In summary, our results revealed that the NiV phosphoprotein behaves as a nanometer-size octopus-like protein that uses its long flexible arms to expose binding sites for multiple viral and cellular partners and to orchestrate their participation in virus replication, in hijacking cellular machines and in interfering with the interferon response. disordered prediction. The D-score was calculated as described in [START_REF] Gerard | Modular Organization of Rabies Virus Phosphoprotein[END_REF]. The threshold to distinguish between ordered and disordered region is set at 0. with standard deviations. The hydrodynamic radius (Rh) was determined from the elution volume by calibrating the column with standard globular proteins (Table 1).

(

  Figure1A) plots shows that in several instances, short regions with an intermediate D-score (moderately above or below 0.5) are transiently folded into helical conformation and could correspond to α-MoRE involved in recruiting different partners, in particular the polo-like kinase 1, the exportin or the viral nucleoprotein (Supplementary FigureS8). The comparison of the NMR spectra from the different N-terminal fragments with the full-length phosphoprotein shows that most resonances overlap (Figure4D), thereby showing that the structural properties of fragments are the same in the full-length protein. In other words, the overall disorder of N-terminal region is not a result of our divide-and-conquer approach, but is an intrinsic property of this region of the protein. This view is also confirmed by the ensemble modeling, which shows that an ensemble model of full-length phosphoprotein,

  virion and might occupy an important part of the particle volume if not otherwise compacted.With respect to the nucleocapsid, the length of the disordered C-terminal linker (70 aa) allows an extension of the order of 50 nm (2 x 70 x 0.35 nm) between two X domains, which would allow to connect nucleoproteins at least 10 subunits apart along the nucleocapsid, and probably even more distant if the possible extension of the C-terminal disordered region of the nucleoprotein (NTAIL) is taken into account. In the virion, the nucleocapsids have a diameter of 18-19 nm and an average pitch of 5 nm[START_REF] Gutsche | Near-atomic cryo-EM structure of the helical measles virus nucleocapsid[END_REF], and thus one phosphoprotein could connect nucleoproteins across the nucleocapsid. The larger size of tetrameric NiV phosphoprotein may thus be of importance in the packaging of the viral particle and in the functioning of the polymerase machine, catching at long distance and directing partners to their site of use.The architecture of the phosphoprotein is conserved among Mononegavirales, including the localization of functional and structural modules, but the length of the protein varies considerably between families and even between genera within the same family. As illustrative examples, VSV in the family Rhabdoviridae is 267 aa long, whereas measles virus and NiV phosphoproteins in the family Paramyxoviridae are 507 and 709 aa long, respectively. The reason why NiV phosphoprotein is so long remains enigmatic, considering that VSV phosphoprotein, for example, perfectly assumes similar functions in the life cycle of the virus. In a previous study, we compared the structure of the C-terminal N-binding domain of the phosphoprotein from different Mononegavirales and discovered that the variation in length of this protein domain correlated with the capacity to perform additional functions, suggesting a model where the protein evolves by accretion of functional modules[START_REF] Martinez | Structure of the C-terminal domain of lettuce necrotic yellows virus phosphoprotein[END_REF].Henipavirus phosphoproteins are significantly longer than the phosphoproteins from other genera of Paramyxoviridae (106). Because the L polymerases of the Mononegavirales have a high error-rate, the genome of these viruses has the capacity to rapidly evolve and trim superfluous parts of the genome, suggesting that the very existence of additional lengths in NiV phosphoprotein sequence is a sign that they perform additional functions or are required for the fitness of the virus. We can therefore speculate that the larger size of the N-terminal domain correlates with its implication in other functions, and that the transient α-helical elements evidenced by NMR spectroscopy and predicted by sequence analysis represent short linear motifs that recruit different and yet unidentified partners. Some of them clearly encompass regions of the protein known to bind viral or cellular proteins (Supplementary

5 .

 5 The shaded areas indicate the known positions of the folded tetramerization domain and X domain. (B) Modular organization of NiV phosphoprotein. The upper part shows the structural organization of the phosphoprotein encoded in the main open reading frame (0 ORF). Boxes indicate the localization of folded domains, undulated lines the localization of predicted MoRE and lines the localization of intrinsically disordered regions (IDR 1-3). The green dots show two identified phosphorylation sites. In the middle part, the lines show the location of the known functional regions. In the lower part, the blue lines show the position of the C gene and of the regions coding for the specific parts of the V and W proteins in the alternative open reading frames (+1 ORF and +2 ORF). (C) Divide-and-conquer strategy. The lines indicate the position of the different fragments of the phosphoprotein used in this study.

FIGURE 2 .

 2 FIGURE 2. Molecular masses and size of full length phosphoprotein (PFL in black) and of different fragments (P1-580 in red; P471-709 in green; P471-580 in blue) in solution. Size exclusion chromatography was performed at 20 °C on a TSK G4000 column equilibrated with 20 mM Tris/HCl pH 7.5 containing 150 mM NaCl using a flow rate of 0.8 mL.min -1 . The elution was monitored on-line by multi-angle laser light scattering and differential refractometry. The lines show the chromatograms monitored by differential refractive index measurements. The crosses indicate the molecular mass calculated from static light scattering across the elution peak and the numbers indicate the weight-averaged molecular mass (kDa)

FIGURE 3 .

 3 FIGURE 3. Crystal structure of NiV phosphoprotein tetramerization domain (PMD). (A) Cartoon representation of tetrameric NiV PMD (4GJW.pdb). The different chains are shown in different colors and the overall dimensions are indicated (black arrows). The N and Cterminal end are indicated for each chain and the three α-helices, α1 to α3, are shown for chain D (in red). The position of the stammer in the long coiled-coil is shown and the dotted lines indicate the location of the a-layers displayed in panels D and E. (B) Contacts between

  PYMOL.

FIGURE 4 .

 4 FIGURE 4. NMR characterization of NiV phosphoprotein N-terminal region (PNTR), Cterminal linker and X domain. (A) Secondary structure propensities (SSP) of the N-terminal region of the phosphoprotein obtained on the basis of experimental 13 Cα and 13 Cβ chemical shifts. The propensities were obtained by combining spectral assignments of individual subconstructs of this region. (B) Secondary structure propensities of the C-terminal intrinsically disordered linker region of the phosphoprotein (PCT-LINK) obtained from experimental 13 Cα and 13 Cβ chemical shifts. (C) Secondary structure propensities of the C-terminal X domain of the phosphoprotein (PXD) obtained from experimental 13 Cα and 13 Cβ chemical shifts. (D) 1 H-15 N HSQC spectrum of full-length NiV phosphoprotein (red) superimposed with HSQC spectra (blue) of individual sub-constructs of the protein (P1-100, P92-190, P173-240, P223-319, P300-401, P387-479).

FIGURE 5 .

 5 FIGURE 5. Ensemble structure of NiV phosphoprotein. (A) Multiple-curve fitting of SAXS data. The upper panel shows experimental SAXS curves of the three tetrameric proteins: PFL (in red), P1-580 (in blue) and P471-709 (in orange), with experimental standard deviations shown as error bars. The black lines show the multiple-curve fit obtained for the three SAXS curves for a selected ensemble of 10 conformers. The reduced χ 2 value is 1.53 over all three constructs. The lower panels show the reduced residuals (∆I/ σ). (B) R g distribution for an ensemble of 10 conformers of full-length phosphoprotein. The thin black

Table 1 .

 1 Mass and size of the different phosphoprotein constructsNumber of amino acids in the used constructs including C-terminal His tag and linker (LEHHHHHH) and in some cases a N-terminal M. Isoelectric point of the used constructs calculated using the webserver ProtParam. In parentheses, isoelectric point calculated without the C-terminal His tag. In parentheses, calculated mass for a tetramer.

	Protein	Naa 1	pIcalc 2	MMcalc 3 (kDa)	MMexp 4 (kDa)	Rh,folded 5 (nm)	Rh,unfolded 5 (nm)	Rh,exp 6 (nm)	Rg,theo 7 (Bernado) (nm)	Rg,theo 8 (Fitskee) (nm)	Rg,exp 9 (nm)	Dmax 10 (nm)
	P1-100	108	5.1	12.347	13.5 ± 1.5	1.8	3.1	2.6 ± 0.2	2.9	3.4	3.2 ± 0.1	12
	P92-190	108	4.9	11.875	12.0 ± 0.5	1.8	3.0	2.6 ± 0.2	2.9	3.4	3.1 ± 0.2	12
	P173-240	78	6.4	8.691	8.0 ± 2	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	P92-250	168	4.6	18.583	21.0 ± 2.0	n.d.	n.d.	n.d.	3.7	4.5	4.0 ± 0.3	15
	P223-319	106	4.5	11.536	12.9 ± 1.5	1.8	3.0	3.1 ± 0.2	2.9	3.4	3.1 ± 0.1	11.5
	P300-401	111	5.9	12.421	11.5 ± 1.5	n.d.	n.d.	n.d.	2.9	3.5	n.d.	n.d.
	P387-479	102	7.1	11.151	12.4 ± 1.5	1.8	3.0	2.5 ± 0.2	3.3	3.3	3.2 ± 0.1	12.5
	P471-580	119	5.2	13.757 (55.028)	58 ± 6	3.1	6.7	4.3 ± 0.2	n.r.	n.r.	n.d.	n.d.
	P655-709	63	5.3	7.364	8 ± 1	n.d.	n.d.	n.d.	2.2	2.5	1.6 ± 0.1	5.5
	P588-650	72	5.3	8.289	8 ± 2	n.d.	n.d.	n.d.	2.4	2.7	2.6 ± 0.1	10
	P588-709	131	4.9	15.041	15.3 ± 1	1.6	2.5	1.9 ± 0.2	n.d.	n.d.	n.d.	n.d.
	P1-580	588	4.7	64.756 (259.024)	250 ± 25	5.5	14.8	9.2 ± 0.3	n.r.	n.r.	10.8 ± 0.3	41
	P471-709	248	4.9	28.369 (113.476)	113 ± 10	4.1	9.8	7.8 ± 0.3	n.r.	n.r.	6.3 ± 0.1	22
	PFL	717	4.7	79.367 (317.468)	306 ± 30	6.0	16.8	9.3 ± 0.2	n.r.	n.r.	10.9 ± 0.1	39

Table 3

 3

	. Crystallographic data collection and refinement statistics
	Parameter
	Resolution (Å)	48.75 -3.00 (3.06-3.00)
	Space group	P1 21 1
	Unit cell
	a, b, c (Å)	59.5, 85.60, 122.6
	α, β, γ (°) Total reflections Unique reflections 1	90.0, 94.7, 90.0 195091 47924
	Rfree test set Multiplicity	1257 4.1 (1.9)
	Completeness (%)	99.5 (99.9)
	I/σ(I)	5.3 (2.27)
	Rwork	0.175 (0.253)
	Rfree	0.229 (0.286)
	RMSD (bond lengths) (Å) 0.013
	RMSD (angles) (°)	1.16
	Ramachandran favored (%)	98.04
	Ramachandran allowed (%)	1.83
	Ramachandran outliers (%)	0.13
	Average B factor (Å 2 )	55
	Values in parentheses are for the highest-resolution shell. 1 Friedel's pairs unmerged

Table 4 .

 4 Isothermal titration calorimetry data.

	Ligand	n	Kd (µM)	∆G (kcal.mol -1 )	∆H (kcal.mol -1 )	-T∆S (kcal.mol -1 )	∆S (cal.mol -1 .K -1 )
	wt	1.19	100 ± 15 -5.4 ± 0.8 -9.5 ± 1.2 4.2 ± 1.2	-14 ± 4.0
	Y116A	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	H117A/D118 A/H119A	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	G120E	1.23	70 ± 9	-5.7 ± 0.7 -8.4 ± 0.6 2.7 ± 0.6	-8.9 ± 2.1
	G125E	1.31	210 ± 90 -5.0 ± 2.0 -7.5 ± 2.6 2.5 ± 2.7	-8.4 ± 9.2
	T = 298 K n.d. : not determined

Modular architecture of Nipah virus phosphoprotein

. (A) Consensus

AUTHOR CONTRIBUTIONS

MRJ, FY, JMB, VV, MB and MJ conceived the experiments; MRJ, FY, GC, EC, CM, VVa, JMB performed the experiments; MRJ, FY, CM, NT, JMB, VV, MB and MJ analyzed and interpreted the data; MRJ, JMB, MB and MJ wrote the paper.

ACCESSION CODES

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4GJW. SAXS data have been deposited in the Small Angle Scattering Biological Data Bank under accession code SASDHT4.

ACKOWLEDGMENTS

We thank Dr. Rob W. Ruigrok (IBS, Grenoble, France) for helpful discussions and Dr. Daniel Panne (EMBL, Grenoble, France) for the gift of a plasmid coding for human STAT1. F.Y. was supported by a predoctoral fellowship from the Région Rhône-Alpes. The work was supported by grants from the French Agence Nationale de la Recherche to M.J. and M.B.

(ANR BSV8-2012-NNViPol) and to M.J. and V.V. (ANR-18-CE11-0014-02), from the Fond de la Recherche Médicale (FRM) (grant "Equipe 2017" DEQ20170336754) to M.J. and from the FINOVI Fundation to M.J. and V.V. We acknowledge the European Synchrotron Radiation Facility for the allocation of beamtime on ID14-3 and ID23-1 and the Synchrotron SOLEIL for the allocation of beamtime on Proxima 1. We acknowledge the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003).

All experiments involving live NiVs were carried out in the INSERM Jean Merieux BSL4 laboratory in Lyon, France. We thank the biosafety team members for their support in conducting experiments. 4 Measured by SEC-MALLS. 5 The theoretical Rh values are calculated from Rh values of folded and chemically unfolded standard proteins taken from Uversky et al. (1997) and using the experimental molecular mass determined by SEC-MALLS. 6 Measured from SEC data by calibrating the system with standard globular proteins. 7 Calculated using the power law and parameters defined by Bernado et al. 8 Calculated using the power law and parameters defined by Fitskee et al. 9 Measured from SAXS data at low Q using the Guinier approximation. 10 Determined from SAXS data using the program PRIMUS. n.d. = not determined n.r. = not relevant Gray shaded lines show proteins that were found to be tetrameric in solution.