The Hendra and Nipah viruses (HeV and NiV) are zoonotic biosafety level-4 pathogens belonging to the Paramyxoviridae family. We previously showed that their W protein, a key player in the evasion of the host antiviral response, forms highly flexible, curved fibrils in vitro. Here, we show that the cysteine oxidation state acts as a molecular switch controlling the formation of either amorphous aggregates or flexible fibrils, and that residues 2 to 29 are essential for fibrillation. We also uncover that the HeV W protein (WHeV) can also self-assemble in cellula. WHeV forms distinct types of nuclear condensates that exhibit different dependencies on the cysteine redox state. While deletion of residues 2-29 prevents formation of nuclear filaments, cysteine-to-serine substitution mainly impairs the formation of non-filamentous condensates. Both infection and WHeV ectopic expression trigger oxidative stress, presumably favorable to WHeV condensation. Finally, we show that impaired ability to form redox-sensitive, non-filamentous condensates is associated with a reduced W ability to inhibit the NF-κB pathway, while it conversely enhances W ability to repress the interferon response pathway by enhancing W binding to STAT1.

Hendra virus (HeV) is a biosafety level 4 human pathogen belonging to the Henipavirus genus within the Paramyxoviridae family. In HeV, the phosphoprotein-encoding gene also drives the synthesis of the V and W proteins that are two major players in the host innate immune response evasion. These three proteins share a common intrinsically disordered N-terminal domain (NTD) and have distinct C-terminal domains. We recently reported the ability of a short region (i.e., PNT3), located within the shared NTD, to form fibrils. We subsequently identified a PNT3 motif (EYYY) critically involved in fibrillation and deciphered the contribution of each tyrosine to the process. Herein, we combined mutational studies with various biochemical and biophysical approaches to further investigate the molecular mechanisms underlying PNT3 fibrillation. The results show that (i) lysine residues play a critical role in driving fibrillation, (ii) hydrophobic residues affect the nucleation step, and (iii) charge distribution strongly affects the fibrillation propensities. Vibrational Raman spectroscopy data further validated the role of lysine residues in promoting fibrillation and enabled documenting the formation of cross-β amyloid structures. Altogether, these results illuminate the molecular mechanisms involved in fibril formation and pave the way towards the rational design of inhibitors.

The RNA genome of measles virus is encapsidated by the nucleoprotein within a helical nucleocapsid that serves as a template for both transcription and replication. The intrinsically disordered domain of the nucleoprotein (NTAIL) is essential for binding the polymerase complex responsible for viral transcription and replication. As for many IDPs, binding of NTAIL occurs through a short molecular recognition element (MoRE) that folds upon binding, with the majority of NTAIL remaining disordered. Although NTAIL regions far from the MoRE influence the binding affinity, interactions between them and the MoRE have not been investigated in depth. Relying on photo-induced electron transfer (PET) experiments between tryptophan and cysteine pairs placed at different positions in the protein under varying salt and pH conditions, combined with analytical models, simulations, and coevolutionary analysis, we identified transient interactions between two disordered regions distant in sequence, which dominate NTAIL dynamics, and regulate the conformational preferences of both the MoRE and the entire NTAIL domain. We propose mechanisms by which these non-local interactions may regulate binding to the measles phosphoprotein, polymerase recruitment, and ultimately viral transcription and replication. Our findings may be extended to other IDPs, where non-local intra-protein interactions affect the conformational preferences of intermolecular binding sites. The measles virus relies on the intrinsically disordered domain of its nucleoprotein, NTAIL, to bind the polymerase complex responsible for viral transcription and replication, but the role played by disordered regions away from the binding site is not clearly understood. Here, through a combination of experiments and simulations, the authors show that transient and non-local interactions between disordered regions distant in sequence influence the conformational preferences of the binding sites and the folding and availability of its molecular recognition element, affecting viral replication kinetics.

The Hendra (HeV) and Nipah (NiV) viruses are high-priority, biosafety level-4 pathogens that cause fatal neurological and respiratory disease. Their P gene encodes not only the P protein, an essential polymerase cofactor, but also the virulence factors V and W. We previously showed that the W protein of HeV (WHeV) forms amyloid-like fibrils and that one of its subdomains, PNT3, fibrillates in isolation. However, the fibrillation kinetics is much faster in the case of the full-length WHeV compared to PNT3, suggesting that another WHeV region contributes to the fibrillation process. In this work, we identified the region spanning residues 2-110 (PNT1) as the crucial region implicated in WHeV fibrillation. Through site-directed mutagenesis, combined with thioflavin T binding experiments and negative-staining transmission electron microscopy, we showed that a predicted cryptic amyloidogenic region (CAR) within PNT1 is the main driver of fibrillation and deciphered the underlying molecular mechanism. Using FTIR, we showed that PNT1 fibrils are enriched in cross β-sheets. Sequence alignment revealed conservation of the CAR across the Henipavirus genus and enabled the identification of a hitherto never reported pro-amyloidogenic motif. The ability to form fibrils was experimentally shown to be a common property shared by Henipavirus PNT1 proteins. Overall, this study sheds light on the molecular mechanisms underlying WHeV fibrillation and calls for future studies aimed at exploring the relevance of the newly identified pro-amyloidogenic motif as a valuable target for antiviral approaches.

The Hendra and Nipah viruses (HeV and NiV) are zoonotic biosafety level-4 pathogens within the Paramyxoviridae family. We previously showed that their W proteins form amyloid-like fibrils in vitro . Here, we demonstrate that W also forms fibrils in cellula and that cysteine residues are crucial in dictating the ability of W proteins to fibrillate. The cysteine oxidation state acts as a switch to generate either amorphous aggregates or flexible fibrils. Ectopic expression of WHeV induces an oxidative stress and WHeV fibrils were observed in the nuclei of different cell lines, with fibrillation being impaired by cysteine substitutions. Finally, nuclear fibrils are associated with an impairment of the NF-κB pathway in WHeV transfected cells. This work provides experimental evidence for the ability of Henipavirus W proteins to fibrillate in transfected cells and the first clues on their functional impact.Significance Statement Nipah and Hendra viruses are severe pathogens infecting humans and livestock, classified among the 8 highest priorities for research by the WHO. The W protein, along with the V protein, is a virulence factor responsible for antiviral response inhibition and we demonstrate here that its fibrillation into amyloid-like fibrils occurs in the nucleus of transfected cells, with their formation being dependent of the redox state of the W cysteine residues. The sole transfection of W provokes the production of reactive oxygen species, creating a suitable environment for the fibrils to form. Finally, we show that W fibrils enhance the repression of the antiviral response, thus pointing to W fibrillation as a new promising antiviral target.### Competing Interest StatementThe authors have declared no competing interest.

The Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N-terminal intrinsically disordered domain (NTD) and possess distinct C-terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid-like fibrils. However, structural information on W CTD (CTDW) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTDWS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTDW interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTDW favors the formation of fibrils by the NTD both in cis and in trans. Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies.

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

The Measles virus nucleocapsid is made of thousands of nucleoprotein (N) repeats, which hold the viral RNA in a helical structure. The last 125 amino acids of each N repeat (NTAIL) are intrinsically disordered and protrude radially outward from the nucleocapsid. NTAIL promotes virus replication by binding to the XD domain of the phosphoprotein P (PXD), which in turn brings the viral polymerase close to the nucleocapsid, where it transcribes and replicates the viral RNA. Only 18 amino acids of NTAIL directly bind to PXD via coupled folding and binding. The majority of NTAIL, on either side of this molecular recognition region (MoRE), remains disordered. While it has been shown that these disordered regions dampen the binding affinity, interactions involving these regions, and their possible functional role have not been identified.

The split-green fluorescent protein (GFP) reassembly assay is a well-established approach to study protein-protein interactions (PPIs). In this assay, when two interacting proteins X and Y, respectively fused to residues 1-157 and to residues 158-237 of GFP, are co-expressed in E. coli, the two GFP halves are brought to sufficient proximity to reassociate and fold to recreate the functional GFP. At constant protein expression level, the intensity of fluorescence produced by the bacteria is proportional to the binding affinity of X to Y. We hypothesized that adding a third partner (Z) endowed with an affinity for either X or Y would lead to an in vivo competition assay. We report here the different steps of the set-up of this competition assay, and define the experimental conditions required to obtained reliable results. Results show that this competition assay is a potentially interesting tool for screening libraries of binding inhibitors, Z being either a protein or a chemical reagent.

Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid-liquid phase separation and fibrillation.

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein–protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan’s lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results.

NTAIL is the intrinsically disordered C-terminal domain of the measles virus nucleoprotein N. NTAIL protrudes from the nucleocapsid where the viral RNA is packaged. It recruits the polymerase and enables viral replication upon binding to a folded domain of protein P (PXD). The binding occurs via a coupled folding and binding mechanism, in which a small NTAIL region acts as binding recognition motive and folds into an κ-helix (κ-MoRE). The remaining portion of NTAIL remains disordered upon binding.

The ASR protein family has been discovered thirty years ago in many plant species and is involved in the tolerance of various abiotic stresses such as dehydration, salinity and heat. Despite its importance, nothing is known about the conserved ABA-Water Deficit Stress Domain (ABA-WDS) of the ASR gene family. In this study, we characterized two ABA-WDS domains, isolated from durum wheat (TtABA-WDS) and barley (HvABA-WDS). Bioinformatics analysis shows that they are both consistently predicted to be intrinsically disordered. Hydrodynamic and circular dichroism analysis indicate that both domains are largely disordered but belong to different structural classes, with HvABA-WDS and TtABA-WDS adopting a PreMolten Globule-like (PMG-like) and a Random Coil-like (RC-like) conformation, respectively. In the presence of the secondary structure stabilizer trifluoroethanol (TFE) or of increasing glycerol concentrations, which mimics dehydration, the two domains acquire an α-helical structure. Interestingly, both domains are able to prevent heat- and dehydration-induced inactivation of the enzyme lactate dehydrogenase (LDH). Furthermore, heterologous expression of TtABA-WDS and HvABA-WDS in the yeast Saccharomyces cerevisiae improves its tolerance to salt, heat and cold stresses. Taken together our results converge to show that the ABA-WDS domain is an intrinsically disordered functional domain whose conformational plasticity could be instrumental to support the versatile functions attributed to the ASR family, including its role in abiotic stress tolerance. Finally, and after validation in the plant system, this domain could be used to improve crop tolerance to abiotic stresses.

The measles virus nucleoprotein N possesses an intrinsically disordered domain, NTAIL, that protrudes from the nucleocapsid and binds to the P protein, thereby recruiting the polymerase and enabling viral replication. A small NTAIL region (18 out of 125 aa) acts as a binding motive (α-MoRE), folding upon binding to the folded X domain of P (PXD). The rest of NTAIL remains disordered. Although the remaining disordered regions of NTAIL have been shown to dampen the binding affinity, the underlying mechanism and their role in NTAIL dynamical reconfiguration upon binding is not clear. We studied the conformational dynamics of full-length NTAIL, in the free and PXD-bound states, using photo-induced electron transfer (PET) between a single tryptophan and a cysteine, placed at different labelling positions, spanning different regions of the protein. We find that full-length NTAIL exhibits significant dynamical heterogeneity, as measured by contact formation rates. These rates can be directly compared to rates generated from polymer models, coarse-grained and all-atom simulations. By comparing PET and CD measurements of full-length NTAIL and α-MoRE peptides under various conditions (e.g. pH, salt, and helix inducing solvents), with analytical polymer models, and with coarse-grained simulations, we find that neither local properties of the α-MoRE (secondary structure sampling), nor charge interactions alone can quantitatively explain our experimental observations. All atom simulations of full-length NTAIL, and of the fragment free in solution, suggest a significant slowing down of contact formation dynamics due to non-local interactions involving the flanking disordered regions. We discuss the role of charge, local secondary structure interactions, and non-local interactions, in the observed dynamical slowing down in the α-MoRE region of NTAIL. Our results highlight the potential role of flanking regions in the function and binding of NTAIL to PXD.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

The nucleoprotein from the measles virus (MeV) possesses a C-terminal intrinsically disordered domain, referred to as NTAIL, which is essential for virus replication. NTAIL binds to the folded X domain of phosphoprotein P (PXD), enabling recruitment of the polymerase complex and fine regulation of viral messenger synthesis. NTAIL is highly disordered in the unbound state, and partially folds upon binding to PXD. However, the majority of NTAIL remains disordered in the bound state. The folding upon binding of NTAIL has been characterized using a large number of experimental techniques, revealing many important aspects of binding. However, the role of dynamical reconfiguration of NTAIL disordered regions, and the mechanism by which these contribute to binding remains elusive. To characterize the large-scale dynamical reconfigurations of NTAIL in solution, in the bound and unbound states, we use photo-induced electron transfer (PET) between tryptophan (W) and cysteine (C). This technique allows probing both the W-C distance and the rate of W-C contact formation in IDPs, without using prosthetic dyes. Measured PET rates can be directly compared to results from molecular simulations, providing a more detailed description of IDP conformational ensembles and dynamics. By generating a series of NTAIL W-C mutants, we use PET to probe different regions of the protein, and their modifications upon binding. We compare our experimental results to molecular simulations of NTAIL in the free and bound states. The combined results provide insights into the role of disordered regions, and their conformational dynamics in IDP binding.

Intrinsically Disordered Proteins (IDPs) are a class of protein that exert their function despite lacking a well-defined three-dimensional structure, which is sometimes achieved only upon binding to their natural ligands. This feature implies the folding of IDPs to be generally coupled with a binding event, representing an interesting challenge for kinetic studies. In this review, we recapitulate some of the most important findings of IDPs binding-induced folding mechanisms obtained by analyzing their binding kinetics. Furthermore, by focusing on the interaction between the Measles virus NTAIL protein, a prototypical IDP, and its physiological partner, the X domain, we recapitulate the major theoretical and experimental approaches that were used to describe binding induced folding.

In this review, we detail the most common experimental approaches to assess and characterize protein intrinsic structural disorder, with the notable exception of NMR and EPR spectroscopy, two ideally suited approaches that will be described in depth in two other reviews within this special issue. We discuss the advantages, the limitations, as well as the caveats of the various methods. We also describe less common and more demanding approaches that enable achieving further insights into the conformational properties of IDPs. Finally, we present recent developments that have enabled assessment of structural disorder in living cells, and discuss the currently available methods to model IDPs as conformational ensembles.