Liquid-Liquid Phase Separation Modifies the Dynamic Properties of Intrinsically Disordered Proteins.

Liquid-liquid phase separation of flexible biomolecules has been identified as a ubiquitous phenomenon underlying the formation of membraneless organelles that harbor a multitude of essential cellular processes. We use nuclear magnetic resonance (NMR) spectroscopy to compare the dynamic properties of an intrinsically disordered protein (measles virus NTAIL) in the dilute and dense phases at atomic resolution. By measuring 15N NMR relaxation at different magnetic field strengths, we are able to characterize the dynamics of the protein in dilute and crowded conditions and to compare the amplitude and timescale of the different motional modes to those present in the membraneless organelle. Although the local backbone conformational sampling appears to be largely retained, dynamics occurring on all detectable timescales, including librational, backbone dihedral angle dynamics and segmental, chainlike motions, are considerably slowed down. Their relative amplitudes are also drastically modified, with slower, chain-like motions dominating the dynamic profile. In order to provide additional mechanistic insight, we performed extensive molecular dynamics simulations of the protein under self-crowding conditions at concentrations comparable to those found in the dense liquid phase. Simulation broadly reproduces the impact of formation of the condensed phase on both the free energy landscape and the kinetic interconversion between states. In particular, the experimentally observed reduction in the amplitude of the fastest component of backbone dynamics correlates with higher levels of intermolecular contacts or entanglement observed in simulations, reducing the conformational space available to this mode under strongly self-crowding conditions.

Multivalent Dynamic Colocalization of Avian Influenza Polymerase and Nucleoprotein by Intrinsically Disordered ANP32A Reveals the Molecular Basis of Human Adaptation.

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remains poorly understood. Here we characterize ternary complexes formed between ANP32A, FluPol, and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted FluPol, containing the E627 K mutation, simultaneously bind the identical extended linear motif on human ANP32A in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.

Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

Destabilization of the human RED-SMU1 splicing complex as a basis for host-directed antiinfluenza strategy.

New therapeutic strategies targeting influenza are actively sought due to limitations in current drugs available. Host-directed therapy is an emerging concept to target host functions involved in pathogen life cycles and/or pathogenesis, rather than pathogen components themselves. From this perspective, we focused on an essential host partner of influenza viruses, the RED-SMU1 splicing complex. Here, we identified two synthetic molecules targeting an α-helix/groove interface essential for RED-SMU1 complex assembly. We solved the structure of the SMU1 N-terminal domain in complex with RED or bound to one of the molecules identified to disrupt this complex. We show that these compounds inhibiting RED-SMU1 interaction also decrease endogenous RED-SMU1 levels and inhibit viral mRNA splicing and viral multiplication, while preserving cell viability. Overall, our data demonstrate the potential of RED-SMU1 destabilizing molecules as an antiviral therapy that could be active against a wide range of influenza viruses and be less prone to drug resistance.

Human viruses, ancient, recent and zoonosis: a never ending story? / Virus humains anciens, récents et zoonotiques : une histoire sans fin ?

For the past three years, the nature and evolution of human viruses have been taught in University Grenoble-Alpes without relying on the systematic list of all virus families. A «historical¼ approach allows to define three main categories of viruses following if they have co-evolved with humans for a very long time (ancient human viruses), if they began to infect humans in the Neolithic or later (recent human viruses) or if they are still animal viruses that are transmitted to humans sporadically (zoonotic viruses). We present below the principles and some examples of this pedagogic separation which has not the pretention to replace the classical taxonomic classification based on morphological and sequence similarity (ICTV classification) or on the form and replication mode of the viral genome (Baltimore classification). It helps grouping of viruses with similar effects even if their evolution is different. We show where human viruses come from and how they can cause human diseases. This approach was tested with Biology students, and then extended to Medicine and Pharmacy students to ensure that teaching was based on the same concepts in the three Faculties. In the end, all the students were very receptive and interested in this approach. Of course, different teaching methods can work, but this way of presenting things is also more fun for teachers and promotes cooperation between speakers.

Depuis trois ans, une expérience pédagogique est menée à l'université Grenoble-Alpes pour enseigner la nature et l'évolution des virus humains, sans se baser sur la liste systématique de toutes les familles de virus. Le choix a été fait d'une approche « historique ¼ des virus chez l'homme, permettant de définir trois grandes catégories de virus selon qu'ils aient co-évolué avec l'homme pendant très longtemps (virus humains anciens), ou qu'ils l'aient infecté plus récemment au Néolithique ou plus tard (virus humains récents) ou enfin qu'ils évoluent à partir de virus animaux transmis à l'homme de manière sporadique (virus zoonotiques). Nous exposons ci-dessous les principes et quelques exemples de cette distinction pédagogique alternative qui n'a pas la prétention de remplacer les classifications taxonomiques classiques basées sur les similarités morphologiques et de séquences (classification ICTV) ou sur la forme et le mode de réplication du génome viral (classification de Baltimore). Elle permet de faciliter le regroupement de virus ayant des effets similaires même si leur divergence évolutive est importante. Nous montrons ainsi l'origine des virus humains et comment ils peuvent entraîner des maladies humaines. Cette approche a été expérimentée avec les étudiants de biologie, puis étendue aux étudiants de médecine et de pharmacie, pour que l'enseignement soit basé sur les mêmes concepts dans les trois UFR. Au final, tous les étudiants ont été très réceptifs et intéressés par cette approche. Bien sûr, différentes méthodes d'enseignement peuvent fonctionner, mais cette façon de présenter les choses est également plus ludique pour les enseignants et favorise la coopération entre les intervenants.

Structural insight into cap-snatching and RNA synthesis by influenza polymerase.

Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Identification of Dynamic Modes in an Intrinsically Disordered Protein Using Temperature-Dependent NMR Relaxation.

The dynamic modes and time scales sampled by intrinsically disordered proteins (IDPs) define their function. Nuclear magnetic resonance (NMR) spin relaxation is probably the most powerful tool for investigating these motions delivering site-specific descriptions of conformational fluctuations from throughout the molecule. Despite the abundance of experimental measurement of relaxation in IDPs, the physical origin of the measured relaxation rates remains poorly understood. Here we measure an extensive range of auto- and cross-correlated spin relaxation rates at multiple magnetic field strengths on the C-terminal domain of the nucleoprotein of Sendai virus, over a large range of temperatures (268-298 K), and combine these data to describe the dynamic behavior of this archetypal IDP. An Arrhenius-type relationship is used to simultaneously analyze up to 61 relaxation rates per amino acid over the entire temperature range, allowing the measurement of local activation energies along the chain, and the assignment of physically distinct dynamic modes. Fast (τ ≤ 50 ps) components report on librational motions, a dominant mode occurs on time scales around 1 ns, apparently reporting on backbone sampling within Ramachandran substates, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. Extending the study to three protein constructs of different lengths (59, 81, and 124 amino acids) substantiates the assignment of these contributions. The analysis is shown to be remarkably robust, accurately predicting a broad range of relaxation data measured at different magnetic field strengths and temperatures. The ability to delineate intrinsic modes and time scales from NMR spin relaxation will improve our understanding of the behavior and function of IDPs, adding a new and essential dimension to the description of this biologically important and ubiquitous class of proteins.

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR.

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function.

Monomeric nucleoprotein of influenza A virus.

Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.

Atomic resolution description of the interaction between the nucleoprotein and phosphoprotein of Hendra virus.

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, N(TAIL), of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N(TAIL) domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N(TAIL) and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of N(TAIL) upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to N(TAIL) without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.

The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

Insights into the structure and dynamics of measles virus nucleocapsids by 1H-detected solid-state NMR.

(1)H-detected solid-state nuclear magnetic resonance (NMR) experiments are recorded on both intact and trypsin-cleaved sedimented measles virus (MeV) nucleocapsids under ultra-fast magic-angle spinning. High-resolution (1)H,(15)N-fingerprints allow probing the degree of molecular order and flexibility of individual capsid proteins, providing an exciting atomic-scale complement to electro microscopy (EM) studies of the same systems.

Structure of the tetramerization domain of measles virus phosphoprotein.

The atomic structure of the stable tetramerization domain of the measles virus phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai virus phosphoprotein, which has a hydrophilic interface, the measles virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.

Structure of the C-terminal domain of lettuce necrotic yellows virus phosphoprotein.

Lettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules.

Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase.

It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.

Intrinsic disorder in measles virus nucleocapsids.

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N(TAIL)) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N(TAIL) that binds P is situated 90 amino acids from the folded RNA-binding domain (N(CORE)) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N(TAIL) in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N(TAIL) is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N(CORE). We present a model in which the first 50 disordered amino acids of N(TAIL) are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N(CORE) helical turns. The model provides a structural framework for understanding the role of N(TAIL) in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza A virus.

The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.

Structure of the vesicular stomatitis virus N°-P complex.

Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N(Δ21)) and a peptide of 60 amino acids (P(60)) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N(0)). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.

Transcription and replication complex of influenza virus, a new antiviral drugs target.

Transcription and replication by influenza virus are carried out by protein-RNA complexes named RNPs. There are eight of these complexes, each containing one of the eight segments of viral RNA, multiple copies of the viral nucleoprotein and each complex carries a copy of the viral RNA-dependent RNA polymerase. The polymerase itself is a complex of three subunits: PB1, PB2 and PA. Through an effort by laboratories from all over the world, atomic structures have been determined of nucleoproteins of several viral strains and of protein domains of PA and PB2. For PB1, only the structures of the small interfaces with PA and PB2 have been determined. Even though a full understanding of the fundamental processes in the viral life cycle is still lacking, the structures have revealed how nucleoprotein can oligomerize and binds to RNA, how PB1 binds to PA and how the polymerase binds to capped cellular pre-messenger RNA (mRNA) and cleaves this RNA in order to make a capped primer for its own mRNAs (cap-snatching mechanism). The structures also stimulated structure-aided drug design efforts and first generation inhibitors against nucleoprotein oligomerization, binding of PB1 to PA and the cap-snatching activity have been published. Such inhibitors may be developed into new anti-influenza drugs.