Comparative analysis of human, rodent and snake deltavirus replication.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

Structure of the N-RNA/P interface indicates mode of L/P recruitment to the nucleocapsid of human metapneumovirus.

Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies.

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long ß-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

Orthoparamyxovirinae C Proteins Have a Common Origin and a Common Structural Organization.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.

Structure and Dynamics of the Unassembled Nucleoprotein of Rabies Virus in Complex with Its Phosphoprotein Chaperone Module.

As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (NNT-ARM), and a peptide encompassing the N0 chaperon module of the P protein. We showed that the chaperone module undergoes a disordered-order transition when it assembles with N0 and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N0 molecules.

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins.

Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

An automated platform for structural analysis of membrane proteins through serial crystallography.

Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Cryo-electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex.

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

Ammonium salts are a reservoir of nitrogen on a cometary nucleus and possibly on some asteroids.

The measured nitrogen-to-carbon ratio in comets is lower than for the Sun, a discrepancy which could be alleviated if there is an unknown reservoir of nitrogen in comets. The nucleus of comet 67P/Churyumov-Gerasimenko exhibits an unidentified broad spectral reflectance feature around 3.2 micrometers, which is ubiquitous across its surface. On the basis of laboratory experiments, we attribute this absorption band to ammonium salts mixed with dust on the surface. The depth of the band indicates that semivolatile ammonium salts are a substantial reservoir of nitrogen in the comet, potentially dominating over refractory organic matter and more volatile species. Similar absorption features appear in the spectra of some asteroids, implying a compositional link between asteroids, comets, and the parent interstellar cloud.

The LC8-RavP ensemble Structure Evinces A Role for LC8 in Regulating Lyssavirus Polymerase Functionality.

The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely nonlethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 colocalize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.

Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy.

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (Pcore) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of Pcore with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

Structural insights into adiponectin receptors suggest ceramidase activity.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs.

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.

Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone.

Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs.

Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein.

Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid (the viral RNA genome packaged by the nucleoprotein N) we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals.

Calcium-controlled conformational choreography in the N-terminal half of adseverin.

Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1-A3) and the Ca(2+)-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca(2+)-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1-A3 provided insights into Ca(2+)-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca(2+)-independent F-actin severing by A1-A3, albeit at a lower efficiency than observed for gelsolin domains G1-G3. Together, these data address the calcium dependency of A1-A3 activity in relation to the calcium-independent activity of G1-G3.

Large heterogeneities in comet 67P as revealed by active pits from sinkhole collapse.

Pits have been observed on many cometary nuclei mapped by spacecraft. It has been argued that cometary pits are a signature of endogenic activity, rather than impact craters such as those on planetary and asteroid surfaces. Impact experiments and models cannot reproduce the shapes of most of the observed cometary pits, and the predicted collision rates imply that few of the pits are related to impacts. Alternative mechanisms like explosive activity have been suggested, but the driving process remains unknown. Here we report that pits on comet 67P/Churyumov-Gerasimenko are active, and probably created by a sinkhole process, possibly accompanied by outbursts. We argue that after formation, pits expand slowly in diameter, owing to sublimation-driven retreat of the walls. Therefore, pits characterize how eroded the surface is: a fresh cometary surface will have a ragged structure with many pits, while an evolved surface will look smoother. The size and spatial distribution of pits imply that large heterogeneities exist in the physical, structural or compositional properties of the first few hundred metres below the current nucleus surface.