Rabies Virus Phosphoprotein Exhibits Thermoresponsive Phase Separation with a Lower Critical Solution Temperature.

Rabies virus (RABV) generates membrane-less liquid organelles (Negri bodies) in the cytoplasm of its host cell, where genome transcription and replication and nucleocapsid assembly take place, but the mechanisms of their assembly and maturation remain to be explained. An essential component of the viral RNA synthesizing machine, the phosphoprotein (P), acts as a scaffold protein for the assembly of these condensates. This intrinsically disordered protein forms star-shaped dimers with N-terminal negatively charged flexible arms and C-terminal globular domains exhibiting a large dipole moment.

Mono- and Bi-specific Nanobodies Targeting the CUB Domains of PCPE-1 Reduce the Proteolytic Processing of Fibrillar Procollagens.

The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs.

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).

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 (N), and a peptide encompassing the N chaperon module of the P protein.

Borna Disease Virus 1 Phosphoprotein Forms a Tetramer and Interacts with Host Factors Involved in DNA Double-Strand Break Repair and mRNA Processing.

Determining the structural organisation of viral replication complexes and unravelling the impact of infection on cellular homeostasis represent important challenges in virology. This may prove particularly useful when confronted with viruses that pose a significant threat to human health, that appear unique within their family, or for which knowledge is scarce. Among , bornaviruses (family ) stand out due to their compact genomes and their nuclear localisation for replication.

Dynamics of the secreted frizzled related protein Sizzled and potential implications for binding to bone morphogenetic protein-1 (BMP-1).

Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl.

Structural Dynamics of the C-terminal X Domain of Nipah and Hendra Viruses Controls the Attachment to the C-terminal Tail of the Nucleocapsid Protein.

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (P) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV P adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (N), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV P were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α is detached from the other two.

Vesicular Stomatitis Virus Phosphoprotein Dimerization Domain Is Dispensable for Virus Growth.

The phosphoprotein (P) of the nonsegmented negative-sense RNA viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (P), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, P, for viral gene expression and virus growth.

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.

Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells.

The ligand activated transcription factor, aryl hydrocarbon receptor (AhR) emerged as a critical regulator of immune and metabolic processes in the gastrointestinal tract. In the gut, a main source of AhR ligands derives from commensal bacteria. However, many of the reported microbiota-derived ligands have been restricted to indolyl metabolites.

Structural analysis of the complex between influenza B nucleoprotein and human importin-α.

Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility.

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.

Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5.

The genome of influenza A virus (IAV) comprises eight RNA segments (vRNA) which are transcribed and replicated by the heterotrimeric IAV RNA-dependent RNA-polymerase (RdRp). RdRp consists of three subunits (PA, PB1 and PB2) and binds both the highly conserved 3'- and 5'-ends of the vRNA segment. The IAV RdRp is an important antiviral target, but its structural mechanism has remained largely elusive to date.

Type I procollagen C-propeptide defects: study of genotype-phenotype correlation and predictive role of crystal structure.

The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data.

Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone.

Nipah virus (NiV) is a highly pathogenic emergent paramyxovirus causing deadly encephalitis in humans. Its replication requires a constant supply of unassembled nucleoprotein (N(0)) in complex with its viral chaperone, the phosphoprotein (P). To elucidate the chaperone function of P, we reconstituted NiV the N(0)-P core complex and determined its crystal structure.

Procollagen C-proteinase enhancer grasps the stalk of the C-propeptide trimer to boost collagen precursor maturation.

Tight regulation of collagen fibril deposition in the extracellular matrix is essential for normal tissue homeostasis and repair, defects in which are associated with several degenerative or fibrotic disorders. A key regulatory step in collagen fibril assembly is the C-terminal proteolytic processing of soluble procollagen precursors. This step, carried out mainly by bone morphogenetic protein-1/tolloid-like proteinases, is itself subject to regulation by procollagen C-proteinase enhancer proteins (PCPEs) which can dramatically increase bone morphogenetic protein-1/tolloid-like proteinase activity, in a substrate-specific manner.

Structural basis of fibrillar collagen trimerization and related genetic disorders.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III.

Transcription et réplication des Mononegavirales : une machine moléculaire originale.

Viruses with a non-segmented negative-sense RNA genome, or Mononegavirales, are important pathogens for plants, animals and humans with major socio-economic and health impacts. Among them are well-known human pathogens such as measles, mumps and respiratory syncytial virus. Moreover, animal reservoirs appear much larger than previously thought, hence broadening the risk of emergence of life-threatening zoonotic viruses such as Rabies, Ebola, Marburg, Nipah or Hendra related viruses.

Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases.

Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only.

Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner.

The crystal structure of the human co-chaperone P58(IPK).

P58(IPK) is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58(IPK), expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58(IPK), and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58(IPK) to 3.

Crystal structure of the LMAN1-CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII.

LMAN1 is a glycoprotein receptor, mediating transfer from the ER to the ER-Golgi intermediate compartment. Together with the co-receptor MCFD2, it transports coagulation factors V and VIII. Mutations in LMAN1 and MCFD2 can cause combined deficiency of factors V and VIII (F5F8D).

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein.

In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices.

Structure of human glycolate oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole.

Glycolate oxidase, a peroxisomal flavoenzyme, generates glyoxylate at the expense of oxygen. When the normal metabolism of glyoxylate is impaired by the mutations that are responsible for the genetic diseases hyperoxaluria types 1 and 2, glyoxylate yields oxalate, which forms insoluble calcium deposits, particularly in the kidneys. Glycolate oxidase could thus be an interesting therapeutic target.