Electrostatic Interactions between Hendra Virus Matrix Proteins Are Required for Efficient Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus belonging to the genus Henipavirus HeV is highly pathogenic, and it can cause severe neurological and respiratory illnesses in both humans and animals, with an extremely high mortality rate of up to 70%. Among the genes that HeV encodes, the matrix (M) protein forms an integral part of the virion structure and plays critical roles in coordinating viral assembly and budding. Nevertheless, the molecular mechanism of this process is not fully elucidated. Here, we determined the crystal structure of HeV M to 2.5-Å resolution. The dimeric structural configuration of HeV M is similar to that of Newcastle disease virus (NDV) M and is fundamental to protein stability and effective virus-like-particle (VLP) formation. Analysis of the crystal packing revealed a notable interface between the α1 and α2 helices of neighboring HeV M dimers, with key residues sharing degrees of sequence conservation among henipavirus M proteins. Structurally, a network of electrostatic interactions dominates the α1-α2 interactions, involving residues Arg57 from the α1 helix and Asp105 and Glu108 from the α2 helix. The disruption of the α1-α2 interactions using engineered charge reversal substitutions (R57E, R57D, and E108R) resulted in significant reduction or abrogation of VLP production. This phenotype was reversible with an R57E E108R mutant that was designed to partly restore salt bridge contacts. Collectively, our results define and validate previously underappreciated regions of henipavirus M proteins that are crucial for productive VLP assembly.IMPORTANCE Hendra virus is a henipavirus associated with lethal infections in humans. It is classified as a biosafety level 4 (BSL4) agent, and there are currently no preventive or therapeutic treatments available against HeV. Vital to henipavirus pathogenesis, the structural protein M has been implicated in viral assembly and budding, as well as host-virus interactions. However, there is no structural information available for henipavirus M, and the basis of M-driven viral assembly is not fully elucidated. We demonstrate the first three-dimensional structure of a henipavirus M protein. We show the dimeric organization of HeV M as a basic unit for higher-order oligomerization. Additionally, we define key regions/residues of HeV M that are required for productive virus-like-particle formation. These findings provide the first insight into the mechanism of M-driven assembly in henipavirus.

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Šresolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

Synthesis, binding and bioactivity of gamma-methylene gamma-lactam ecdysone receptor ligands: advantages of QSAR models for flexible receptors.

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or 'attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for gamma-methylene gamma-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.

Cellular munc18c levels can modulate glucose transport rate and GLUT4 translocation in 3T3L1 cells.

Munc18c has been shown to bind syntaxin 4 and to play a role in GLUT4 translocation and glucose transport, although this role is as yet poorly defined. In the present study, the effects of modulating the available level of munc18c on glucose transport and GLUT4 translocation were examined. Over-expression of munc18c in 3T3L1 adipocytes inhibited insulin-stimulated glucose transport by approximately 50%. Basal glucose transport rates were also decreased by approximately 25%. In contrast, microinjection of a munc18c polyclonal antibody stimulated GLUT4 translocation by approximately 60% over basal levels without affecting insulin-stimulated GLUT4 levels. Microinjection of a control antibody had no effect. These data are consistent with the likelihood that antibody microinjection sequesters munc18c enabling translocation/fusion of GLUT4 vesicles. Mutagenesis of a potential proline-directed kinase phosphorylation site in munc18c, T569, that in previous studies of its neuronal counterpart munc18a caused its dissociation from its complex with syntaxin 1a, had no effect on munc18c's association with syntaxin 4 or its inhibition of glucose transport, indicative that phosphorylation of this residue is not important for insulin regulation of glucose transport. The over-expression and microinjection sequestration data support an inhibitory role for munc18c on translocation/fusion of GLUT4 vesicles. They further show that altering the level of available munc18c in 3T3L1 cells can modulate glucose transport rates, indicating its potential as a target for therapeutics in diabetes.

Snares for GLUT4--mechanisms directing vesicular trafficking of GLUT4.

Fusion of GLUT4 vesicles with the plasma membrane is a key terminal step in insulin-regulated glucose transport. This fusion event is mediated by SNARE proteins, syntaxin 4, SNAP23 and VAMP2, through a process regulated by accessory proteins whose roles are still unclear. Munc18c is a key regulatory protein of syntaxin, and possibly of whole-SNARE complex cycling. Rab GTPases, through interactions with tethering molecules and SNAREs, provide targeting specificity in the GLUT4 secretory pathway. We review here insulin-mediated signaling of glucose transport, and particularly the role played by SNAREs and their accessory proteins.