The molecular basis of host range restriction of avian influenza virus polymerases in mammalian hosts

Influenza A virus adaptation to humans is a rare but recurrent event that can result in a pandemic. The influenza polymerase, composed of the viral proteins PB1, PB2 and PA, is responsible for transcription and replication of the viral genome. Typical avian-origin influenza polymerases are restricted in human cells and polymerase subunit PB2 is particularly involved in this restriction. Residue 627 of PB2 is a glutamic acid in almost all avian influenza viruses, and mutation to a lysine is a potent enhancer of avian polymerase function in human cells. However, the underlying molecular mechanism and the cellular factors modulating avian-origin influenza replication in mammals are not known. Using a cell based replication assay in heterokaryons formed between avian and human cells, we concluded that the restriction is not due to a dominant human inhibitory factor. We also showed that supply of avian factors to human cells stimulated the activity of an avian-origin influenza polymerase and a functional screen was set up to attempt to isolate such chicken co-factor(s). We hypothesised that PB2 E627K mutation enhances the viral polymerase activity by optimizing an interaction with a human co-factor. A biochemical approach was used to try to identify this factor. Pigs are thought to be more susceptible to avian influenza than other mammalian species and are a supposed “mixing vessel” for influenza A viruses. To compare the replicative capacity of different influenza polymerases in pig, human and avian cells, an influenza polymerase assay in pig cells was set up. This assay was also used to study the impact in pig cells of known PB2 mammalian adaptive mutations. No obvious difference in the capacity of pig and human cells to support influenza polymerase activity was found, questioning the suggested susceptibility of pigs to influenza. Viruses from the avian H9N2 G1 lineage have been responsible for some human infections. Despite having none of the known mammalian signatures, we found the H9N2 G1 polymerase was active in human and pig cells. This study identified H9N2 G1 PA protein as being particularly mammalian adapted, highlighting the role of PA in influenza mammalian adaptation

Species difference in ANP32A underlies influenza A virus polymerase host restriction.

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals

A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates

Evidence for Avian and Human Host Cell Factors That Affect the Activity of Influenza Virus Polymerase▿ †

Typical avian influenza A viruses do not replicate efficiently in humans. The molecular basis of host range restriction and adaptation of avian influenza A viruses to a new host species is still not completely understood. Genetic determinants of host range adaptation have been found on the polymerase complex (PB1, PB2, and PA) as well as on the nucleoprotein (NP). These four viral proteins constitute the minimal set for transcription and replication of influenza viral RNA. It is widely documented that in human cells, avian-derived influenza A viral polymerase is poorly active, but despite extensive study, the reason for this blockade is not known. We monitored the activity of influenza A viral polymerases in heterokaryons formed between avian (DF1) and human (293T) cells. We have discovered that a positive factor present in avian cells enhances the activity of the avian influenza virus polymerase. We found no evidence for the existence of an inhibitory factor for avian virus polymerase in human cells, and we suggest, instead, that the restriction of avian influenza virus polymerases in human cells is the consequence of the absence or the low expression of a compatible positive cofactor. Finally, our results strongly suggest that the well-known adaptative mutation E627K on viral protein PB2 facilitates the ability of a human positive factor to enhance replication of influenza virus in human cells

Segregation and twinning in the rare-earth doped KPb₂Cl ₅ laser crystals

International audienceWe present the Er3+:KPb2Cl5 lattice parameters thermal expansion and twinning characterization, pure KPb 2Cl5 crystals room temperature static dielectric constant measurements, and equilibrium segregation coefficients determination of Er 3+,Ho3+, and Pr3+ defects in KPb 2Cl5. The two main current limitations which still hinder the development of this crystal as a laser material, namely, the Er3+ cations segregation and low solubility limit as well as the crystal twinning, are discussed

The interferon inducible isoform of NCOA7 inhibits endosome-mediated viral entry

International audienceInterferons (IFNs) mediate cellular defence against viral pathogens by upregulation of IFN-stimulated genes whose products interact with viral components or alter cellular physiology to suppress viral replication1-3. Among the IFN-stimulated genes that can inhibit influenza A virus (IAV)4 are the myxovirus resistance 1 GTPase5 and IFN-induced transmembrane protein 3 (refs 6,7). Here, we use ectopic expression and gene knockout to demonstrate that the IFN-inducible 219-amino acid short isoform of human nuclear receptor coactivator 7 (NCOA7) is an inhibitor of IAV as well as other viruses that enter the cell by endocytosis, including hepatitis C virus. NCOA7 interacts with the vacuolar H+-ATPase (V-ATPase) and its expression promotes cytoplasmic vesicle acidification, lysosomal protease activity and the degradation of endocytosed antigen. Step-wise dissection of the IAV entry pathway demonstrates that NCOA7 inhibits fusion of the viral and endosomal membranes and subsequent nuclear translocation of viral ribonucleoproteins. Therefore, NCOA7 provides a mechanism for immune regulation of endolysosomal physiology that not only suppresses viral entry into the cytosol from this compartment but may also regulate other V-ATPase-associated cellular processes, such as physiological adjustments to nutritional status, or the maturation and function of antigen-presenting cells

Segregation and twinning in the rare-earth doped KPb₂Cl ₅ laser crystals

We present the Er3+:KPb2Cl5 lattice parameters thermal expansion and twinning characterization, pure KPb 2Cl5 crystals room temperature static dielectric constant measurements, and equilibrium segregation coefficients determination of Er 3+,Ho3+, and Pr3+ defects in KPb 2Cl5. The two main current limitations which still hinder the development of this crystal as a laser material, namely, the Er3+ cations segregation and low solubility limit as well as the crystal twinning, are discussed

A cationic order-disorder phase transition in KPb2Cl5

The phase transition occurring at Tt = 528 K in the natural mineral and synthetic laser host compound KPb2Cl5 has been characterized by means of both single-crystal X-ray diffraction (XRD) as a function of temperature and differential scanning calorimetry (DSC). The K+ and Pb2+(2) cations order on passing from the high-temperature orthorhombic phase (space group Pmcn; a = 8.951, b = 8.015, c = 12.683 Å, Z = 4 at 623 K) to the low-temperature monoclinic one (space group P21/c; a = 8.849, b = 7.918, c = 12.472 Å, = 90.11°, Z = 4, at room temperature), leading to a group-subgroup first-order phase transition displaying moderately fast kinetics and low entropy production upon thermal cycling around Tt. The positional entropy calculated from our crystal structure models corresponds to the transition entropy measured by DSC within the experimental error, and amounts to about 0.34 R. The driving force for the phase transition, A 22 J mol-1, remains negligible with respect to both the thermal energy at Tt (RTt 4.4 kJ mol-1) and the transition latent heat (H 790 J mol-1), thereby suggesting that this phase transition is a close-to-equilibrium process

Yb:CaF2 grown by liquid phase epitaxy

International audienceYtterbium doped CaF2 crystalline layers have been grown for the first time from high temperature solutions at controlled atmosphere by using the liquid phase epitaxy technique. Doped layers having thicknesses between a few microns to a hundred of microns have been grown onto non-oriented and (1 1 1) oriented CaF2 substrates. The Yb3+:CaF2 layers show structural properties very close to the undoped substrate without any further thermal treatment. Registration of room temperature emission spectra and fluorescence lifetime measurements performed with epitaxial layers corresponding to different ytterbium concentrations show similar spectroscopic properties as in the bulk crystals