Unit of Virus Host Cell Interactions

About: Unit of Virus Host Cell Interactions is a facility organization based out in Grenoble, France. It is known for research contribution in the topics: RNA & Binding site. The organization has 199 authors who have published 248 publications receiving 14467 citations.

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

Abstract: 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.

Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs.

Abstract: Summary Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1–PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F2 plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with Kd values of 1.1 μm or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.

The abscisic acid receptor PYR1 in complex with abscisic acid

Abstract: The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone-receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.

The Structural Basis for CAP Binding by Influenza Virus Polymerase Subunit Pb2.

Abstract: Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.