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The exact role of the influenza polymerase PA subunit remains unclear, although it has been implicated in cap-binding, endonuclease activity, viral RNA binding and replication (9,13-15). PA is separable by [...], 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 (1). The PB2 subunit binds the 5' cap of host pre-mRNAs (2-4), which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PBI (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 (6,7). Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease (8). 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 (9). These results will facilitate the optimisation of endonuclease inhibitors (10-12) as potential new anti-influenza drugs.

Natively unfolded proteins play key roles in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. We present a description of the intrinsic conformational sampling of unfolded proteins based on residue-specific [phi]/[psi] propensities from loop regions of a folded protein database and simple volume exclusion. This approach is used to propose a structural model of the 57-aa, natively disordered region of the nucleocapsid-binding domain of Sendal virus phosphoprotein. Structural ensembles obeying these simple rules of conformational sampling are used to simulate averaged residual dipolar couplings (RDCs) and small-angle x-ray scattering data. This protein is particularly informative because RDC data from the equally sized folded and unfolded domains both report on the unstructured region, allowing a quantitative analysis of the degree of order present in this part of the protein. Close agreement between experimental and simulated RDC and small-angle x-ray scattering data validates this simple model of conformational sampling, providing a precise description of local structure and dynamics and average dimensions of the ensemble of sampled structures. RDC data from two urea-unfolded systems are also closely reproduced. The demonstration that conformational behavior of unfolded proteins can be accurately predicted from the primary sequence by using a simple set of rules has important consequences for our understanding of the structure and dynamics of the unstructured state. NMR | randomcoil | protein folding | protein dynamics conformational sampling

Résumé: Depuis trois ans, une expérience pédagogique est menée à l'université Grenoble-Alpes pour enseigner la nature et l'évolution des virus humains, sans se baser sur la liste systématique de toutes les familles de virus. Le choix a été fait d'une approche « historique » des virus chez l'homme, permettant de définir trois grandes catégories de virus selon qu'ils aient co-évolué avec l'homme pendant très longtemps (virus humains anciens), ou qu'ils l'aient infecté plus récemment au Néolithique ou plus tard (virus humains récents) ou enfin qu'ils évoluent à partir de virus animaux transmis à l'homme de manière sporadique (virus zoonotiques). Nous exposons ci-dessous les principes et quelques exemples de cette distinction pédagogique alternative qui n'a pas la prétention de remplacer les classifications taxonomiques classiques basées sur les similarités morphologiques et de séquences (classification ICTV) ou sur la forme et le mode de réplication du génome viral (classification de Baltimore). Elle permet de faciliter le regroupement de virus ayant des effets similaires même si leur divergence évolutive est importante. Nous montrons ainsi l'origine des virus humains et comment ils peuvent entraîner des maladies humaines. Cette approche a été expérimentée avec les étudiants de biologie, puis étendue aux étudiants de médecine et de pharmacie, pour que l'enseignement soit basé sur les mêmes concepts dans les trois UFR. Au final, tous les étudiants ont été très réceptifs et intéressés par cette approche. Bien sûr, différentes méthodes d'enseignement peuvent fonctionner, mais cette façon de présenter les choses est également plus ludique pour les enseignants et favorise la coopération entre les intervenants. For the past three years, the nature and evolution of human viruses have been taught in University Grenoble-Alpes without relying on the systematic list of all virus families. A «historical» approach allows to define three main categories of viruses following if they have co-evolved with humans for a very long time (ancient human viruses), if they began to infect humans in the Neolithic or later (recent human viruses) or if they are still animal viruses that are transmitted to humans sporadically (zoonotic viruses). We present below the principles and some examples of this pedagogic separation which has not the pretention to replace the classical taxonomic classification based on morphological and sequence similarity (ICTV classification) or on the form and replication mode of the viral genome (Baltimore classification). It helps grouping of viruses with similar effects even if their evolution is different. We show where human viruses come from and how they can cause human diseases. This approach was tested with Biology students, and then extended to Medicine and Pharmacy students to ensure that teaching was based on the same concepts in the three Faculties. In the end, all the students were very receptive and interested in this approach. Of course, different teaching methods can work, but this way of presenting things is also more fun for teachers and promotes cooperation between speakers. [ABSTRACT FROM AUTHOR]