Modelos tridimensionales de la ribonucleoproteína y la polimerasa recombinantes del virus de la gripe

Biblos-e Archivo/Manakin Repository

Show simple item record

dc.contributor.advisor Ortín Montón, Juan (dir.) es
dc.contributor.author Area Gómez, Estela es
dc.contributor.other UAM. Departamento de Biología Molecular es_ES
dc.date.accessioned 2015-01-26T13:10:31Z
dc.date.available 2015-01-26T13:10:31Z
dc.date.issued 2004-02-16
dc.identifier.uri http://hdl.handle.net/10486/663358 en
dc.description Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 16-02-2004 es_ES
dc.description.abstract Within Negative Strand RNA Viruses, Influenza A are distinct in having a segmented genome and replicating in the cell nucleus. Thus, their genome consists of a set of 8 ribonucleoproteins (RNPs) of various lengths that are transported to the nucleus inmediately &er virus cell entry. Independent transcription and replication of each RNP takes place in this cellular compartment by action of the vira1 polymerase, that is a heterotrimer compcsed by the PBI, PB2 and PA subunits. Mutational studies showed that PB1 subunit harbours the polymerase activity, as it contains specific sequence domains whose mutation abolish activity. PB2 subunit is a capbinding protein involved in the iniciation of transcription, but the endonuclease activity was located in PB1 protein. PA subunit is an acidic protein wich has protease activity located in its amino terminal region. Our knowledge about the structure of the Influenza virus RNP and its polymerase is very limited. Classical electron microscopy studies identified the RNPs as supercoiied ribbon structures with the polymerase localted at one end of the supercoil. We have carried out RNP reconstitution in vivo by transfection of recombinant proteins and a short model vRNA. This technique has allow us to obtain biologically active mini-RNPs similar to natural ones but more rigid and homogeneus, and thereby more accessible to the image proccessing tools. By electron microscopy of negative-stained RNPs, we selected individual images from which a three dimensional stmctural model was obtained. This structure has a circular s h w with a rine of 9 NP monomers and the polymerase complex connected to two adjacent NP mole~ules. The NP ring shows a clear vorticity that may be at the basis of the superhelicity of the full size RNPs. The RNP purification method was improved by introducing tags for affinity chromatography,with those a three dimensional volume of the polymerase at a resoiution of 23 A. was obtained. The reconstruction reflects that influenza wly- merase is a very compact structure that does not represent the subunit-subunit contacts seen by mutational studies. This new improved volume has two areas of contact with the NP ring by two adjacent NP monomen, that show different intensity and couid represent the interaction of the NP ring with PB 1 and PB2 subunits. The localization of the polymerase subunits was attempted by generation of RNPslabeled with monoclonal antibodies specific for PB2 and PA subunit. The three dimensional reconstruction of these labeled particles showed the positions of domains of each subunits in the heterotrimer of the polymerase. Since we had no available monoclonal antibody specific for PBl, we detennined its localization by three dimensional reconstruction of RNPs tagged with TAP in the C-terminus of PB1. This TAP tag has a molecuiar weight of 20 kD and it was visible protruding out of the polymerase complex in this three dimensional reconstruction. en
dc.format.extent 132 pag. es_ES
dc.format.mimetype application/pdf en
dc.language.iso spa en
dc.subject.other Virus de la gripe-Tesis doctorales es_ES
dc.title Modelos tridimensionales de la ribonucleoproteína y la polimerasa recombinantes del virus de la gripe es_ES
dc.type doctoralThesis en
dc.subject.eciencia Biología y Biomedicina / Biología es_ES
dc.rights.accessRights closedAccess en


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record