Caracterización estructural y funcional de la ARN polimerasa dependiente de ARN del virus de Thosea asigna

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dc.contributor.advisor Rodríguez Aguirre, José Francisco (dir.)
dc.contributor.advisor Verdaguer Massana, Nuria (dir.)
dc.contributor.author Ferrero, Diego Sebastián
dc.contributor.other UAM. Departamento de Biología Molecular es_ES
dc.date.accessioned 2014-06-03T14:37:43Z
dc.date.available 2014-06-03T14:37:43Z
dc.date.issued 2013-07-22
dc.identifier.uri http://hdl.handle.net/10486/660511
dc.description Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-07-2013 es_ES
dc.description.abstract Thosea asigna virus (TaV) is an insect-restricted virus with a monosegmented positivesense, single stranded RNA (ssRNA) genome enclosed within an icosahedral T = 4 capsid. TaV belongs to the recently created Permutotetraviridae family, which includes non-enveloped viruses with a particular organization of sequence motifs within their RNA dependent RNA polymerases (RdRp). All polymerases are broadly conserved in viruses following a right hand architecture with fingers, palm and thumb subdomains, and also conserved six ordered sequence motifs (A-B-C-D-E-F), four of them located into the palm subdomain (A to D) and two (E-F) that are only present in RdRps. Previous sequence analyses showed that, similarly to what had been found for members of the Birnaviridae family, the TaV RpRd does not follow the canonical organization (C-A-B-D) of the palm subdomain. Given the rarity of these atypical polymerases, the structural and biochemical information about them is rather scarce. For this reason, we performed a comprehensive study of the RdRp of TaV. We have solved the structure of a recombinant version of the TaV RdRp domain purified from insect cells by X-ray crystallography. The exhaustive analysis of the atomic structure allowed the identification of the conservation of the overall fold of the polymerase which shows a surprisingly high degree of similarity to its Flavivirus counterpart. We have also identified several structural elements that might play a role in the regulation of the polymerase activity, i.e. the 30 N-terminal residues and the extense loop that block the active site cavity that may act as negative regulator elements. This finding suggested that these elements may undergo a structural rearrangement, thus allowing the polymerase to be active by a de novo initiation mechanism as confirmed by biochemical analyses presented here. The use of mutant versions of the TaV RdRp provided insight into general mechanisms controlling RdRp activity and about the regulatory role played by their own structural elements. In addition, electron microscopy images led to the generation of a three-dimensional reconstruction of the complete protein encoded by TaV ORF1. This provided relevant structural information concerning the C-terminal domain of the polypeptide. Results gathered by confocal microscopy analysis of a series of mutant versions of the protein expressed in insect cell indicate that the C-terminal domain of the TaV ORF protein is responsible for the formation of veshicle-like structures, and probably for its interaction with an as yet unindentified membranous compartment, within the cell cytoplasm where the viral replication machinery is located. According to the results presented here, TaV can be related to Flaviviridae family considering the structural and functional similarity en_US
dc.format.extent 115 pag. es_ES
dc.format.mimetype application/pdf es_ES
dc.language.iso spa es_ES
dc.subject.other Insectos - Parásitos - Tesis Doctorales es_ES
dc.subject.other ARN polimerasas - Tesis Doctorales es_ES
dc.title Caracterización estructural y funcional de la ARN polimerasa dependiente de ARN del virus de Thosea asigna es_ES
dc.type doctoralThesis en
dc.subject.eciencia Biología y Biomedicina / Biología es_ES
dc.rights.cc Reconocimiento – NoComercial – SinObraDerivada es_ES
dc.rights.accessRights openAccess en


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