Tesis doctoral de Patricia Resa Infante
Replication and transcription of influenza a virus is carried out by ribonucleoprotein complexes (rnps) in the nucleus of infected cells. These rnps are composed of a genomic rna segment associated to the nucleoprotein and the polymerase complex (formed by the pb1, pb2 and pa subunits). To start the rna transcription, the pb2 subunit in the polymerase binds the 5¿ cap of host pre-mrnas, which are then cleaved 10¿13 nucleotides downstream by the pa subunit. in this doctoral thesis, we have expressed, purified and analysed structurally and functionally for the first time, polymerase-rna template complexes obtained after in vivo replication. These complexes were generated by co-transfection of plasmids expressing the three-polymerase subunits and a genomic plasmid expressing a minimal template of either positive- or negative-polarity. Their generation in vivo was strictly dependent on the polymerase activity, they contained mainly negative-polarity viral rna and were able to transcribe and replicate in vitro. In vivo replication intermediates appeared as either monomers or dimers of the polymerase, most of the latter being sensitive to rnase treatment. The 3d structure of the monomeric polymerase-vrna complexes was similar to the rnp-associated polymerase. Structural analyses of the dimeric complexes indicated the presence of tight dimers compatible with a defined interaction between two monomers. These results provide structural support for the trans/cis model for influenza rna replication and transcription. on the other hand, we have identified an independently folded domain of pb2 that possesses specific cap binding activity. The x-ray structure of the domain with bound cap analog reveals a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirmed that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. finally, we have studied the relevance of the interaction between pb2 and ¿-importins for virus replication. The pb2 nls was mutated and the phenotype of mutant subunits, polymerase complexes and rnps was analysed. While mutant pb2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally. However, mutant rnps generated with a viral cat replicon showed up to hundred-fold reduced cat accumulation. Rescue of nuclear localisation of mutant pb2 by insertion of an additional sv40 tag-derived nls did not revert the mutant phenotype of rnps. Furthermore, determination of recombinant rnp accumulation in vivo indicated that pb2 nls mutations drastically reduced virus rna replication. These results indicate that, above and beyond its role in nuclear accumulation, pb2 interaction with ¿-importins is required for virus rna replication.
Datos académicos de la tesis doctoral «Analisis estructural y funcional de la polimerasa del virus de la gripe«
- Título de la tesis: Analisis estructural y funcional de la polimerasa del virus de la gripe
- Autor: Patricia Resa Infante
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 26/05/2010
Dirección y tribunal
- Director de la tesis
- Juan Ortin Monton
- Tribunal
- Presidente del tribunal: Juan Antonio Garcia alvarez
- Antonio Mas lópez (vocal)
- José Alcami pertejo (vocal)
- gustavo Del real sodevilla (vocal)