Análisis estructural de la ribonucleoproteína nativa del virus de la gripe

Resumen

The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. These viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein (NP) and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication constituting, therefore, the most important functional complex of the virus. The complete nucleocapsid of the virus is composed of 8 RNPs and they are closely packed forming bundles. Despite their importance, information about the structure of these RNPs is scarce. In this Doctoral Thesis the three-dimensional structure of a native RNP has been determined using a combination of single molecule cryo-electron microscopy and cryo-electron tomography as well as image processing and docking assays. The native RNPs isolated from virions show a double helical conformation in which two NP strands of opposite polarity are associated each other along the helix. Both strands are connected by a short loop constituted by three NP monomers at one end of the particle and interact with the polymerase complex at the other end. The double-helical structure is defined by a minor groove between the connected NP strands and a major groove between the NP strands not physically in contact. The docking of the atomic structure of NP into the helix has allowed us to define the interactions between the functional elements of RNPs and to propose the location of the viral RNA. Additionally, our results revealed the existence of two conformations of the polymerase complex but their biological relevance is not clear at the moment. Finally, electron tomography assays have shown that the structure of the RNPs packed inside virions is the same than the isolated ones. In this Doctoral Thesis has provided the first model for a native negative-stranded RNA virus ribonucleoprotein complex. This structure will be relevant for unraveling the mechanism of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.