RESEARCH SUMMARY

In the last two years, our group has continued the study of the structure and function of the influenza virus ribonucleoproteins (RNP) and the included polymerase complex. We have purified recombinant RNP as well as viral polymerase complex expressed in human cells.

The structure of these viral complexes was studied by electron microscopy of negative-stained or frozen samples and three-dimensional reconstruction, in collaboration with Jaime Martí­n-Benito, José María Valpuesta (CNB) and Óscar Llorca (CIB).

On the one hand, we established the first structure of an influenza RNP by cryo-EM at 12-18 Ã resolutions that describes the interactions among NP monomers as well as the interactions of NP with the polymerase complex. The structure of the polymerase contained in the RNP is the best information available so far on any negative-strand RNA virus polymerase, and has allowed the localisation of specific domains of the included subunits. In addition, we described the structure of the polymerase in association to the template RNA but devoid of NP. These complexes were generated by replication in vivo of a micro-RNA template and show similarities, but also differences, with the polymerase integrated in a functional RNP.

The mechanism of viral RNA replication and transcription was analysed by genetic trans-complementation tests. We used highly efficient procedures for the generation and purification of recombinant RNP, and also characterised polymerase mutants defective in either RNA replication or transcription, to determine whether the polymerase associated to the parental RNP is responsible for the synthesis of the progeny RNA and/or its encapsidation into progeny RNP.

The results are compatible with a new model for virus replication whereby a polymerase complex distinct from that present in the parental RNP carry out RNA synthesis in trans and yet another polymerase complex is in charge of encapsidating the progeny RNA. In contrast, the polymerase complex associated to the parental RNP carry out RNA transcription in cis.