The Structure of a Biologically Active Influenza Virus Ribonucleoprotein Complex

Abstract

The influenza viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication. Despite their importance, information about the structure of these RNPs is scarce. We have determined the three-dimensional structure of a biologically active recombinant RNP by cryo-electron microscopy. The structure shows a nonameric nucleoprotein ring (at 12 Å resolution) with two monomers connected to the polymerase complex (at 18 Å resolution). Docking the atomic structures of the nucleoprotein and polymerase domains, as well as mutational analyses, has allowed us to define the interactions between the functional elements of the RNP and to propose the location of the viral RNA. Our results provide the first model for a functional negative-stranded RNA virus ribonucleoprotein complex. The structure reported here will serve as a framework to generate a quasi-atomic model of the molecular machine responsible for viral RNA synthesis and to test new models for virus RNA replication and transcription.

Author Summary

The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics that constitute a major public-health issue. The recent spillover of avian H5N1 and H1N1 swine influenza viruses to humans poses a serious threat of a new pandemic. These viruses contain a segmented RNA genome, which forms independent ribonucleoprotein particles including the polymerase complex and multiple copies of the nucleoprotein. Each of these ribonucleoprotein particles are replicated and express the encoding virus genes independently in the virus-infected cells. To better understand how these processes take place we have determined the three-dimensional structure of a model ribonucleoprotein particle that only contains 248 nucleotides of virus RNA but is biologically active in vitro and in vivo. The structure shows a circular appearance and includes 9 nucleoprotein monomers, two of which are associated to the polymerase complex. Docking of the available atomic structures of the nucleoprotein and domains of the polymerase complex has permitted us to propose a quasi-atomic model for this ribonucleoprotein particle and some of the predictions of the model have been confirmed experimentally by site-directed mutagenesis and phenotype analysis in vitro and in vivo.