Influenza Revealed: Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng ). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631 , published online 22 November) and Arranz et al. (p. 1634 , published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription.

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. 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.

Three‐dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the α and β isoforms) using five specific CCT subunits. The CCT–tubulin interaction has a different geometry to the CCT–actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT‐bound conformation suggests a common mode of chaperonin–substrate interaction. CCT stabilizes quasi‐native structures in both proteins that are open through their domain‐connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP‐1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD‐bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three‐dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.