Research Article: The Structure of a Biologically Active Influenza Virus Ribonucleoprotein Complex

Date Published: June 26, 2009

Publisher: Public Library of Science

Author(s): Rocío Coloma, José M. Valpuesta, Rocío Arranz, José L. Carrascosa, Juan Ortín, Jaime Martín-Benito, Félix A. Rey.

http://doi.org/10.1371/journal.ppat.1000491

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.

Partial Text

The influenza A viruses belong to the family Orthomyxoviridae and are genetically and antigenically heterogeneous. They are responsible for annual epidemics of respiratory disease and represent an important public-health problem [1]. All viral subtypes can be found in their natural reservoir, that comprises several wild avian aquatic and terrestrial species. From this reservoir, influenza viruses can occasionally infect mammalian species, including man, by either gene reassortment with already established mammalian viruses or by direct adaptation [2], and thus produce a pandemic. Since 1997, transmissions of avian H5N1 influenza viruses to humans have originated hundreds of highly pathogenic infections and generated fears for a new pandemic of unprecedented impact [2],[3]. The recent transmission of swine H1N1 influenza viruses to humans could represent the first time that a new pandemic can be followed on line (http://www.who.int/csr/disease/swineflu/en/index.html). The genome of the influenza A viruses comprise eight single-stranded RNA molecules of negative polarity with partially complementary ends that form a closed structure. The native ribonucleoprotein (RNP) particles are formed by the association of these single-stranded RNAs to multiple monomers of nucleoprotein (NP) and a single copy of the polymerase, a heterotrimer composed by the PB1, PB2 and PA subunits [4],[5]. Such RNPs are independent molecular machines responsible for transcription and replication of each virus gene. When analysed structurally by electron microscopy, virion RNPs appear as flexible, supercoiled structures [6],[7]. The helical organization of the RNPs is determined by the structure of the NP, as complexes of NP and unrelated RNA also adopt helical structures [8], and purified NP can form RNP-like helical particles in the absence of RNA [9]. The polymerase complex binds the vRNA promoter, that is formed by the partially complementary 5′- and 3′-terminal sequences [10]–[12], and determines the superhelical arrangement of natural virus RNPs [13]. Although the RNPs are the essential elements for virus replication and gene expression, their structural analysis has been hampered by their heterogeneity and flexibility. However, in vivo replication of recombinant model-RNPs indicated that helical-, elliptic- or circular-shaped structures could be generated with RNA templates of diminishing lengths [14]. The clone 23 model-RNP, which represents the smallest efficient replicon, was circular in shape and showed sufficient structural rigidity to be analysed by electron microscopy and image processing after negative staining [15]. Here we report the purification of recombinant clone 23 RNPs to near homogeneity and their structural analysis by cryo-electron microscopy (cryo-EM). It is important to stress that the RNPs analysed were the final products of in vivo replication, as no RNP accumulation was observed when NP or polymerase negative mutants were used for in vivo reconstitution. The final structure shows a resolution of 12 Å for the NP and 18 Å for the polymerase complex and represents the first structure of a functional influenza virus RNP and indeed of the RNP from any negative-stranded RNA virus.

In this report we have presented the three-dimensional structure of an active influenza virus RNP, as determined by cryo-EM. In fact, this represents the first structure of a biologically active RNP from any negative-stranded RNA virus. Two technical developments have allowed this breakthrough: (i) the generation of recombinant RNPs that are efficient replicons and have sufficient structural rigidity [14] and (ii) the optimisation in the purification protocols of RNPs amplified in vivo. As compared to full-length virion RNPs, the structure reported here would represent a minimal RNP in which the helical section has been deleted and only the promoter region bound to the polymerase complex and the terminal loop remains. The structure obtained for the polymerase complex present in the RNP is compatible with those reported earlier by negative-staining [16],[18] and represents the most accurate model for a complex polymerase of a negative-stranded RNA virus thus far reported. The correlation with the sites previously mapped [16] and the docking of the atomic structure of specific domains permitted the rough localisation of the polymerase subunits (Fig. 3). Unfortunately, the other polymerase domains whose atomic structure is known [22]–[24] are not large and conspicuous enough to allow unambiguous docking in the cryo-EM structure.

 

Source:

http://doi.org/10.1371/journal.ppat.1000491