Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

Abstract

Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.

Designed Nanoparticles Elicit Cross-Reactive Antibody Responses To Conserved Influenza Virus Hemagglutinin Stem Epitopes

“…Electron microscopy. Negative-staining electron microscopy of nanoparticles was similar to that reported previously for other samples (78), with the exception that the samples were stained with 1.5 % phosphotungstic acid. Images were collected on a Tecnai-12 electron microscope with LaB6 filament operating at 100 kV (FEI, Hillsboro, OR) at a nominal magnification of 52,000x.…”

Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication

“…About half of the low-NP regions are predicted to form secondary and tertiary RNA structures, based on computational analysis, and mutations designed to alter the stability of these predicted RNA structures resulted in attenuated virus infection (summarized in Table 1 ). The presence of RNA structures in RNP complexes may explain the pleomorphic nature of RNPs 28 . Several, mostly shorter low-NP-binding regions were not predicted to form stable structures.…”

Structure and Function of the Influenza Virus Transcription and Replication Machinery

“…Studies aimed at obtaining highresolution vRNP structures are hampered by the heterogeneity in vRNA length (890-2341 nucleotides for influenza A virus; Fig. 1A) and high-plasticity of the NP-NP interactions, which leads to the formation of curves and kinks in vRNPs, and complicates computational processing of cryogenic electron microscopy (cryo-EM) data (Gallagher et al 2017). NP is a crescent-shaped molecule that is comprised of a head and body domain and a 28 amino acid-long tail loop.…”