The structure of the influenza A virus genome

Abstract

Influenza A viruses (IAVs) constitute a major threat to human health. The IAV genome consists of eight single-stranded viral RNA segments contained in separate viral ribonucleoprotein (vRNP) complexes that are packaged together into a single virus particle. The structure of viral RNA is believed to play a role in assembling the different vRNPs into budding virions^1-8 and in directing reassortment between IAVs⁹. Reassortment between established human IAVs and IAVs harboured in the animal reservoir can lead to the emergence of pandemic influenza strains to which there is little pre-existing immunity in the human population^10,11. While previous studies have revealed the overall organization of the proteins within vRNPs, characterization of viral RNA structure using conventional structural methods is hampered by limited resolution and an inability to resolve dynamic components^12,13. Here, we employ multiple high-throughput sequencing approaches to generate a global high-resolution structure of the IAV genome. We show that different IAV genome segments acquire distinct RNA conformations and form both intra- and intersegment RNA interactions inside influenza virions. We use our detailed map of IAV genome structure to provide direct evidence for how intersegment RNA interactions drive vRNP cosegregation during reassortment between different IAV strains. The work presented here is a roadmap both for the development of improved vaccine strains and for the creation of a framework to 'risk assess' reassortment potential to better predict the emergence of new pandemic influenza strains.

Figure 1 |. Analysis of the influenza A virus (IAV) genome structure using SHAPE-MaP.

a, Schematic showing different samples used for SHAPE-MaP analysis. b, Median SHAPE reactivities of different WSN vRNA segments in virio. Medians were calculated over 50 nucleotide windows and plotted relative to the global median of a given segment. c, SHAPE reactivity distributions in different samples; **** P ≤ 2.2×10⁻¹⁶, two-sided Wilcox Rank-Sum Test, n = 13581 nucleotides per sample, in virio data is an average of 3 biologically independent samples, ivtRNA data is an average of 2 biologically independent samples, and nkvRNA data is from a single biological sample. d, Base-pairing probability distributions in different vRNA samples, calculated using SHAPE-informed partition function. e, Secondary RNA structure of the NS segment. Upper black arcs indicate the maximum expected accuracy RNA structure; only interactions associated with greater than 80% base pairing probabilities are shown. Lower coloured arcs indicate base-pairing probabilities. Dashed rectangle highlights the position of the hairpin shown in f. All sequence positions are annotated as 5′- 3′ in vRNA sense. nkvRNA, naked viral RNA; ivtRNA, in vitro transcribed RNA; MEA, maximum expected accuracy.

Figure 2 |. Inter-segment RNA interactions in the IAV genome.

a, Schematic summarizing the SPLASH method. b, The most prevalent inter-segment RNA interactions identified using SPLASH for the WSN (H1N1) IAV strain. The eight vRNA segments are shown on the perimeter of the circle and the links indicate the regions involved in inter-segment base-pairing. The links are shaded by interaction frequencies based on the chimeric read contact matrix (see Methods). Dotted arrows point to SHAPE-guided RNA structure predictions of three representative interactions. c, Distribution of the ΔG energies associated with the interactions identified by SPLASH versus a permutated interaction dataset; **** P ≤ 1×10⁻¹⁶, two-sided Wilcox Rank-Sum Test, n = 611 interactions in common between two biologically independent experiments.

Figure 3 |. RNA interactions form a redundant, plastic network to accommodate variation and reassortment.

a-b, Inter-segment RNA interaction maps for two parent strains, PR8 (H1N1) and Udorn (H3N2), and c, a reassortant of PR8 that bears the Udorn PB1 and NA gene segments, PR8::Udorn(PB1+NA) (H1N2). Interactions are coloured according to the parent strain that donated the interaction and shaded according to their interaction frequency, as indicated above (see Figure 2). PR8, A/Puerto Rico/8/34; Udorn, A/Udorn/307/72.

Figure 4 |. Inter-segment RNA interactions drive IAV segment co-segregation during reassortment.

a, Inter-segment RNA interaction map for the Udorn virus, with the interaction between PB1 and NA segments highlighted in dark blue. Structure prediction for the highlighted PB1-NA interaction is shown on the right. Circled nucleotides highlight the bases that differ between the Udorn and Wyo03 strains. b, Competitive reverse-engineering of influenza viruses. Six plasmids encoding H1N1 background segments are transfected together with an H3N2 NA segment-encoding plasmid and PB1 segment-encoding plasmids from both H1N1 and H3N2 strains. The origin of the PB1 segment in the progeny viruses is determined using RT-qPCR. c, Co-segregation between H1N1 or H3N2 PB1 segments and H3N2 NA segments; P values as indicated, ANOVA with Sidak correction for multiple testing, n = 5 (Ud-NA), n = 3 (Mem71-NA and PC73-NA), and n = 8 (Wyo03-NA) biologically independent experiments; bar plot centre represents the mean, error bars indicate SEM. d, Preferential Wyo03 PB1 and NA segment co-segregation is recovered by substituting the 4 nucleotides that differ in Wyo03 NA from those in the PB1-interacting region of Udorn NA (highlighted in a) for a Udorn-like sequence; P values as indicated, ANOVA with Sidak correction for multiple testing, n = 7 (PR8 PB1 vs. Wyo03 PB1 competitions) and n = 8 (PR8 PB1 vs. Udorn PB1 competitions) biologically independent experiments; bar plot centre represents the mean, error bars indicate SEM. e, Substitution of the 4 Udorn-like nucleotides regenerates a strong inter-segment RNA interaction between the H3N2-origin PB1 and NA segments in reassortant viruses. Mem71, A/Memphis/1/71; Wyo03, A/Wyoming/3/03; PC73, A/Port Chalmers/73; Udorn, A/Udorn/307/72; PR8, A/Puerto Rico/8/34.