At the beginning of 2025, influenza is on the rise in all age groups, and this year’s epidemic looks set to be a major one. The disease is caused by a virus with a segmented RNA genome. The first observations of the influenza genome date back to 1969, with a first model proposed in 1972 [1]. It had been proposed that each RNA segment was covered by multiple copies of the nucleoprotein (NP) and capped by the RNA polymerase, these entities are called vRNPs and form a helical structure made of two antiparallel strands (Figure 1.A). The X-ray structure of the RNA-free NP was published in 2006 and until now, nobody had been able to observe any RNA attached to the NP nor to envisage how the protein-RNA complex could organize into an antiparallel double-stranded helix.
The Nuclear Replication Viruses team (formerly headed by Rob Ruigrok) has been studying the nucleocapsids (viral genomes encapsidated by nucleoproteins) of negative strand RNA viruses for over four decades. Regarding influenza virus, the authors have partaken with the group of Stephen Cusack (EMBL) in the epic story of deciphering the structure of the RNA polymerase. In parallel, the team set out to unlock the secret of the helical nucleocapsid. A few years ago, the authors identified conditions to reconstitute nucleocapsid-like particles starting from recombinant NP and small RNA probes [2]. These in vitro assembled particles resemble to vRNPs extracted from the virus (Figure 1). Florian Chenavier, a Gral-2 PhD student shared with the Methods and Electron Microscopy group (IBS), used this strategy to obtain a cryo-EM reconstruction (dataset collected on CM01, ESRF). This model was the first subnanometer structure of the helical NP-RNA complex [3]. However, compared to the vRNPs, this helical nucleocapsid-like structure showed a parallel double-stranded conformation. This result was nonetheless a proof of concept that a high-resolution structure of such a complex could be obtained using cryo-EM. In a first attempt, by slightly shortening the protein construct used, a dataset was collected on a Glacios microscope (IBS) and the resolution of the model was increased to 3.3 Å resolution [4]. The construct, having a greater propensity to assemble into less flexible helices, allowed more substrates of systematically increasing the length of RNA and a series of cryo-EM reconstructions to be obtained. For one, the helical structure attained a resolution of 3.0 Å, and is made up of two antiparallel strands (Figure 2). The NP-RNA complex adopts two different conformations on the opposing strands, making the RNA molecule of strand-1 less exposed to the solvent than strand-2.
This result is a major advance to understand the organization of the influenza genome. They detail for the first time the whole RNA pathway across NP as well as NP-NP interactions that drive the helical assemblies. They show that the surface of NP can harbour several conformations of the RNA and provide details to further understand the inherent flexibility of influenza vRNPs. They also represent a new alternative to design molecules targeting NP-RNA interactions and so influenza genome encapsidation.
A. Ballandras-Colas, T. Crépin and G. Schoehn (IBS)
[1] Compans RW, Content J, Duesberg PH (1972) J Virol, 10, 795-800.
[2] Labaronne A, Swale C, Monod A et al. (2016) Viruses, 8, 247.
[3] Chenavier F, Estrozi LF, Teulon J-M et al. (2023) Sci Adv, 9:eadj9974.
[4] Chenavier F, Zarkadas E, Freslon LL et al. (2025) Nucleic Acids 53: gkae1211.
Figure 1: Influenza vRNPs and the nucleocapsid-like particles. Electron microscopy observations (negative staining) of the (A) vRNPs extracted from the virus preparation or (B) nucleocapsid-like particles assembled in vitro. The scale bar represents 50 nm.
Figure 2: The antiparallel double-stranded nucleocapsid-like particle. (A) Helical symmetrized representation from the atomic model of one protomer per strand. Each protomer is shown in surface representation coloured as gradients of pink or cyan for Strand-1 and Strand-2, respectively, that follow the 5’ to 3’ direction of the RNA. The RNA is shown as a yellow ribbon. (B) and (C) RNA conformation on the two strands. A single NP protomer is shown in surface with the RNA in ribbon. The colour code is the same as (A).
