A model for the genome organisation of the virus responsible for influenza is proposed on the basis of a tool developed from recombinant viral nucleoprotein and synthetic RNA. In an article published in the journal Science Advances, scientists used cryo-electron microscopy to study the assembly of the influenza nucleoprotein into a helix, thereby providing details of the protein-protein and protein-RNA interactions within the nucleocapsid.
Since October 2021, Europe is suffering the most devastating epizootic of highly pathogenic avian influenza virus (HPAI) it has ever known. A highly contagious viral disease affecting both domestic and wild birds, the spread of the virus appears now to be running out of steam (the French Ministry of Agriculture recorded a total of 401 confirmed outbreaks on farms between August 2022 and July 2023, more than a third fewer than in the previous year). In accordance with the provisions of the World Organisation for Animal Health (formerly known as OIE), France regained its HPAI-free status on 14 August 2023. Nevertheless, the virus remains under very close surveillance because of its classification by the WHO as a pathogenic agent with high pandemic potential.
The influenza A virus genome is made up of eight single-stranded RNA molecules of negative polarity (A). Each RNA molecule is coated with multiple copies of viral nucleoproteins (NP) and their 3’ and 5’ ends interact with an RNA polymerase to form the ribonucleoprotein complex (RNP), a functional entity for viral proliferation. Extracted directly from the virus and observed by electron microscopy, RNPs appear to be complex, extremely flexible and highly dynamic architectures (B). Until now, studies of RNPs using cryo-electron microscopy (cryo-EM) had only provided a molecular envelope in which the NP molecules had been positioned imprecisely, and without providing any details of their interaction with the viral RNA.
In this article published in the journal Science Advances, IBS scientists have decided not to use RNPs purified from viruses. Instead, based on their expertise acquired over the last ten years in the expression and purification of recombinant NP protein, they have developed a protocol to self-assemble RNP-like particles in vitro from recombinant NP proteins and small RNA probes (C). By developing this approach, they were able to produce significantly more biological material than by starting with viruses, an essential condition for conducting a high-resolution cryo-ME study. The optimum experimental conditions for cryo-EM were developed on the ISBG’s electron microscopy platform (UMS3518) before obtaining a dataset of 27,000 films using the Titan Krios electron microscope on ESRF’s CM01 line.
During data processing, the straightest filaments (RNPs) were selected and extracted in silico into overlapping segments. Once sorted, the 227,000 highest quality segments were classified and averaged to generate an initial three-dimensional reconstruction at nanometric resolution. As this model is affected by the inherent flexibility of the RNP particles produced, a focused refinement was carried out to generate a final model at 5 Å resolution (D).
Scientists now have a high-resolution 3D reconstruction of these particles, which are highly similar to influenza RNPs. This enables us to understand how the nucleoprotein molecules interact with each other within this complex and flexible architecture. This mode of interaction is very similar to that observed in the three-dimensional structures obtained by X-ray diffraction from nucleoproteins alone. However, this cryo-EM 3D reconstruction at sub-nanometric resolution enables for the first time the visualisation of the RNA placement within RNPs. Although RNA appears to be involved in structuring this complex architecture, it can slide freely on the surface of proteins, most likely to promote RNA accessibility to RNA polymerase during the viral cycle of the influenza virus (replication and translation of genetic information at the RNP level).
Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation. Florian Chenavier, Leandro F. Estrozi, Jean-Marie Teulon, Eleftherios Zarkadas, Lily-Lorette Freslon, Jean-Luc Pellequer, Rob W.H. Ruigrok, Guy Schoehn, Allison Ballandras-Colas, Thibaut Crépin. Science Advances 2023 ; 9(50):eadj9974.
doi : 10.1126/sciadv.adj9974
Contact : Thibaut Crepin (IBS/Viral Replication Machines Group)