Avian influenza viruses represent a recurring threat to human health. In particular highly pathogenic zoonotic avian strains, such as the currently circulating H5N1 subtype, can adapt to infect humans with high mortality, posing a catastrophic pandemic threat, in addition to global decimation of wild and domestic bird populations.
Host adaptation is necessary for efficient replication and sustained human-to-human transmission. Amongst other adaptations, replication in human cells requires mutations on the surface of the viral polymerase – the machine responsible for creating new copies of its genetic material. These mutations, located in the terminal domains of one of the subunits of the polymerase (PB2) are known to compensate differences in a host protein, ANP32A, that the virus somehow exploits in the infected cell and that is required for replication.
This enigmatic protein has a long disordered tail, that is 30% longer in birds than in humans, and highly negatively charged, comprising around 70% of acidic amino acids. The molecular origin of the associated compensatory mechanism remains unclear. A recent study from researchers at the IBS, in collaboration with the EMBL, Pasteur Institute and ENS Paris sheds new light on the question.
The Blackledge group at the IBS used high field NMR spectroscopy to demonstrate that avian ANP32A colocalizes two viral proteins, the polymerase domain PB2, and the nucleoprotein (NP). ANP32A interacts with the two proteins using two separate interaction sites on its disordered tail, an interaction that could position copies of NP in close proximity to the newly synthesized RNA as it exits the viral polymerase, allowing for rapid and efficient encapsidation of the viral genome. Such a step is thought to be essential to protect the viral RNA from the host immune system.
Camacho-Zarco and Yu et al noticed however that this mechanism would not be possible in human cells, because the disordered tail of the host protein would be too short to accommodate two separate interaction sites and thereby colocalize the NP and the polymerase.
They then demonstrated, using NMR relaxation and exchange, that the mutations present in the viral polymerase provide a new interaction mechanism that allows both viral proteins to simultaneously bind exactly the same stretch of the disordered tail of human ANP32A, forming a dynamic ternary complex [1].
The negatively charged flexible tail colocalises the nucleoprotein and the polymerase via highly dynamic and electrostatic interactions [2], rapidly, and crucially multivalently, fluctuating between positively charged surfaces on the two viral proteins. Indeed, the adaptive mutations, in particular PB2 E627K, the signature of all 20th century human flu pandemics, complete the positively charged distribution on the surface of the polymerase domain, thereby promoting the observed multivalency and providing a mechanism to overcome the collapse of the two binding sites that are present in the avian form of the protein.
On this basis, and in comparison with the recent structure of the folded domain of ANP32A in complex with a recently determined structure of a dimer of polymerases of the related influenza C, allowed the authors to speculate on the position of the disordered domain relative to the interacting PB2 domains and the colocalised NP [3].
This remarkable piece of molecular engineering on the part of influenza virus underlines the mechanistic plasticity of the infectious agent to overcome species barriers and achieve zoonosis, but also reveals completely new avenues for the development of potent inhibitory strategies against the ever-present pandemic threat of avian influenza.
A. Camacho-Zarco, L. Yu, M. Blackledge (IBS)
[1] A. R. Camacho-Zarco, L. Yu, T. Krischuns, S. Dedeoglu, et al. (2023). J. Am. Chem. Soc., 145, 20985–21001..
[2] A. R. Camacho-Zarco, S. Kalayil, D. Maurin, N. Salvi, et al. (2020). Nat. Commun., 11, 3656.
[3] L. Carrique, H. Fan, A. P. Walker, J. R. Keown, et al. (2020). Nature, 587, 638–643.
Figure: Top left – Avian ANP32A colocalizes the polymerase domains (PB2(C)) and nucleoprotein (NP) via two distinct interaction sites on the disordered tail. Bottom left – In humans the two sites have collapsed. In response the virus mutates residues on the surface of PB2, to promote multivalent, electrostatic interactions that colocalize the two proteins via the identical disordered interface. Bottom right -Model of the possible colocalization of NP and the PB2(C) domains in the context of the structure of the folded ANP32A bound to influenza C polymerase dimer [3].
