MEMBRANE REMODELING AND VIRAL REPLICATION
Viruses can't replicate on their own, and once they've entered the host cell, each type of virus has to develop its own replication strategy to survive and spread. Membrane remodeling is an essential aspect of the propagation strategy of many human pathogenic viruses. Some RNA viruses are known to use this process, mobilizing and modifying host cell membranes to build specialized intracellular structures that serve as replication factories for the viral genome. This is the case for coronaviruses in general, and SARS-CoV-2 in particular, for which the process of membrane remodeling leads to the formation of compartments called double-membrane vesicles (DMVs). The viral RNA synthesized within these membrane compartments must then be exported to the host cell cytoplasm for translation into viral proteins or incorporation into new viral particles. This export takes place through crown-shaped molecular pores recently discovered in DMV membranes.
NSP3, A VIRAL PROTEIN INVOLVED IN MEMBRANE REMODELING
Among the SARS-CoV-2 proteins involved in DMV biogenesis, the non-structural protein 3 (nsp3) plays a predominant role, and is thought to be the major component of these pores, with 6 molecules present per pore. The N-terminal ends of nsp3 are oriented towards the cytoplasm (crown tips), while its C-terminal parts point towards the interior of DMVs (see figure). Nsp3 thus appears to be a therapeutic target of choice, but the size and complexity of this long protein of 1945 amino acids make it difficult to study its structure.
The aim of the present work was to consider the C-terminal region of nsp3, which contains the transmembrane segments, in order to understand how it is organized and how it contributes to the arrangement of molecular pores. In order to obtain sufficient quantities of material, this part of the protein (noted nsp3C) was expressed in a cell-free system before being reconstituted in lipid nano-disks for observation by electron microscopy. The images revealed objects with an oligomeric ring-like organization with a central pore. By combining these experimental data with structural predictions obtained using AlphaFold AI software, the authors highlighted the protein's self-association capabilities and were able to position it in a model of molecular pores (figure).
Diagram illustrating the presence of double-membrane vesicles (violet) associated with endoplasmic reticulum membranes (blue). These vesicles have crown-shaped molecular pores on their surface (gray). Two perspectives of a 3D reconstruction of the nsp3C envelope reconstituted in lipid nanodiscs (yellow) are shown: a side view and a front view. Top left: positioning of the envelope on the molecular pore. Bottom right: nsp3C model obtained with AlphaFold.
Babot et al, Med Sci (Paris), 2024
The experimental and biocomputational approach used in this work provides unprecedented clues to the structural organization of nsp3, main component of the molecular pores present on the surface of double-membrane vesicles and necessary for the export of newly synthesized viral RNA into the cytoplasm of host cells. By revealing the oligomeric organization of a key player in the biogenesis of SARS-CoV-2 DMVs, these results provide a solid basis for the design of future antiviral strategies.
Contact : virginie.gervais@i2bc.paris-saclay.fr or marion.babot@universite-paris-saclay.fr