Background

Enteroviruses are non-enveloped RNA viruses that cause a variety of diseases, from poliomyelitis to hand, foot, and mouth disease, and meningitis. Their approximately 30-nanometer icosahedral capsids protect the positive-sense single-stranded RNA, and for infection to begin, this genome must exit the capsid and reach the cytoplasm. However, it has been unclear how far the rigid protein shell opens within actual cells and what pathway the RNA takes to escape.

Previous structural studies mainly involved inducing uncoating by applying acidic conditions or heat to purified viruses. These in vitro experiments observed that one to three pentamers, which make up the capsid of echovirus 18 (E18), detach. However, it remained unclear whether the same phenomenon occurs in infected cells. Average structure analysis using icosahedral symmetry also has the limitation of potentially missing asymmetric openings that occur at only one location on each particle.

Key Findings

The researchers observed E18-infected African green monkey kidney-derived COS-7 cells using cryo-electron tomography (cryo-ET) and single-particle cryo-electron microscopy (cryo-EM). Intracellular viral particles containing RNA were reconstructed at a resolution of 4.3 Ångströms, and intact particles, empty capsids with the genome released, and partially opened capsids were all identified.

E18 bound to the neonatal Fc receptor (FcRn) on the cell surface. At this time, a 'pocket factor' that stabilized the hydrophobic pocket inside the capsid protein VP1 partially shifted, changing the particle into a state suitable for RNA release. Subsequently, adjacent capsid pentamers 1–3 detached, creating a large opening, and the RNA exited through this gap. The fact that incomplete capsids that had lost their genomes were directly found in infected cells supports the idea that capsid opening is a physiological uncoating pathway, rather than a simple in vitro degradation.

In contrast, 'activated intermediates' in which the capsid had expanded but the RNA remained were not detected in cells. This suggests that the process of pocket factor detachment and capsid opening after FcRn binding does not last long enough to be captured by structural analysis. The researchers interpreted that the genome release of E18 proceeds very quickly, and that the actual phenomenon is better explained by the large-scale structural collapse of pentamers than by the previously proposed model in which only a small channel is formed in the intact capsid, allowing RNA to move one strand at a time.

Significance and Outlook

This study is significant in that it validates the 'pentamer detachment model' proposed in purified particles within infected cells. By connecting receptor binding, pocket factor release, capsid opening, and RNA release as a single continuous process, the structural outline of the early stages of enterovirus infection has also been clarified. In particular, the fact that genome release can be blocked by stabilizing the pocket factor binding site or the interface between pentamers broadens the range of targets for antiviral drugs.

However, the study was limited to E18 in cultured COS-7 cells. It is still unknown whether other enteroviruses create the same size of opening, or whether uncoating proceeds in the same order in human intestinal, respiratory, and nerve tissues. The fact that activated intermediates were not detected is also more of an indirect indication that they have a short lifespan than evidence that they do not exist. Time-resolved structural analysis and human-derived organoid studies are needed to determine the time window in which each step can be blocked with drugs.