Viruses are ingenious, infectious agents, capable of replicating inside the living cells of a host organism. Enterovirus, a common viral pathogen, is responsible for a range of diseases from mild colds to severe conditions, including viral meningitis, myocarditis, and paralysis.
Enterovirus is a ‘naked’ virus that houses its RNA replication factory within a protein-shelled capsid. The RNA provides the pattern to create new viral particles.
Once in the host cell, the Enterovirus uses the viral protein, 3CD, to recognize two RNA elements—the cloverleaf RNA at the end of the nucleic acid strand and the internal cis-acting replication element. Researchers know this protein binds to both RNA elements, but the molecular mechanism underlying this process remains unclear. A new study sheds light on how enteroviruses use structured RNA elements and multifunctional proteins to coordinate viral replication efficiently using minimal genetic material.
“This work highlights that RNA is not just a messenger of genetic code, but an active participant in viral regulation,” said Dimagi Dias-Solange, a graduate student in Kay Choi’s lab at Indiana University, Bloomington and first author on the paper. “The fact that an RNA structure can direct protein assembly emphasizes the sophistication of viral genome architecture and could influence how we study RNA in other systems, including human biology.”
The two elements of RNA regulate different steps of the replication cycle. It plays a role in the assembly of additional viral RNA. It also provides the scaffolding for the viral replication complex that involves both viral and host proteins.
The team used Beamline 8.2.2 at the Advanced Light Source (ALS) to perform x-ray protein crystallography to obtain high-resolution diffraction patterns to resolve the structure of the RNA cloverleaf-protein complex.
“Imagine a virus as a tiny robot with very limited instructions, but it still needs to make copies of itself inside our bodies,” said Dias-Solange. “Instead of using a lot of tools, it uses very clever tricks to get the job done.”
The cloverleaf plays a critical role in viral replication by switching between synthesizing proteins (translation) and making an identical copy of the virus (replication). One protein, 3Cpro, proved critical in this process. 3Cpro acts like a Swiss army knife, assembling into different shapes that are tailored to function at different stages of viral replication process.
“What surprised us is that the RNA acts like a mold,” said Dias-Solange. “It brings two of these proteins together in just the right way to start the virus’s replication process.”
According to Dias-Solange, the findings present new molecular targets for antiviral drug and biochemical assay development. This work could be used to disrupt how the virus replicates. In addition, this work can contribute to the broader field of RNA biology and synthetic biology that could yield programmable biomolecular devices in the future.
“The RNA-guided assembly mechanism we uncovered may not be limited to enteroviruses,” said Dias-Solange. “This work opens up a new way of thinking about how RNA shapes, not just sequences, control biological processes and could inspire new research into non-coding RNA functions, synthetic RNA devices, or RNA-targeting therapeutics.”
Dimagi Dias-Solange et al., Structure of coxsackievirus cloverleaf RNA and 3Cpro dimer establishes the RNA-binding mechanism of enterovirus protease 3Cpro. Sci. Adv. 11, eads6862(2025). doi:10.1126/sciadv.ads6862