Researchers at the University of Maryland, Baltimore County (UMBC) have uncovered a crucial step that enteroviruses use to reproduce inside human cells. The findings, published in Nature Communications, explain how viruses responsible for illnesses such as polio, encephalitis, myocarditis, and even the common cold take control of cellular machinery to copy themselves. Scientists say the discovery could eventually help researchers create a new generation of antiviral drugs capable of targeting many enteroviruses at once.
The study was led by Deepak Koirala, associate professor of chemistry and biochemistry at UMBC, along with recent Ph.D. graduate Naba Krishna Das. Their work helps answer longstanding questions about how these viruses launch replication once they invade a cell.
“My lab has been really motivated to understand how RNA viruses produce their proteins inside the cell and multiply their genome to make more virus particles,” Koirala says. Earlier work from the team identified an important cloverleaf shaped structure within the virus’s RNA. The new study shows how that structure recruits proteins needed to build the viral replication machinery.
How Enteroviruses Reproduce Inside Cells
Enteroviruses carry very small RNA genomes that must perform two jobs at once. The viral RNA has to direct the production of viral proteins while also serving as the template for creating new copies of the virus.
Most of the viral genome contains instructions for structural proteins, but it also encodes several specialized proteins required for replication. One of the most important is a fusion protein called 3CD.
The 3C portion cuts long chains of amino acids into the separate proteins the virus needs. The 3D portion acts as an RNA polymerase, an enzyme that copies viral RNA so the virus can reproduce. Human cells do not naturally contain this type of polymerase, meaning the virus has to supply its own version.
“We previously determined the structure of the RNA alone, and other groups determined the structure of 3C and 3D, but now we’ve captured the structure of the RNA and proteins together, so we know how they are interacting,” Koirala explains. “We found that it’s the 3C domain of 3CD that binds to the RNA in the viral genome, and then it recruits the other components, such as host protein PCBP2, to assemble the replication complex.”
The researchers also found that this molecular complex functions like a switch. When 3CD is attached, the virus copies its RNA genome. When the protein detaches, the RNA becomes available for producing viral proteins instead.
Scientists Resolve a Longstanding Viral Mystery
To examine these interactions in detail, the team used X-ray crystallography to visualize the RNA cloverleaf and the 3CD protein together. They also relied on isothermal titration calorimetry (ITC), which measures the heat released when molecules bind, and biolayer interferometry (BLI), which uses changes in light interference to track how long molecules stay attached.
The experiments helped settle an ongoing scientific debate. The researchers showed that two full 3CD molecules, each carrying its own RNA polymerase, bind side by side on the viral RNA. Earlier research had proposed that the proteins formed a single fused pair instead.
Scientists still do not fully understand why two copies are required, but the new study provides a much clearer picture of how the replication process begins.
Potential for Broad Spectrum Antiviral Drugs
One of the most promising findings was how similar the mechanism appeared across all seven enteroviruses examined in the study. The viruses shared nearly identical RNA cloverleaf structures and binding behavior.
That level of similarity suggests the RNA structure is extremely important to viral survival. Significant mutations would likely disrupt replication, making the structure a potentially stable drug target across many enteroviruses.
Researchers say this raises the possibility of developing broad spectrum antiviral drugs that could work against an entire family of viruses rather than a single pathogen.
Scientists are already developing drugs that interfere with the 3C and 3D proteins, but the new findings reveal another possible strategy.
Drugs disrupting 3C and 3D activity are already in development, but “now we have another layer to test,” Koirala says. “What if we target the RNA, or the RNA-protein interface, so that we break the interaction? That is another opportunity. Now that we have high-resolution structures, you can precisely design drug molecules to target them.”
Koirala says the study highlights how surprisingly sophisticated viruses can be despite their tiny genomes.
“Viruses are so, so clever. Their entire genome is equivalent to about one mRNA sequence in humans, yet they are so effective,” Koirala says. His latest work demonstrates “why we need to investigate this basic science — so that it can be translated into developing drugs targeting pathogens that cause so many harmful diseases.”