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.”

Lysosomes function as the cell’s internal recycling centers. They break down proteins, nucleic acids, carbohydrates, and lipids, helping cells dispose of waste and reuse materials for essential biological processes. They also store nutrients that can be released when needed. Because of these roles, lysosomes are critical for maintaining cellular metabolism, including both catabolism (breaking down complex molecules to simple ones) and anabolism (building complex molecules from simpler ones).

The research team focused on hematopoietic stem cells (HSCs), which are rare, long-lasting stem cells found in the bone marrow that generate all blood and immune cells in the body. The study was led by Saghi Ghaffari, MD, PhD, Professor of Cell, Developmental, and Regenerative Biology at the Icahn School of Medicine and a member of the Black Family Stem Cell Institute.

As people grow older, these stem cells gradually lose their ability to repair and replenish the blood system. This decline weakens immune defenses and contributes to the increased vulnerability to infections seen in older adults. Aging HSCs are also linked to clonal hematopoiesis, an asymptomatic condition considered a premalignant state that raises the risk of blood cancers and inflammatory diseases. The condition becomes much more common with age.

According to the American Cancer Society, age and smoking are the two strongest risk factors associated with the five-year risk of developing cancer. Data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results report show that the median age at cancer diagnosis is 67.

Restoring Old Stem Cells to a Youthful State

The researchers discovered that lysosomes in aged HSCs become excessively acidic, damaged, depleted, and abnormally active. These changes disrupt both the metabolic balance and epigenetic stability of the stem cells.

Using single-cell transcriptomics and functional testing, the team found that blocking this excessive lysosomal activity with a vacuolar ATPase inhibitor restored lysosomal health and improved the function of aging blood stem cells.

After treatment, the old stem cells began behaving more like young, healthy cells again. They regained the ability to regenerate effectively, produce balanced blood and immune cells, and generate additional healthy stem cells. The treated cells also showed improved metabolism and mitochondrial performance, healthier epigenetic patterns, reduced inflammation, and fewer harmful inflammatory signals that can damage tissues throughout the body.

“Our findings reveal that aging in blood stem cells is not an irreversible fate. Old blood stem cells have the capacity to revert to a youthful state; they can bounce back,” said Dr. Ghaffari. “By slowing down the lysosomes and reducing their acidity, stem cells became healthier and could make new balanced blood cells and new stem cells much more effectively. By targeting lysosomal hyperactivity, we were able to reset aged stem cells to a younger, healthier state, improving their ability to regenerate blood and immune cells.”

Major Increase in Blood-Forming Capacity

The researchers also tested an ex vivo treatment approach (when cells are removed from the body, modified in a laboratory, and returned to the body). Treating old stem cells with the lysosomal inhibitor increased their blood-forming ability in living animals by more than eightfold, highlighting the powerful regenerative effects of correcting lysosomal dysfunction.

The improvement also reduced damaging inflammatory and interferon-related pathways. According to the researchers, this occurred because healthier lysosomes improved the processing of mitochondrial DNA and lowered activation of the cGAS-STING immune signaling pathway, which appears to play a major role in stem cell inflammation and aging.

Potential for Anti-Aging and Blood Disorder Therapies

The findings could open the door to new treatments aimed at preventing or reversing age-related blood disorders. They may also improve stem cell transplantation outcomes in older patients and enhance conditioning methods used in gene therapy.

“Lysosomal dysfunction emerges as a central driver of stem cell aging,” added Dr. Ghaffari. “Targeting this pathway may one day help maintain healthy blood and immune systems in the elderly, improve their stem cells for transplantation, and reduce the risk of age-associated blood disorders and perhaps have an effect on overall aging.”

The team is now investigating whether lysosomal dysfunction in aging stem cells contributes to the development of leukemic stem cells, potentially connecting normal stem cell aging with cancer formation.

The research involved collaboration with Mickaël Ménager, PhD, and colleagues at the Imagine Institute and INSERM UMR 1163 at Université de Paris Cité in Paris. Funding was provided by the National Institutes of Health, New York State Stem Cell Science, INSERM, and the Agence Nationale de la Recherche.