NIAID Scientists Explore Pathways of Viral Evolution

Viruses are known to evolve quickly, in part because one generation of viral growth occurs in just minutes—an instant compared to the decades taken for a human generation to occur. This evolution happens at a vast scale, too. There are more viruses on the planet than stars in the universe. That’s trillions of trillions of viruses, all evolving at breakneck speed—and yet, researchers have found that viruses often evolve to arrive at similar genetic features. NIAID scientists recently developed new tools to understand this process in an important group of viruses called enteroviruses, which are responsible for several dangerous diseases, including polio; hand, foot, and mouth disease (HFMD); and acute flaccid myelitis (AFM). Their findings have implications for the evolution of other viral pathogens, as well.

The enterovirus that causes polio, Poliovirus, may be the most well-known. It infects the nervous system, leading to paralysis and death in severe cases. Although vaccination against polio has resulted in eradication of the virus from many parts of the world, the virus continues to emerge in some places, posing a threat to public health. HFMD is caused by different enteroviruses from a group often referred to as non-polio enteroviruses, including enterovirus A71 (EV-A71) and coxsackie virus A6 (CV-A6). HFMD is very contagious and often results in a fever, sore throat, and rash, with symptoms more commonly occurring in children than adults. Although uncommon, HFMD can result in serious complications, and so large outbreaks can raise public health concerns. In rare cases, non-polio enteroviruses such as EV-A71 and EV-D68 can also lead to AFM, a dangerous disorder of the nervous system that can result in permanent impairment. Despite the risks they pose, many enteroviruses and the diseases they cause are not well understood. It’s important for scientists to uncover how enteroviruses grow and evolve so they can develop new countermeasures against these diseases.

Patrick Dolan, Ph.D., chief of the Quantitative Virology and Evolution Unit in the Division of Intramural Research, is investigating aspects of enterovirus evolution. His research group developed a new method called SEARCHLIGHT (short for scRNA-seq–enabled acquisition of mRNA and consensus haplotypes linking individual genotypes and host transcriptomes). The method homes in on individual viruses within a rapidly evolving viral population. Using cutting-edge technology, the scientists examined the communities of viruses in single cells during viral infection. This method allows for the exploration of the concept of “viral quasi-species”—a term coined by scientists to describe how populations of viruses rapidly evolve when faced with new environmental pressures. The researchers observed how, in specific conditions, viral populations explore their “genetic neighborhood”—the possible ways they could adapt—through mutation to find more successful genetic strategies. In addition, they observed how EV-D68 and EV-A71, two distantly related members of the enterovirus family, followed common routes of evolution under similar conditions, suggesting that the routes viruses travel to explore their genetic neighborhood are shaped by common features of the viruses. Insights from this research may allow researchers to predict how new viral variants emerge and develop strategies to counter them.

To further explore features of enterovirus evolution, Dr. Dolan’s team used an innovative method to map all the sites of the genome known to foster insertions and deletions, or InDels. InDels are major evolutionary events in which short genetic sequences can be removed or added, as opposed to single substitutions in the genetic sequence, which are minor events that generally have more modest effects on gene function. The team examined tens of thousands of mutations in EV-A71, finding that InDels were only tolerated at specific hotspots, many of which appear to be critical for adaptions in viral function and immune evasion. For instance, the researchers observed many InDels in the regions of the genome dedicated to forming the viral capsid—the outer shell of the virus that interacts with the body’s machinery to enter cells, as well as triggering an immune response. This suggests that InDels in this region provide a mechanism for the virus to adapt new ways to infect cells and trick the immune system, which are critical functions for viral propagation. Based on these findings, InDels contribute significantly to the evolution and diversification of viruses. This new map of InDel sites may provide novel avenues for investigation of viral functions and interventions against them.

These studies demonstrate the power of advanced genetic technology to explore important questions in enterovirus evolution. Because enteroviruses have genetic similarities to many other viruses, this research has further implications towards discovering mechanisms of adaptation in a range of pathogens. A deepened understanding of viral evolution will provide researchers with new ways to develop targets for vaccines, therapeutics, and other countermeasures. Additionally, this research highlights the importance of investing in basic research to into how viruses adapt and function, fostering a body of knowledge researchers can draw upon for the development of interventions against infectious diseases. 

References:

• N Dábilla N, PT Dolan. Structure and dynamics of enterovirus genotype networks. Science Advances DOI: 10.1126/sciadv.ado1693 (2024).
• W Bakhache et al. Deep mutation, insertion and deletion scanning across the Enterovirus A proteome reveals constraints shaping viral evolution. Nature Microbiology DOI: 10.1038/s41564-024-01871-y (2024).

For NIAID scientist Hans Ackerman, M.D., DPhil, and his fellow malaria researchers it was a story several years in the making and involving multiphoton molecular imaging of live human arteries; gene expression and immunoprecipitation studies; computational modelling of the interactions between two proteins—hemoglobin and the enzyme nitric oxide synthase (NOS); and functional studies of vasoregulation in isolated human arteries. What emerged from their efforts is a new and unexpected understanding of the role played by hemoglobin in regulating constriction and dilation of human blood vessels as well as insights into the links between hemoglobin gene variants, including the one that gives rise to Sickle Cell Disease, and protection from severe malaria. Their findings appear online in the journal Circulation.

Malaria as a vascular disease

Malaria parasites infect red blood cells, and it is the interaction of these infected cells with the vascular endothelium (the layer of cells lining blood vessels) that gives rise to disease symptoms and helps characterize malaria as a vascular disease, notes Dr. Ackerman. In the healthy endothelium, nitric oxide (NO, which is made by the enzyme NOS) is produced and then quenched in a carefully regulated manner, allowing blood vessels to dilate and constrict smoothly. During a malaria infection, in contrast, a series of pathological forces combine in the blood vessels, resulting in dysregulation of vasodilation, breakdown of tight junctions between cells, and leakage of fluid into the surrounding tissue. When these damaging effects occur in the brain, they may lead to cerebral malaria, coma and death. 

“We don’t currently have malaria treatments that target endothelial dysregulation, but the concept of modulating nitric oxide signaling is appealing because it could potentially help restore vascular function and integrity,” says Dr. Ackerman. In studies in mice with cerebral malaria, direct administration of NO improved survival; however, the same effect was not seen in clinical trials involving children with cerebral malaria who were treated with inhalable NO. “So, we were interested in obtaining a better understanding of endothelial NO signaling with the aim of finding ways to modulate it,” Dr. Ackerman said.

Looking inside live arteries

To do this, Dr. Ackerman and his colleagues first used multiphoton microscopy to visualize hemoglobin inside live human artery samples that were obtained from healthy volunteers or from patients who were undergoing abdominal surgery. “I expected that endothelial hemoglobin would simply pervade the vessel in a kind of mist,” said Dr. Ackerman. “To our surprise, the imaging revealed that hemoglobin molecules were situated regularly throughout the stretchy elastin fibers separating the endothelial layer from the surrounding layer of smooth muscle around the artery,” he said. A 3D rendering showed hemoglobin clusters sitting in “pores” where endothelial cells connect to smooth muscle cells. These junctions are where cell-to-cell signaling, including the production of NO, happens, explains Dr. Ackerman. “It looks like hemoglobin is organized in a way to put it very close to the site of NO production, suggesting that it may play a role in regulating NO production, thus helping to reduce any off-target effects of NO,” he added.

A second surprise emerged from the multiphoton imaging: in contrast to mice, where only the alpha subunit of hemoglobin is found in the endothelium, human arteries express both the alpha and beta subunits of hemoglobin, which exists as a tetrameric—four-part—molecule. The team used immunoprecipitation to determine that alpha and beta hemoglobin subunits form a complex with endothelial NOS (along with another enzyme) that is embedded in the artery wall. When NOS produces nitric oxide, it immediately encounters the hemoglobin and is transformed into a relatively inactive chemical called nitrate that is unable to leave the endothelial cell. When hemoglobin is displaced from the endothelial NOS, however, then NO is produced and can send a signal to the smooth muscle cells that allows the vessel to dilate. Taken together, the team’s findings through these studies identified hemoglobin as a direct regulator of vasodilation and constriction in the endothelium.

Human hemoglobin variants

The investigators next sought to understand how variant forms of hemoglobin changed its ability to regulate NO production. In mice, noted Dr. Ackerman, it would be possible to selectively “knock out” the genes responsible for either alpha or beta hemoglobin subunits and observe the effects.  Since this is not possible in people, the team turned to naturally occurring conditions in which either the alpha or beta hemoglobin subunit gene is mutated. First, they used live artery imaging to view blood vessel samples taken from people who have a partial deletion of the alpha globin gene, which limits the amount of alpha hemoglobin they can produce. Compared to arteries from healthy volunteers, the arteries containing the alpha globin deletion dilated more when subjected to identical constriction-causing stimuli. The interpretation is that lower levels of the alpha subunit mean the hemoglobin complex does not function to fully inhibit NO production and the NO that is produced has a greater vasodilating effect relative to that seen in healthy arteries. 

The investigators then employed a 15-amino-acid-long peptide replicating the part of the alpha hemoglobin subunit that interacts with NOS. When introduced into the vessel wall, the manufactured peptide binds to the NOS enzyme, preventing the full hemoglobin-NOS complex from forming and catalyzing the chemical reaction that transforms NO to nitrate. Without the reaction, the NO produced by NOS can leave the cell and stimulate dilation. 

Beta globin gets in the picture

The information about the role played by alpha subunit hemoglobin had previously been sketched out in mice, explained Dr. Ackerman, and their studies extended that understanding to humans. The next phase of the research used computational modelling to develop a picture of how alpha-beta hemoglobin forms the complex with NOS. The model shows portions of the alpha and beta amino acid chains where they come into contact with key portions of NOS enzyme. One of the most important sites of contact, the investigators saw, was where a positively charged amino acid of NOS meets the negatively charged amino acid glutamic acid in position 6 of the beta globin chain forming a stabilizing ‘salt bridge’ between the two molecules. 

“We next generated a molecular simulation model of the complex, which shows how tightly the hemoglobin nestles into a half-moon shape of NOS,” said Dr. Ackerman. “However, if we simulate the shape of the complex after removing that single, negatively charged glutamic acid from position 6 of the beta chain, we see a gap open up between hemoglobin and NOS.”

Removing glutamic acid from position 6 of hemoglobin’s beta chain exactly replicates the situation that arises in people with Sickle Cell Disease. People who inherit two copies of the mutated gene coding for beta hemoglobin produce a form of hemoglobin, called sickle or hemoglobin S, where a different amino acid is substituted for glutamic acid in position 6, leading hemoglobin S molecules to aggregate, which distorts red blood cells into a sickle shape. People who have sickle cell trait (those have inherited a single mutated gene), which is most common in Africans and people with African ancestry, produce both normal and hemoglobin S (and do not have symptoms of Sickle Cell Disease). People with sickle cell trait are known to have some protection from severe malaria. It’s possible, says Dr. Ackerman, to explain this advantage by reference to the picture they’ve developed of the interaction between hemoglobin S and NOS: in theory, a somewhat loose connection between hemoglobin S and NOS would allow production of NO and subsequent vessel dilation, lessening the vasodilation dysregulation that characterizes severe malaria. 

Next steps: finding ways to target the hemoglobin-NOS complex

Dr. Ackerman and his colleagues also synthesized a peptide that replicates the part of the beta chain where the sickle cell trait amino acid substitution occurs that alters the way hemoglobin interacts with NOS and studied its effects in isolated human arteries. They found that the mimetic peptide disrupted the hemoglobin-NOS complex, which permits NO to be produced and leave the cell, resulting in vasodilation. Unlike NO-producing nitroglycerin pills, which dilate blood vessels in a non-specific way, the synthetic beta globin peptide made by the investigators targets just the cell junctions where NO is produced, explains Dr. Ackerman. It’s possible, he adds, that such a mimetic peptide could be further developed into a treatment that could restore normal vasodilation and could be envisioned as an intervention for cases of severe malaria and other diseases where NO signaling is insufficient.

Reflecting on the odyssey of research that culminated in their new paper, Dr. Ackerman said, “These discoveries were elevated by the participation of Black individuals who were interested in learning about alpha thalassemia and sickle trait and who wanted to contribute by participating in research. Our research team included members who came from communities where thalassemia and sickle cell disease are common. This helped our team engage with study participants and develop a strong sense of purpose around our research that was essential in producing this new understanding about fundamental workings of the human body.”

Reference: SD Brooks et al. Sickle trait and alpha thalassemia increase NOS-dependent vasodilation of the human arteries through disruption of endothelial hemoglobin-eNOS interactions. Circulation DOI: 10.1161/CIRCULATIONAHA.123.066003 (2025).