ABOVE: Fluorescent epithelial and neuronal cells in culture connected by tunneling nanotubes Anna Pepe, Institut Pasteur

SARS-CoV-2 usually infects cells by binding with the angiotensin-2 converting enzyme receptor. But although many cells—including neurons and cells that make up the blood-brain barrier—lack this protein, bits of the virus have been found in the brains of infected people post-mortem. Scientists have wondered how the virus is able to enter such unwelcoming tissues. Now, a study published yesterday (July 20) in Science Advances suggests that the virus may be shuttling itself through tiny tubes that extend from infected host cells.

“It’s a pretty exciting study,” Viabhav Tiwari, a virologist at Midwestern University who wasn’t involved in the research, tells The Scientist. “They are saying that the virus can be transferred and it’s most likely through these bridges. . . . Totally fascinating.”

See “Cancer Cell Nanotubes Hijack Mitochondria from Immune Sentinels

Tunneling nanotubes (TNTs) are delicate, hairlike structures that sprout from the cell body and pierce through neighboring cell membranes when cells are stressed, including when they’re low on oxygen or during infection. Through the tubes, which are made of the protein actin, cells can send and receive RNA, nutrients, even entire organelles—and, unfortunately, viruses. From previous work, Pasteur Institute cell biologist Chiara Zurzolo knew that some viruses use nanotubes to spread from cell to cell. And given the fact that SARS-CoV-2 was infecting such a broad array of cell types, she thought maybe the coronavirus could similarly exploit TNTs.

“This virus is a beast. It infects everything,” Zurzolo says. “It spreads very fast throughout the brain and we think this is a possible mechanism” of how it does so.

To test this line of thinking, the researchers cultured Vero E6 cells, which model the cells that line our skin, organs, and blood vessels—and express angiotensin-2 converting enzyme (ACE2). Separately, the team also cultured SH-SY5Y, which model human neuronal cells and lack the ACE2 receptor. As predicted, the coronavirus easily infected the epithelial cells, but not the neurons. But when the scientists cultured infected epithelial cells and the neurons alongside one another, they detected viral proteins within the neurons after just one day. Furthermore, the researchers found that when ACE2 receptors were blocked, the virus was still able to find its way from infected epithelial cells to noninfected ones. 

See “SARS-CoV-2 Can Spread Via Cell-to-Cell Transmission

miscroscopy images of tunneling nanotubes
Fluorescent (top) and cryo-electron (bottom) microscopy images showing SARS-CoV-2 inside and on top of tunneling nanotubes
Anna Pepe, Institut Pasteur

Using a combination of fluorescence confocal microscopy and cryo-electron microscopy (cryo-EM)—a technique that involves flash-freezing samples and bombarding them with electrons, allowing researchers to capture 3D images of minuscule molecules—the scientists observed viral proteins and RNA within TNTs that were bridging cells. The TNTs also contained double-membrane vesicles, which are factories that churn out viral RNA. The researchers considered these findings strong evidence that the TNTs were acting as conduits for viral transmission, likely allowing the virus to bypass the blood-brain barrier and get into the brain.

However, Tiwari points out that while the study did show a potential way that neurons could be infected, the researchers didn’t show evidence that ACE2-positive cells could infect the types of epithelial cells that compose the blood-brain barrier. They also didn’t directly show that blood-brain barrier cells could form TNTs and transfer the virus to neurons. “Are blood-brain barrier cells capable of inducing these bridges?” he asks. “They didn’t really answer that.”

Avindra Nath, a neurologist at the National Institutes of Health who was not involved in the study, similarly notes that while many cells make TNTs in culture, such structures may not occur in vivo. 

“Further studies are necessary to establish if the same mechanism [of TNTs shuttling SARS-CoV-2] operates in the animal or human brain,” says Margolzata Kloc, a biochemist at Houston Methodist Medical Research Institute who was not involved in the study. “This can be very challenging because TNTs are ephemeral structures and catching them in action can be difficult.”

Studying TNTs in humans requires difficult-to-find, high-quality post-mortem tissue, which then needs to be imaged at super-high resolution. And since TNTs are made of just actin, there are very few biomarkers for these tiny structures, making them difficult to study and distinguish from other actin-based protrusions. As a result, TNT-mediated viral infection in vivo just “isn’t well documented,” explains Tiwari. The only in vivo studies involving TNTs Tiwari is aware of have happened in the eye.

Nath also points out that it remains unclear whether viral entry to the brain is actually an important part of COVID-19 pathology. Though the virus is seen in brain tissues, it may not be responsible for the neurological symptoms of COVID-19, since the amounts seen in neurological samples are low compared to what’s seen in lung tissues. “That small amount of virus cannot explain the pathology,” he says.

Still, Zurzolo and colleagues speculate that if SARS-CoV-2 does use nanotubes to make its way to the brain or elsewise, blocking TNT formation may be a way to stop the virus’s early spread and reduce infection severity overall. “We definitely need to stop the spread of virus initially,” Zurzolo says, in order to prevent it from wreaking havoc throughout the body. 

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