New microscope technique reveals how COVID-19 virus replicates inside cells

Viral RNA, labeled with a fluorescent dye, clusters around the nucleus of a cell infected with SARS-CoV-2, as captured through super-resolution microscopy. Credit: Nature Communications (2024).

Researchers at Stanford University have developed a groundbreaking method to observe the SARS-CoV-2 virus, which causes COVID-19, replicating inside human cells at an incredibly detailed level.

This new technique, described in the journal Nature Communications, could significantly enhance drug development for treating the virus.

The team, led by Stanford associate professor of bioengineering Stanley Qi, used advanced super-resolution microscopy to capture crisp images of the virus’s RNA and its replication structures.

These images show the virus forming spherical shapes around the cell’s nucleus, providing unprecedented insight into the virus’s behavior.

“We have not seen COVID infecting cells at this high resolution and known what we are looking at before,” said Qi. “Being able to know what you are looking at with this high resolution over time is fundamentally helpful to virology and future virus research, including antiviral drug development.”

To spread, viruses like SARS-CoV-2 take over cells, turning them into virus-producing factories. These factories create special replication organelles where the virus’s RNA duplicates itself.

The Stanford scientists aimed to reveal this replication process in the sharpest detail possible.

They labeled the viral RNA and associated proteins with fluorescent molecules of different colors.

However, imaging glowing RNA alone would result in fuzzy blobs with conventional microscopes. To overcome this, they added a chemical that temporarily suppresses the fluorescence, causing the molecules to blink on and off randomly.

This allowed them to pinpoint the location of individual molecules.

Using lasers, powerful microscopes, and a camera taking photos every 10 milliseconds, the researchers captured snapshots of the blinking molecules.

By combining these images, they created highly detailed photos showing the viral RNA and replication structures within the cells.

“We have highly sensitive and specific methods and also high resolution,” said Leonid Andronov, co-lead author and Stanford chemistry postdoctoral scholar. “You can see one viral molecule inside the cell.”

The resulting images, with a resolution of 10 nanometers, reveal the most detailed view yet of how the virus replicates inside a cell. The images show magenta-colored RNA forming clumps around the cell’s nucleus, which accumulate into large, repeating patterns.

“We are the first to find that viral genomic RNA forms distinct globular structures at high resolution,” said Mengting Han, co-lead author and Stanford bioengineering postdoctoral scholar.

These clusters show how the virus evades the cell’s defenses. “They’re collected together inside a membrane that sequesters them from the rest of the cell, so that they’re not attacked by the cell’s defenses,” explained W. E. Moerner, the paper’s co-senior author and Harry S. Mosher Professor of Chemistry.

Compared to electron microscopes, this new technique offers more precise information about where virus components are located in a cell, thanks to the blinking fluorescent labels. It can also reveal nanoscale details of cellular processes that traditional biochemical assays cannot detect.

“The conventional techniques are completely different from these spatial recordings of where the objects actually are in the cell, down to this much higher resolution,” said Moerner. “We have an advantage based on the fluorescent labeling because we know where our light is coming from.”

Understanding exactly how the virus stages its infection holds great promise for medicine. Observing how different viruses take over cells may help explain why some cause mild symptoms while others are life-threatening. This super-resolution microscopy can also aid in drug development. “This nanoscale structure of the replication organelles can provide some new therapeutic targets for us,” said Han. “We can use this method to screen different drugs and see their influence on the nanoscale structure.”

The team plans to repeat the experiment and observe how the viral structures change in the presence of antiviral drugs like Paxlovid or remdesivir. If a drug can suppress the viral replication step, it suggests the drug is effective at inhibiting the virus, making it easier for the host to fight the infection.

The researchers also aim to map all 29 proteins that make up SARS-CoV-2 and understand their roles throughout the infection. “We hope that we will be prepared to really use these methods for the next challenge to quickly see what’s going on inside and better understand it,” said Qi.

This innovative research could lead to significant advancements in our understanding and treatment of viral infections, providing new hope in the fight against diseases like COVID-19.

Source: Stanford University.