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A new, nano-scale look at how the SARS-CoV-2 virus replicates in cells may offer greater precision in drug development, a Stanford University team reports in Nature Communications. Using advanced microscopy techniques, the researchers produced what might be some of the most crisp images available of the virus’s RNA and replication structures, which they witnessed form spherical shapes around the nucleus of the infected cell.

“We have not seen COVID infecting cells at this high resolution and known what we are looking at before,” said Stanley Qi, Stanford associate professor of bioengineering in the Schools of Engineering and Medicine and co-senior author of the paper. “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.”

The work illuminates molecular-scale details of the virus’ activity inside host cells. In order to spread, viruses essentially take over cells and transform them into virus-producing factories, complete with special replication organelles. Within this factory, the viral RNA needs to duplicate itself over and over until enough genetic material is gathered up to move out and infect new cells and start the process again.

The Stanford scientists sought to reveal this replication step in sharp detail. To do so, they first labeled the viral RNA and replication-associated proteins with fluorescent molecules of different colors. Imaging glowing RNA alone would result in fuzzy blobs in a conventional microscope. They added a chemical that temporarily suppresses fluorescence; molecules would then blink back on at random times, making it easier to pinpoint their locations.

Using lasers, powerful microscopes, and a camera snapping photos every 10 milliseconds, researchers gathered snapshots of blinking molecules. Combining sets of these images created finely detailed photos showing viral RNA and replication structures in 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 have a resolution of 10 nanometers. They reveal what might be the most detailed view yet of how the virus replicates inside a cell. The images show magenta RNA forming clumps around the nucleus of the cell, which accumulate into a large repeating pattern.

“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.

Video footage shows different colored fluorescent labels blinking on and off, revealing more precise locations for individual molecules.

The clusters help show how the virus evades cell defenses by being collected together inside a membrane that sequesters them from other parts of the cell. W.E. Moerner, co-senior author and Harry S. Mosher Professor of Chemistry in Stanford’s School of Humanities and Sciences noted: “They’re collected together inside a membrane that sequesters them from the rest of the cell so that they’re not attacked by other parts.”

Compared to electron microscopes, this imaging technique allows researchers greater certainty about where virus components are within a cell thanks to blinking fluorescent labels. It also provides nanoscale details invisible through traditional biochemical assays.

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

Observing exactly how viruses stage infections holds promise for medicine as understanding why some pathogens produce mild symptoms while others are life-threatening could benefit drug development efforts.

“This nanoscale structure... can provide some new therapeutic targets for us,” said Han. The team plans further experiments to observe how viral structures shift when exposed to drugs like Paxlovid or remdesivir—potentially indicating effectiveness if replication steps are suppressed.

Additionally, researchers plan to map all 29 proteins comprising SARS-CoV-2 throughout an infection cycle using these methods for rapid insights into future challenges.

Acknowledgements: Additional Stanford co-authors include postdoctoral scholar Yanyu Zhu; PhD student Ashwin Balaji; former PhD student Anish Roy; postdoctoral scholar Andrew Barentine; research specialist Puja Patel; Jaishree Garhyan—director In Vitro Biosafety Level-3 Service Center; Moerner—Stanford Bio-X member/Wu Tsai Neurosciences Institute faculty fellow/Sarafan ChEM-H fellow; Qi—Bio-X member/Cardiovascular Institute/Maternal & Child Health Research Institute/Stanford Cancer Institute/Wu Tsai Neurosciences Institute/Sarafan ChEM-H institute scholar/Chan Zuckerberg Biohub – San Francisco Investigator

This research was funded by National Institute General Medical Sciences/National Institutes Health with support from Stanford University Cell Sciences Imaging Core Facility

Taylor Kubota – tkubota@stanford.edu

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