A new way to see viruses in action: super-resolution microscopy offers a nanoscale look

A new nanoscale study of how the SARS-CoV-2 virus replicates in cells could provide greater precision in drug development, a Stanford University team reports in Natural communications. Using advanced microscopy techniques, the researchers produced what may be some of the sharpest images available of the virus’s RNA and replication structures, which they witnessed as spherical shapes around the core of the virus. infected cell.

“We didn’t see COVID infecting cells at this high resolution and we knew what we were observing before,” said Stanley Qi, associate professor of bioengineering at Stanford in the schools of engineering and medicine and co -main author of the article. “Being able to know what you’re looking at with this high resolution over time is fundamentally useful to virology and future virus research, including antiviral drug development.”

Flashing RNA

The work illuminates molecular-level details of virus activity inside host cells. To spread, viruses take over cells and turn them into virus-producing factories, equipped with special replication organelles. Within this factory, the viral RNA must duplicate itself over and over again until enough genetic material is collected to come out, infect new cells, and start the process again.

Stanford scientists sought to reveal this replication step in the most detail possible to date. To do this, they first labeled viral RNA and replication-associated proteins with fluorescent molecules of different colors. But imaging bright RNA alone would result in blurry spots in a conventional microscope. So they added a chemical that temporarily suppresses fluorescence. The molecules would then reignite at random times, and only a few would light up at a time. This made it easier to localize the flashes, revealing the location of individual molecules.

Using a setup that included lasers, powerful microscopes, and a camera that took photos every 10 milliseconds, the researchers gathered snapshots of the blinking molecules. When they combined sets of these images, they were able to create finely detailed photos showing viral RNA and replication structures in cells.

“We have very sensitive and specific methods as well as high resolution,” said Leonid Andronov, co-senior author and postdoctoral researcher in chemistry at Stanford. “You can see a viral molecule inside the cell.”

The resulting images, with a resolution of 10 nanometers, reveal what could be the most detailed view yet of how the virus replicates inside a cell. The images show magenta RNA forming clumps around the cell’s nucleus, which accumulate into a large repeating pattern. “We are the first to discover that viral genomic RNA forms distinct globular structures at high resolution,” said Mengting Han, co-senior author and postdoctoral researcher in bioengineering at Stanford.

The clusters help show how the virus evades cellular defenses, said WE Moerner, co-senior author of the paper and the Harry S. Mosher Professor of Chemistry in the School of Human Sciences. “They are gathered together inside a membrane that separates them from the rest of the cell, so that they are not attacked by the rest of the cell.”

Nanoscale drug testing

Compared to using an electron microscope, the new imaging technique can allow researchers to know with greater certainty where virus components are located in a cell thanks to the flashing fluorescent tags. It can also provide nanoscale details of cellular processes that are invisible in medical research conducted using biochemical assays.

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

Seeing exactly how the virus develops its infection holds promise for medicine. Observing how different viruses take over cells can help answer questions such as why some pathogens produce mild symptoms while others are life-threatening. Super-resolution microscopy can also benefit drug development. “This nanoscale structure of replication organelles may provide us with new therapeutic targets,” Han said. “We can use this method to test different drugs and see how they influence the nanoscale structure.”

Indeed, this is what the team intends to do. They will repeat the experiment and see how viral structures change in the presence of drugs like Paxlovid or remdesivir. If a drug candidate can suppress the viral replication step, this suggests that the drug is effective in inhibiting the pathogen and making it easier for the host to fight the infection.

The researchers also plan to map the 29 proteins that make up SARS-CoV-2 and see what these proteins do throughout the infection. “We hope we’ll be ready to actually use these methods for the next challenge to quickly see what’s going on inside and understand it better,” Qi said.