New Microscopy Reveals Secrets of SARS-CoV-2 Replication

Recent research published in Nature Communications by a Stanford University team may improve drug development by examining at the nanoscale how the SARS-CoV-2 virus replicates in cells.

By using cutting-edge microscopy techniques, the researchers were able to capture what may be some of the clearest images of the virus's replication and RNA structures, which they saw forming spherical shapes around the infected cell's nucleus.

We have not seen COVID infecting cells at this high resolution and known what we are looking at before. 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.”

Stanley Qi, Associate Professor and Study Co-Senior Author, Department of Bioengineering, Schools of Engineering and Medicine, Stanford University

Blinking RNA

The research sheds light on the molecular-level aspects of the virus's activity within host cells. Viruses take over cells and turn them into factories that produce new viruses, complete with unique replication organelles, to spread.

The viral RNA must repeatedly replicate itself within this factory until sufficient genetic material is accumulated to allow the virus to spread to other cells and recommence the process.

The goal of the Stanford researchers was to provide the most precise explanation of this replication step to date. They first used fluorescent molecules of various colors to label the replication-associated proteins and viral RNA to achieve this. However, a standard microscope would only show fuzzy blobs when imaging glowing RNA alone. Thus, a substance that momentarily suppresses the fluorescence was added.

The molecules would then sporadically blink again, only occasionally lighting up at a time. This made it simpler to identify the flashes and revealed the precise locations of each molecule.

Researchers captured images of the blinking molecules with a system comprising lasers, strong microscopes, and a camera that took pictures every 10 milliseconds. The researchers were able to produce incredibly detailed images that displayed the viral RNA and replication structures in the cells by combining sets of these images.

We have highly sensitive and specific methods and also high resolution. You can see one viral molecule inside the cell.”

Leonid Andronov, Study Co-Lead Author and Chemistry Postdoctoral Scholar, Stanford University

With a resolution of 10 nm, the resulting images provide what may be the most in-depth look at the virus's internal replication process to date. The pictures demonstrate how magenta RNA gathers around the cell nucleus to form clumps that eventually form a sizable repeating pattern.

We are the first to find that viral genomic RNA forms distinct globular structures at high resolution.”

Mengting Han, Study Co-Lead Author and Postdoctoral Scholar, Stanford University

The clusters help show how the virus evades the cell’s defenses, said W. E. Moerner, the paper’s Co-Senior Author and Harry S. Mosher Professor of Chemistry in the School of Humanities and Sciences. “They’re collected together inside a membrane that sequesters them from the rest of the cell, so that they’re not attacked by the rest of the cell.”

Nanoscale Drug Testing

Because the fluorescence labels blink, researchers can more accurately determine the location of virus components within a cell using this new imaging technique than they could with an electron microscope.

Additionally, it can provide nanoscale details of invisible cellular processes in medical research conducted through biochemical assays.

Moerner said, “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. We have an advantage based on the fluorescent labeling because we know where our light is coming from.”

Recognizing the virus's precise steps to infect a host has therapeutic potential. Seeing how various viruses infiltrate cells may provide light on why certain infections cause only minor symptoms while others can be fatal. Drug development can also benefit from super-resolution microscopy.

Han said, “This nanoscale structure of the replication organelles can provide some new therapeutic targets for us. We can use this method to screen different drugs and see its influence on the nanoscale structure.”

The team plans to repeat the experiment to observe any changes in viral structures caused by the presence of medications such as remdesivir or Paxlovid. The ability of a potential medication to inhibit the viral replication stage indicates that the drug is effective in hindering the pathogen and facilitating the host's defense against the infection.

The researchers also plan to map all 29 proteins that make up SARS-CoV-2 and see what those proteins do throughout an 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.