Forget about locating molecules in the blink of an eye, which takes as long as a quarter second—far too long a time to distinguish a sequence of subcellular events. Instead, try doing what Stanford University scientists did in a study of viral organelle formation. They located molecules in the blink of a fluorescent label.

More specifically, they used a super-resolution fluorescence microscopy setup that involved laser excitation, fast-blinking labels, and a high-speed camera to determine the precise locations of key RNA molecules and replication intermediates behind the transformation of host cells infected by SARS-CoV-2. The camera snapped photos every 10 milliseconds, capturing the fluorescent signals of randomly blinking labels attached to the RNA molecules. When the photos were analyzed, patterns emerged showing that viral genomic RNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region. These clusters grow and accommodate more viral genomic RNA molecules as infection time increases.

Detailed findings appeared in Nature Communications, in a paper titled, “Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.” The paper’s senior authors were Stanley Qi, PhD, associate professor of bioengineering at Stanford, and W. E. Moerner, PhD, the Harry S. Mosher Professor of Chemistry.

“This study expands the knowledge of the biology of coronaviruses and opens new possibilities for therapeutics against SARS-CoV-2, considering that clusters of viral genomic RNA have also been reported in SARS-CoV-2 infected interstitial macrophages of human lungs, suggesting their importance in COVID-19,” the article’s authors wrote. “Careful examination of the organization of replication organelles may provide new avenues to target the organelles to disrupt SARS-CoV-2 replication and transcription.”

“Examining localization patterns for different viral variants or in different host cells will be useful to broaden understanding of the viral infection,” they continued. “It will also be important to examine how the structures reported in this study change upon the addition of drug treatments.”

The precise locations of key RNA molecules and replication intermediates have been difficult to determine using electron microscopy. Consequently, the Stanford scientists used multicolor confocal microscopy and super-resolution microscopy, instruments that ultimately revealed the localization patterns of viral RNA, related viral proteins, and altered host cell structures.

“We have not seen COVID-19 infecting cells at this high resolution and known what we are looking at before,” Qi said. “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. 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 over again.

To reveal this replication step in the sharpest detail to date, the scientists first labeled the viral RNA and replication-associated proteins with fluorescent molecules of different colors. But imaging glowing RNA alone would result in fuzzy blobs in a conventional microscope. So, the scientists added a chemical that temporarily suppresses the fluorescence. The molecules would then blink back on at random times, and only a few lit up at a time. That made it easier to pinpoint the flashes, revealing the locations of the individual molecules.

The scientists gathered snapshots of the blinking molecules that had a resolution of 10 nm. Combining these snapshots allowed the scientists to see how the virus replicates itself inside a cell. The images show magenta RNA forming clumps around the nucleus of the cell, which accumulate into a large repeating pattern.

“The clusters help show how the virus evades the cell’s defenses,” Moerner noted. “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.”

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

Seeing exactly how the virus stages its infection holds promise for medicine. Observing how different viruses take over cells may 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 the replication organelles can provide some new therapeutic targets for us,” said Mengting Han, PhD, co-lead author and Stanford bioengineering postdoctoral scholar. “We can use this method to screen different drugs and see its influence on the nanoscale structure.”

Indeed, that’s what the team plans to do. They will repeat the experiment and see how the viral structures shift in the presence of drugs like Paxlovid or remdesivir. If a candidate drug can suppress the viral replication step, that suggests the drug is effective at inhibiting the pathogen and making it easier for the host to fight the infection. The researchers also plan to map all 29 proteins that make up SARS-CoV-2 and see what those proteins do across the span of an infection.

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