Shadowing SARS-CoV-2 Through Mucus and Cilia

Abstract: infectious disease Shadowing SARS-CoV-2 Through Mucus and Cilia Researchers discovered how SARS-CoV-2 invades and spreads through nasal epithelial cells, identifying potential new drug targets to prevent transmission. W BY STEPHANIE DEMARCO, PHD Microvilli escape routes After figuring out how SARS-CoV-2 enters the cells, the researchers wanted to know images of the month CREDIT: CHIEN-TING WU Mucin barriers and cilia ladders To refine their idea of how quickly SARSCoV-2 binds to cilia after infection, the team relied on the lead author, Chien-Ting Wu’s expertise in electron microscopy (EM) to take scanning EM images of the organoids soon after SARS-CoV-2 infection. Wu, who is now a virologist at UT Southwestern, observed virions dotted all over the cilia as soon as six hours after infection. The researchers wondered why the virus took 24 to 48 hours to infect the cells if they reached the cilia in only six hours. In cell culture experiments, Jackson said, “the virus gets into the cell in about 10 minutes.” They hypothesized that the mucus layer, which was absent in cell culture experiments, slowed the virus down. To crack this mystery, the researchers treated the organoids with mucinase, an enzyme that digests the main component of mucus called mucins, and infected the organoids with SARS-CoV-2. The virus invaded the cells much faster than before. “When you accept that the mucins are the barrier, what does the cilia do?” Jackson asked. The team then knocked down cilia in the organoids and found that SARS-CoV-2 couldn’t enter the cells at all. They even showed that other respiratory viruses including respiratory syncytial virus (RSV) and parainfluenza virus (PIV) also couldn’t infect the nasal epithelial cells without cilia. “That confirmed the model that the virus needed these little portals or ladders to crawl through the mucous,” said Jackson. “The cilia are kind of like the ladders that the army in Game of Thrones uses to climb up the walls of the castle and attack.” how the virus climbs back out and spreads to other nasal epithelial cells. To find out, the researchers used transmission EM to visualize the nasal epithelial cells after infection and noticed that emerging SARS-CoV-2 virions clustered around what looked like brand new structures. Normally, cells can form extensions called microvilli that look similar to cilia but are nonmotile and typically shorter. But the researchers saw that the SARS-CoV-2 infected nasal epithelial cells formed extremely long and more branched microvilli than they had ever seen before. The uninfected cells did not form these structures. SARS-CoV-2 virions, shown by nucleocapsid proteins in red and viral spike proteins in green, invade the cilia, represented by acetylated tubulin in white, of human nasal epithelial cells. Cellular DNA appears blue in this immunofluorescence image. Using transmission EM, the researchers observed SARS-CoV-2 virions — the circular structures — exiting abnormally branched and extended microvilli. “When you infect with the virus, the microvilli, they branch, and that’s just crazy. I don’t think anyone had ever observed that.” – Peter Jackson, Stanford University “If you look at some of the EM pictures, they’re really kind of amazing,” said Jackson. “When you infect with the virus, the microvilli, they branch, and that’s just crazy. I don’t think anyone had ever observed that.” The small dots decorating the cilia in this scanning electron microscope image are..