A research team at the College of Arts and Sciences at Indiana University Bloomington is uncovering important details about how the COVID-19 virus behaves inside infected cells, and their findings could help guide the development of future treatments.
A recent study published in the Journal of Biological Chemistry by Ph.D. student Patrick Laughlin, with Distinguished Professor of Molecular and Cellular Biochemistry Adam Zlotnick, focused on one of the virus’s main proteins, called the nucleocapsid protein (known as the “N-protein”), which plays a crucial role in the virus’s survival and ability to spread. Understanding how this protein works may open the door to new ways of stopping the virus in its tracks. Kimberly Young of the Zlotnick Lab, Dr. Giovanni Gonzalez-Gutierrez of the IU Electron Microscopy Center on the Bloomington campus, and Dr. Joseph Wang of Penn State University were also co-authors of the study.
N-protein is a structural component of the SARS-CoV-2 virus, the virus responsible for COVID-19. It performs several key jobs. Its most prominent role is to package the virus’s genetic material, known as RNA, so that it can fit inside each virus particle. This RNA is the blueprint the virus uses to replicate once it enters a person’s body.
Notably, the N protein forms liquid-like droplets called “condensates” inside infected cells, and these droplets are believed to help the virus multiply and evade the body’s immune defenses.
The formation of these droplets happens through a process called “liquid-liquid phase separation.” Akin to mixing oil and water, where oil naturally forms droplets within the water, the N protein can group together with the virus’s RNA inside infected cells forming droplets or condensates. These droplets are important for helping the virus complete its life cycle.
But while scientists have known that these droplets form, they don’t fully understand what causes the droplets to come together or break apart—or how the virus controls this process to its advantage.
“By learning how N-protein droplets are stabilized and destabilized, we can make predictions about how its behavior switches to initiate virus assembly at the right time,” said Laughlin. “If we can control the switch, we may be able to stop the virus.”
In their study, the researchers set out to better understand how the N protein forms these condensates, and what makes them dissolve. Rather than looking at a full, complex virus, they used a simplified system with just the N-protein and a small strand of DNA, which acts like the virus’s RNA in the experiment. The short DNA strand used in the experiment helped the scientists focus on how two N proteins interact with each other without distractions from more complex factors.
The researchers created a specialized tool called a FRET assay, which measures how molecules interact, to examine how these proteins come together into condensates.
By examining these interactions in a controlled environment, the team was able to see how the N protein behaves when it’s on its own, and how it changes when something similar to viral RNA is introduced.
Key discoveries that may help stop the virus
One of the most important findings was that the N protein can form small clusters, or groups, by itself. But when a strand of DNA (similar to RNA) is added to the mix, these small clusters come together to form larger three-dimensional droplets, or condensates. This process is most efficient when the amount of N protein and the amount of DNA are roughly equal.
The researchers discovered that the N protein can link up with other N proteins to form what scientists call “oligomers”—proteins that bind together to form a complex, or a grouping. Moreover, the researchers found, adding the small DNA strand helped these proteins assemble into a more organized, three-dimensional structure, like droplets.
In other words, the N protein and the genetic material it works with need to be balanced for the droplets to form properly. However, if there is too much of either one—too much N protein or too much RNA—the droplets break apart. This balance is delicate, and even a small shift can cause the process to fail.
This finding is significant because it suggests that the virus must maintain a precise balance of its own components to function properly. If the balance is disturbed, the virus may not be able to replicate. This could have important consequences for how the virus survives and spreads inside a person’s body.
The study also found that the concentration of the N protein plays a big role in whether or not these droplets form. The protein needs to be present in excess of a specific amount to trigger the formation of droplets; if there’s too little, the process breaks down.
These findings could help scientists develop new antiviral treatments aimed at disrupting the virus’s ability to form these droplets. If researchers can find a way to interfere with this process—by tipping the balance so the droplets can’t form, for example—they might be able to slow down or stop the virus’s replication.
This approach would target the virus at a very fundamental level, attacking the way it organizes itself inside the cell. By blocking this organization process, scientists may be able to prevent the virus from multiplying as effectively.
This research opens up new possibilities for antiviral drugs that could disrupt the virus’s ability to organize itself within cells. These drugs wouldn’t just stop the virus from replicating—they might also help the body’s immune system fight back more effectively by making the virus more vulnerable.
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