Lukas Flippo, Photo Editor

Yale researchers have figured out how to map the shape of the SARS-CoV-2 — another critical step in the scientific battle against the virus.

Researchers working under the mentorship of Sterling Professor of Molecular, Cellular and Developmental Biology and Professor of Chemistry Anna Marie Pyle used both experimental and computational methods to analyze the different structures present in the SARS-CoV-2 genome. Graduate student Rafael Tavares GRD ’21 led the computational front of the investigation and published the results in the Journal of Virology on Feb. 10. Meanwhile, graduate student Nicholas Huston GRD ’23 carried out the experimental side of the research, publishing his results in Molecular Cell on Jan. 1.

SARS-CoV-2 is a single-stranded RNA virus that causes COVID-19. While most recent studies of the virus have focused on its spike protein, which helps it infect cells and is important for vaccine development, Pyle’s team decided to focus on the shape of the RNA genome to get a better understanding of how the virus infects cells and replicates. Determining where the virus is most highly folded could help prevent false negatives in diagnostic testing and better inform treatment practices.

“It’s important not to think of the SARS-CoV-2 genome as a big string or tape that has the protein-coding information,” Pyle said. “The tape has a life of its own. It is organizing itself into stable structures that is impacting how the genome is read and how it copies itself.”

On the experimental side, led by Huston, researchers used biochemical and sequencing techniques to map the structures present in the viral genome. They analyzed the structural information of the virus while it was infecting monkey cells, rather than observing the virus isolated in a test tube.

For Huston’s team, the large size of the SARS-CoV-2 genome presented a challenge.

“SARS-CoV-2 has the largest genome out of all the single-stranded RNA viruses,” Huston said. “In the past, analyzing the genomes of coronaviruses was difficult because of their great size. However, we were able to use a special enzyme that is able to copy a lot of RNA into DNA in a single pass, something that many commercially available enzymes simply cannot do.”

Huston used a biochemical reaction to probe how flexible the RNA was at each given nucleotide. If that site in the genome was flexible, the biochemical reaction occurred at a faster rate, depositing molecules onto the RNA. When the “special enzyme” ran along the RNA to copy it into DNA, it would introduce a mutation at any site containing the molecules from the biochemical reactions. Thus, researchers could catalog sites of flexibility and rigidity in the genome by observing the mutation frequency at each site. Once they had this information, they were able to use it to guide existing computer algorithms that can predict RNA structure. 

Working at the same time as Huston and in collaboration with the experimental side of the research, Tavares developed a computational approach to identify what regions of the genome are more likely to form complex shapes.

“These shapes don’t happen at random,” Tavares said. “They are functional for the virus, sometimes critical for the virus to survive and replicate. If you know what regions are most likely to form complex shapes, you can do experiments to characterize the shapes, and then use them to develop therapeutics against COVID-19.”

Tavares and Huston found that the part of the genome that codes for proteins is highly folded and adopts many stable structures. Although it is not uncommon for RNA viral genomes to exhibit folding, the level to which the SARS-CoV-2 genome is folded was surprising to the researchers.

According to Pyle, information about how SARS-CoV-2 RNA folds can give scientists some insight into how the virus functions in people’s cells. For example, Pyle noted that the viral RNA is clearly folding tightly into complex structures at specific parts in the genome to avoid host detection.

“Somehow, the ability of the genome to become folded up is likely to be facilitating its ability to resist all the normal cellular mechanisms for detecting and responding to viral RNA,” Pyle said.

Pyle said this new knowledge of the SARS-CoV-2 genome structure may have major implications for how COVID-19 is tested for and treated.

For example, people around the world — including members of the Yale community — are being tested for COVID-19 using the RT-qPCR method. This technique involves converting RNA from a SARS-CoV-2 sample to its complementary DNA sequence in a process called reverse transcription, or RT, and then amplifying the DNA sequence for detection by a process called qPCR. A major part of the kit used in this testing process is the primers, small DNA pieces that are designed to recognize and bind to a specific sequence of the viral RNA. 

“We now know that the SARS-CoV-2 genome contains regions that are highly folded,” Pyle said. “If primers are designed to bind to these locations, they may not be able to interact that well, leading to false negatives. We need to be a little careful about where we design those primers for the test kit.”

In the future, Pyle and her team hope to identify the regions of folded genomic RNA that are the most vulnerable to attack — something Pyle called the “Achilles’ heel” of the virus. If found, these areas can then be disrupted with a number of different technologies to hopefully slow the disease’s progression.

According to the Centers for Disease Control and Prevention, as of Feb. 15 there have been more than 27 million cases of COVID-19 in the United States.

Veronica Lee | veronica.lee@yale.edu