Coronavirus replication protein could be future treatment target

February 22, 2021
Nsp13 helicase (orange) unwinds RNA (blue), the same material as the SARS-CoV-2 genome. Recent findings show that mechanical forces (black arrows) increase nsp13 unwinding efficiency. (Rockefeller University/Keith Mickolajczyk)

Nsp13 helicase (orange) unwinds RNA (blue), the same material as the SARS-CoV-2 genome. Recent findings show that mechanical forces (black arrows) increase nsp13 unwinding efficiency. (Rockefeller University/Keith Mickolajczyk)

Scientists have developed a deeper understanding of the novel coronavirus’ RNA-unwinding helicase, which they identified as a possible target to stop COVID-19 infection because of its resistance to mutations and critical role in viral replication. Their study was one of 10 chosen by the Biophysical Society to be highlighted at its 65th annual meeting and will be presented on Tuesday.

Researchers at Rockefeller University in New York found that the helicase relies on other proteins to properly function and unwinds RNA very slowly when isolated. This contrasts with the helicases of other viruses such as the one that causes hepatitis C, which works at full speed and aggressively drives replication.

The researchers’ in-press paper was made available online in Biological Journal in December as part of the publication’s policy to immediately release COVID-19 research. The paper has been accepted for publication and peer reviewed, but may not be in its finalized version until it is formally published in the scientific journal on March 16. 

The novel coronavirus is made up of only about 29 proteins, and some of them are prone to mutations. Differences in the spike protein, for instance, may be the source of more contagious variants of the virus.

The helicase protein, named nsp13, is much more stable and rarely differs between individual viruses. It was also a 99.9% match with the helicase of SARS coronavirus with just one difference in amino acids, giving the researchers a place to start their investigation.

The Rockefeller researchers isolated nsp13 and measured the speed at which it separated base pairs of RNA and DNA, finding that it separated base pairs very slowly.

They also used optical tweezers — a very precise laser beam that can freeze or move microscopic particles — to “hold” RNA at both ends and pull it taut before adding the helicase. Nsp13 was found to work more than 50 times more effectively when this force was applied, suggesting that it works best in tandem with other proteins.

“What that means is that the protein, in its native state in cells, is most likely working together with partner proteins or gaining on mechanical stress provided by our own cells,” said Keith Mickolajczyk, a postdoctoral researcher at Rockefeller University and the study’s first author. “By itself, it's a very slow and inefficient enzyme that would most likely not be able to unwind the SARS-CoV-2 RNA genome successfully.”

The researchers speculated that nsp13 may receive help from the virus’ RNA-dependent RNA polymerase, which attaches to the helicase and begins the creation of new RNA strands, or takes advantage of proteins found in human cells.

They also found that nsp13 unwound DNA three times as quickly as RNA, despite residing in a virus whose genetic material is entirely composed of RNA. Mickolajczyk’s interpretation is that nsp13 acts very passively and waits for openings that allow it to unwind, and DNA strands are more likely to provide those chance opportunities because they are slightly less stable than RNA strands.

Nsp13 could be a target for future COVID-19 treatments because its structure varies so little between individual viruses, according to Mickolajczyk. The same is true between different coronavirus strains, such as those that cause COVID-19 and SARS, so the helicase could also be the focus of a treatment for future coronaviruses, as, "Coronaviruses probably aren’t going away,” he said.

“These viruses adapt and change and in doing so will elude our best defenses against them, so it's really important to really look at them and figure out which portions of them change the most and which portions of them change the least between these different strains and variants,” Mickolajczyk said. “I think we found a nice target in the nsp13.”

The postdoc, who will be presenting the findings at the Biophysical Society's annual meeting, said future tests could be conducted on the complex nsp13 forms with the RNA polymerase to test whether the proteins work together synergistically. 

The article, “Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase,” was released online Dec. 16 in Biophysical Journal. The authors of the study were Keith Mickolajczyk, Patrick Shelton, Michael Grasso, Sara Warrington, Amol Aher, Shixin Liu and Tarun Kapoor, Rockefeller University; and Xiaocong Cao, Rockefeller University and University of Science and Technology of China. The first author was Keith Mickolajczyk.

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