Cryo-EM Images Reveal Coronavirus Proofreading Enzyme in Atomic Detail

The closest-ever detailed look at a key enzyme inside the virus that causes COVID-19 could lead to more effective treatment of the disease.

Nucleotide analogs are a common type of antiviral medication that mimic the genetic material viruses use to replicate, essentially duping them into inserting faulty building blocks into new copies of the virus. Many nucleotide analogs don't work as well as expected against SARS-CoV-2, the virus that causes COVID-19, because coronaviruses carry an enzyme that identifies and removes genetic errors in its RNA – a "proofreader" called exoribonuclease (ExoN).

A research team led by Yang Yang, an assistant professor in the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology at Iowa State University, used images generated by a cryogenic electron microscope (cryo-EM) to observe the interplay between ExoN and RNA incorporated with antivirals such as remdesivir, sofosbuvir and bemnifosbuvir.

These atomic snapshots provide a lot of insight into exactly what functional groups are interacting and what kind of modifications could make these treatments more effective."

Yang Yang, Assistant Professor in the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University

Zooming In

Yang said the cryo-EM images underpinning the research – described in recent articles published in Nature Communications and the Proceedings of the National Academy of Sciences – achieved a resolution of 2.4 angstroms, a new record for ExoN. An angstrom is equal to one hundred-millionth of a centimeter. Most atoms are 1 to 3 angstroms wide.

The studies, which built on prior research in Yang's lab, relied on his new methods for optimizing cryo-EM. The powerful imaging technology creates 3D depictions at an atomic scale by flash-freezing biological samples, which creates a crystal-free thin ice slice analyzed with an electron beam. Scientists who work with cryo-EM are constantly refining their workflow to zoom in with greater clarity. 

"In terms of hardware, the microscope we used a few years ago compared with the microscope we use nowadays is basically the same," he said. "We've seen this significant bump in resolution because of some major changes we've made in how we prepare cryo-EM samples."

The images used in the research came from one of the three federally funded cryo-EM centers, which have the highest-powered instruments in the U.S. But much of the testing to perfect the protocols came from working with Iowa State's cryo-EM facility, Yang said.

"It takes a lot of trial and error," he said.

Closer Look Suggests Strategies

The new level of detail allowed Yang's team to map how nucleotide analog antivirals change the binding dynamics of the virus' RNA, making it more likely to detach from its replication enzyme and to attract the proofreading ExoN. Having a better understanding of the molecular mechanisms is crucial to overcoming that reciprocal trade-off, Yang said.

"It points us in a clear direction," he said.

One potential solution is modifying a nucleotide analog so the faulty RNA it generates can't be recognized by ExoN due to shape of the modified nucleotide being incompatible, he said. Another option would aim to increase the binding with ExoN, while at the same time reshaping the proofreading enzyme to lock it in an inactive form unable to perform its normal function.

Yang said his lab also is studying other types of commercially available nucleotide analog treatments, looking for signs of ExoN resistance. Finding an existing medicine more capable of withstanding proofreading is likely a quicker route to improving the arsenal of antiviral treatments for COVID-19.

"If we were to go down the road of designing an entirely new generation of nucleotide analogs, we would still have a lot to test," he said.