RNA viruses, such as the coronavirus that causes COVID-19, jump into a life-and-death race the moment they infect a cell.

These viruses have only minutes to establish their replication machinery inside the host cell before the genetic instructions contained in their vulnerable RNA genomes — which are even more fragile than DNA — are destroyed by cellular housekeeping. If they win the race, the viruses can multiply rapidly, going from just a few copies of their RNA genome to a half-million copies incorporated into new infectious particles in less than 12 hours. If they lose, they die.

A UW research team has shed new light on these crucial early stages of virus infection and control. The group, housed at the Morgridge Institute for Research and led by a CALS scientist, has developed new ways to release viral RNA replication complexes from cells and visualize them in sophisticated ways through cryo-electron microscopy (cryo-EM).

A Public-Private Partnership

The Morgridge Institute for Research is a private, nonprofit organization dedicated to improving human health through interdisciplinary biomedical research. It’s housed in UW’s Discovery Building, along with the public Wisconsin Institute for Discovery and the first-floor community space called the Town Center, managed by the Wisconsin Alumni Research Foundation. The Morgridge Institute uses mechanisms unique to a private entity to help recruit top scientific talent and build powerful research collaborations. Many faculty from CALS and across the UW campus serve as Morgridge investigators and affiliates and leverage the institute’s resources to advance their research.

Cryo-EM combines highly advanced imaging with extensive computational analysis to allow scientists to visualize flash-frozen molecules in their native state at molecular to atomic resolution (see A Cold, Hard Look at Macromolecules, Spring 2021). The technology provides revolutionary insights into biological structure, which can be a powerful foundation for developing therapeutics to thwart disease.

Most microbe and host genes function in large protein complexes that operate as molecular machines. The structures of these critical assemblies, however, have largely been unknown, greatly limiting understanding and control of the relevant processes. In 2017, using cryo-EM tomography and an advanced model virus, a research team led by professor Paul Ahlquist provided the first full imaging of a viral RNA replication complex and its striking organization.

The team found the parental viral genomic RNA “chromosome” tightly coiled inside a protective sac in the cell membrane. This vesicle includes a narrow channel leading to the cytoplasm, the dense mixture of diverse proteins and other molecules that fills the inside of a cell. Atop this channel, facing the cytoplasm, they discovered the site of the viral RNA replication machinery — the dynamic, multifunctional engine of genome copying. They also found that this machinery is organized in a previously unknown 12-fold, symmetric ringed complex that they named the “crown.”

Earlier this year, in a paper published in the Proceedings of the National Academy of Sciences, the team presented a further leap by revealing the intricate structure of this molecular crown, including enzyme domains that serve multiple functions, at atomic to near-atomic resolution. These dramatically higher resolution results show how the many distinct parts of this replicative engine are arranged. They also provide an essential basis for working out its assembly and dynamic operation, as well as ways to interfere with both — all of which could lead to better ways to fight viral infection.

“The first visualizations of the crown machinery by our lab in 2017 were like identifying the existence and general outline of a building,” says Hong Zhan, assistant scientist at the Morgridge Institute and first author on the study. “The new 2023 resolution is like showing fine details, such as the electrical wiring and the interior mechanisms of the door locks.”

“In virology, the complexes people have focused on to date mainly are the infectious particles that move between cells, which are relatively easy to purify and study because they release themselves from cells,” says Ahlquist, who is professor of plant pathology, molecular virology, and oncology and director of the Morgridge Institute’s John and Jeanne Rowe Center for Virology. “However, most viral replication processes occur in the complex environment within cells. This is a new chapter, where we’ve been able to reach inside cells to capture and image, in great detail, even more intricate viral machinery that carries out the central events of viral replication.”

The study found that the crown is made of two stacked rings, each containing 12 copies of an enormous viral RNA replication protein whose multiple domains provide all functions required to synthesize new copies of the viral genomic RNA. “However,” says team member Johan den Boon, “the proteins in the upper and lower rings are in dramatically different conformations, with their constituent domains in different positions relative to each other.”

One implication is that the same protein domains operate in distinct ways in the upper and lower rings. Several other features underscore that the crown is not a static structure but a sophisticated, active machine that progresses and cycles through a series of movements to carry out its successive activities. Based on this structure and further targeted experiments, the Morgridge team is clarifying the crown’s functions and conformational gymnastics.

Another valuable finding from these studies is that the lower ring is an assembly precursor, meaning it forms prior to the actual steps of RNA replication. This “proto-crown” then recruits the viral genomic RNA template and other components to initiate synthesis of new RNAs, and it serves as a base to assemble the mature, double-ring replication complex.

Growing evidence suggests that the crown not only synthesizes new copies of the viral RNA genome but also helps deliver these new genomes into downstream processes of gene expression and assembly of new infectious viral particles. Based on these findings, the crown appears to perform major functions for organizing many critical phases throughout infection.

“Just slowing down the assembly and function of RNA replication complexes is enough to kill these viruses,” Ahlquist says. “These new results provide a strong basis for finding new ways to do that.”

Ahlquist and other team members praise the UW–Madison Cryo-EM Research Center (CEMRC) and its leadership as crucial to their progress. The CEMRC was launched in 2020 with funding from a $22.7 million grant from the National Institutes of Health and an initial $15 million-plus investment from several campus units: the Department of Biochemistry and College of Agricultural and Life Sciences, the Morgridge Institute for Research, the Office of the Vice Chancellor for Research and Graduate Education, the School of Medicine and Public Health, and the Departments of Biomolecular Chemistry and Neuroscience. These units continue to partner on the center’s efforts to make the valuable technology accessible to scores of scientists across campus, the nation, and beyond. Led by biochemistry professor Elizabeth Wright, CEMRC provides advanced capabilities in essentially all forms of cryo-EM imaging.

Emerging results from the Morgridge group and other researchers indicate that the principles revealed by these studies are evolutionarily ancient. New evidence also shows that similar crown-like complexes are central to the replication of most, if not all, RNA viruses in this large class. This includes SARS-CoV-2 (the coronavirus that causes COVID-19) and many other pathogens.

According to Ahlquist, what his team and others have discovered might serve as the basis for developing more powerful, broad-spectrum antiviral strategies. These strategies could inhibit infection by not just one but whole groups of viruses.

Meet the Research Team

At CALS and the Morgridge Institute

• Paul Ahlquist, professor of plant pathology, molecular virology, and oncology; director of the John and Jeanne Rowe Center for Virology
• Tim Grant, assistant professor of biochemistry and Morgridge investigator

At the Morgridge Institute

• Hong Zhan, assistant scientist
• Nuruddin Unchwaniwala, former assistant scientist (now a researcher at Assembly Biosciences)
• Johan den Boon, associate scientist
• Janice Pennington, former electron microscopy specialist (now retired)
• Andrea Rebolledo-Viveros BS’16, former specialist (now a graduate student, Tufts University)
• Mark Horswill, senior research specialist
• Roma Broadberry, graduate student, biophysics
• Jonathan Myers, former assistant research specialist