Stunningly detailed blueprint of viral genome replication machinery revealed

RNA viruses, such as the coronavirus that causes COVID-19, are in a race to the death the moment they infect a cell.

These viruses have only a few minutes to establish their replication machinery inside host cells before the genetic instructions contained in their fragile RNA genomes — more fragile than DNA — would otherwise be destroyed by the cell’s housekeepers. If successful, the virus can progress from a few copies of its RNA genome to half a million copies contained in new infectious particles in less than 12 hours. Otherwise, the virus dies.

In a study published online Jan. 24 Proceedings of the National Academy of Sciences (PNAS), scientists at the Morgridge Institute have gained new insight into these critical early stages of viral infection and their control. Researchers have developed new methods to release viral RNA replication complexes from cells and visualize them in complex ways using cryo-electron microscopy (cryo-EM).

Combining highly advanced imaging with extensive computational analysis, cryo-EM enables scientists to visualize snap-frozen molecules in their native state at molecular-to-atomic resolution, providing revolutionary insights into biological structures that could be a key factor in developing therapeutics Ways to stop a strong foundation of disease.

The research team was led by Paul Alquist, director of the Institute’s John and Jeanello Center for Virology and UW-Madison professor of molecular virology and oncology. The team included scientists Hong Zhan, Nuruddin Unchwaniwala and Johan den Boon, Morgridge researcher and UW-Madison assistant professor of biochemistry Tim Grant, and co-authors Andrea Rebolledo-Viveros, Janice Pennington, Mark Horswill, Roma Broadberry and Jonathan Myers.

Most microbial and host genes function in large protein complexes that operate as molecular machines. However, the structure of these key components is largely unknown, greatly limiting the understanding and control of the associated processes. In 2017, using an advanced model virus, the Ahlquist lab provided the first complete imaging of the viral RNA replication complex and its remarkable organization.

They found that the parental viral genomic RNA “chromosome” is tightly coiled within a protective membrane vesicle, and they found that the necked channel of the vesicle to the cytoplasm is the site of the actual viral RNA replication machinery, the dynamic multifunctional engine of genome replication. point unknown 12-fold symmetric ring complex, which they named “Crown”.

Now, in its new paper Member of the National Academy of Sciences, the team took a further leap forward by revealing the complex structure of this molecular corona and its constituent enzymatic domains at atomic to near-atomic resolution. These dramatically increased resolution results show how the many different functional modules of this replication engine are arranged, providing the necessary basis for calculating methods for its assembly, dynamic operation, and interference with both.

For comparison, first author Hong Zhan said: “Our lab visualized the crown machinery for the first time in 2017, like determining the presence and general outline of the building. The new 2023 resolution is like showing fine details, Like wires and door locks.”

“In virology,” Ahlquist said, “the complexes that people have focused on so far are mainly infectious particles that move between cells and are relatively easy to purify and study because they are released from the cells.”

“However, most viral replication processes occur in the complex environment inside cells,” he adds. “This is a new chapter where we have been able to go deep inside the cell to capture and image in great detail the more complex viral machinery that executes the core events of viral replication.”

Team member Johan den Boon notes that, among other results, they discovered that “the crown is a giant viral RNA replication protein consisting of two stacked 12-mer rings, and that its multiple domains provide all that is needed to synthesize new copies of viral genomic RNA.” Function. However, proteins in the upper and lower loops have distinct conformations, with their constituent domains in different positions relative to each other.

One implication is that the same protein domain behaves differently in the upper and lower loops. Several other functions underscore that the crown is not a static structure, but a sophisticated, active machine that progresses and cycles through a series of movements to perform its continuous activity. Based on this structure and further targeted experiments, the Morgridge team is elucidating the functional and conformational gymnastics of the crown.

Another valuable finding of these studies is that the lower 12-mer loop is an assembled precursor that forms before the actual step of RNA replication. This “protocrown” precursor then recruits the viral genomic RNA template and other components to initiate the synthesis of new RNA and serves as the basis for assembly of the mature bicyclic replication complex.

Accumulating evidence suggests that the corona not only synthesizes new copies of the viral RNA genome, but also facilitates the delivery of these new genomes into downstream processes of gene expression and assembly of new infectious virus particles. The corona thus appears to provide the primary function of organizing many key stages throughout the infection process.

“Simply slowing down the assembly and function of the RNA replication complex was enough to kill these viruses,” Ahlquist said. “These new results provide a solid basis for finding new approaches.”

Ahlquist and other team members credit the UW-Madison Cryo-EM Research Center (CEMRC) and its leadership as critical to their progress. CEMRC is making this valuable technology available to many scientists at the UW-Madison campus and, as a national center, goes well beyond that. Led by Professor of Biochemistry Elizabeth Wright, CEMRC provides advanced capabilities for nearly all forms of cryo-EM imaging.

New results from Morgridge’s group and others suggest that the principles revealed by these studies are evolutionarily ancient, and that a similar corona complex is central to the replication of most, if not all, RNA viruses in this broad class. This includes the COVID-19 SARS-CoV-2 coronavirus and many other pathogens.

As a result, Ahlquist said, the conservative fundamentals could become the basis for developing more powerful, broad-spectrum antiviral strategies that can suppress not just one virus, but infection by entire groups of viruses.