Detailed Visualization of Viral Genome Replication Machinery Obtained

A new study sheds light on how positive-strand RNA viruses like coronavirus, alphaviruses, and flaviviruses rapidly establish their replication machinery inside the host cell before the genetic instructions contained in their vulnerable RNA genomes is destroyed by cellular housekeeping. The insights obtained might serve as the basis for developing more powerful, broad-spectrum antiviral strategies that could inhibit infection by not just one but whole groups of viruses. 

Morgridge Institute for Research scientists described their novel methods to release viral RNA replication complexes from cells and visualize them in sophisticated ways by cryo-electron microscopy (cryo-EM) in a paper published earlier this week in PNAS. 

The research team was led by Paul Ahlquist, director of the Institute’s John and Jeanne Rowe Center for Virology and University of Wisconsin-Madison professor of molecular virology and oncology. In 2017, using an advanced model virus, the Ahlquist lab provided the first full imaging of a viral RNA replication complex and its striking organization. They found the parental viral genomic RNA “chromosome” tightly coiled inside a protective membrane vesicle, whose necked channel to the cytoplasm they discovered to be the site of the actual viral RNA replication machinery—the dynamic, multifunctional engine of genome copying—in a previously unknown, 12-fold symmetric, ringed complex that they named the “crown.”

Now, in its new paper in PNAS, the team presents a further leap by revealing the intricate structure of this molecular crown and its component enzyme domains at atomic to near-atomic resolution.  These dramatically higher resolution results show how the many distinct functional modules of this replicative engine are arranged, providing an essential basis for working out its assembly, its dynamic operation, and ways to interfere with both. 

For comparison, according to Hong Zhan, first author of the paper, “The first visualizations of the crown machinery by our lab in 2017 were like identifying the existence and general outline of a building. The new 2023 resolution is like showing fine details, such as the electrical wiring and door locks.”  

“In virology,” Ahlquist says, “the complexes people have focused on to date mainly were the infectious particles that move between cells, which are relatively easy to purify and study because they release themselves from cells.”  

“However, most viral replication processes occur in the complex environment within cells,” he adds.  “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.”  

Team member Johan den Boon notes that, among other results, they find that “the crown is made of two stacked 12-mer rings of an enormous viral RNA replication protein, whose multiple domains provide all functions required to synthesize new copies of the viral genomic RNA.  However, 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.  Multiple 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 elucidating the crown’s functions and conformational gymnastics.  

Another valuable finding from these studies is that the lower 12-mer ring is an assembly precursor that forms prior to the actual steps of RNA replication.  This “proto-crown” precursor then recruits the viral genomic RNA template and other components to initiate synthesis of new RNAs, and 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.  The crown thus appears to provide 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.”