New insight into how viruses form


Rebecca Pool

Tuesday, October 8, 2019 - 14:15
Overview of the bacteriophage MS2 viral capsid studied by  Professor Vinothan Manoharan and colleagues at Harvard. [Garmann et al]
Using interferometric scattering microscopy, researchers have imaged individual virus formation, offering a real-time view into the kinetics of viral assembly.
World-first images provide new insights into how an individual virus protein shell, or viral capsid, self-assembles around its RNA genome.
“Structural biology has been able to resolve the structure of viruses with amazing resolution, down to every atom in every protein,” highlights Professor Vinothan Manoharan from the Harvard John A. Paulson School of Engineering and Applied Sciences.
“But we still didn’t know how that structure assembles itself,” he adds. “Our technique gives the first window into how viruses assemble and reveals the kinetics and pathways in quantitative detail.”
Manoharan and his colleagues set out to study single-stranded RNA viruses, the most abundant type of virus on the planet and responsible for the common cold, West Nile fever, gastroenteritis and more. 
They attached MS2 viral RNA strands to substrates, such as the stems of flowers, flowed proteins over sample surfaces and used their laser interferometry method in wide-field mode to study individual capsids assembling around the RNA. 
The dark spots seen above are individual viruses. The spots grow darker as more and more proteins attach to the RNA strand. (Video courtesy of Manoharan lab) 
As the researchers point out, assembling particles appear as dark, diffraction-limited spots, with the intensity of each spot being approximately linearly proportional to the number of proteins bound to the RNA strand. 
By recording the intensities of these growing spots, they determined how many proteins were attaching to each RNA strand over time.
“One thing we noticed immediately is that the intensity of all the spots started low and then shot up to the intensity of a full virus,” says Manoharan. “That shooting up happened at different times.”
“Some capsids assembled in under a minute, some took two or three, and some took more than five,” he adds. “But once they started assembling, they didn’t backtrack. They grew and grew and then they were done.”
The researchers compared these observations to previous results from simulations, which predicted two types of assembly pathways.
In one type of pathway, the proteins first stick randomly to the RNA and then rearrange themselves into a capsid.
In the second, a critical mass of proteins, called a nucleus, must form before the capsid can grow.
The experimental results matched the second pathway and ruled out the first. The nucleus forms at different times for different viruses but once it does, the virus grows quickly and doesn’t stop until it reaches its right size. 
The researchers also noticed that the viruses tended to mis-assemble more often when there were more proteins flowing over the substrate. 
“Viruses that assemble in this way have to balance the formation of nuclei with the growth of the capsid. If nuclei form too quickly, complete capsids can’t grow,” says Manoharan. “That observation might give us some insights into how to derail the assembly of pathogenic viruses.”
How the individual proteins come together to form the nucleus is still an open question but now that experimentalists have identified the pathway, researchers can develop new models that explore assembly within that pathway.
Those models might also be useful for designing nanomaterials that assemble themselves.
Research is published in PNAS.
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