High-Voltage Electron Microscope Reveals Giant Virus Structure

An international research collaboration has revealed, for the first time, the structure of tokyovirus. Discovered in 2016, the “giant virus” has been difficult to visualize in detail because it is too big for conventional electron microscopy, but too small for optical microscopy used to study larger specimens.

The breakthrough—published in a recent issue of Scientific Reports—was enabled by cryo-high-voltage electron microscopy.

“Giant viruses” are exceptionally large physical size viruses, larger than small bacteria, with a much larger genome than other viruses,” says co-corresponding author Kazuyoshi Murata, project professor, Exploratory Research Center on Life and Living Systems (ExCELLS) and National Institute for Physiological Sciences, the National Institutes of Natural Sciences in Japan. “Few studies have revealed the capsid—the protein shell encapsulating the double-stranded viral DNA—structure of large icosahedral, or 20-sided, viruses in detail. They present special challenges for high-resolution cryo-electron microscopy from their size, which imposes hard limits on data acquisition.”

To overcome the challenge, the researchers used one of the few high-voltage electron microscopy (HVEM) facilities in the world that is equipped to image biological specimens. This type of electron microscope accelerates voltage to theoretically increase the power of the microscope, which allows for thicker samples to be imaged at higher resolutions.

At the Research Center for Ultra-High Voltage Electron Microscopy at Osaka University, the team imaged flash-frozen tokyovirus particles, with the goal of reconstructing a single particle in full detail for the first time.

“Cryo-HVEM on biological samples has not been previously reported for single particle analysis,” Murata says . “For thick samples, such as tokyovirus with a maximum diameter for 250 nanometers, the influence of the depth of field causes an internal focus shift, imposing a hard limit on attainable resolution. Accelerating the voltage, or shortening the wavelength of the emitted electrons, can increase the depth of field and improve the optical conditions in thick samples.”

Prepared with these adjustments, the researchers imaged tokyovirus in detail to clarify the structure of the full virus particle. They achieved a 3D reconstruction at a resolution of 7.7 angstroms, a resolution just a bit lower than the technology could theoretically attain. Murata said that the result of the resolution was limited by the amount of data they could collect.

“Cryo-HVEM currently requires the manual collection of micrographs taken with the microscope,” Murata says. “We identified 1,182 particles from 160 micrographs, which is an extremely small number compared to reports of other giant viruses imaged with less powerful microscopes.”

According to Murata, a lower magnification increases the number of particles included in each micrograph, but the magnification must be high enough to image the particles in detail. Automated acquisition of micrographs has facilitated a significant increase in the number of images captured at high magnification, but manual mode allowed researchers to maintain a better particle count per micrograph while also maintaining a higher sampling frequency.

Even with limited samples and slightly lower resolution, Murata said, the researchers gathered enough information to better understand the giant virus particles structure with more clarity than ever before.

“The cryo-HVEM map revealed a novel capsid protein network, which included a scaffold protein component network,” Murata said, noting that this scaffolding network’s connection between vertices in the icosahedral particle may determine the particle size. “Icosahedral giant viruses, including tokyovirus, have large, uniform sized functional cages created with limited components to protect the viral genome and infect the host cell. We are beginning to learn how this works, including the advanced functions of the structures and how we might be able to apply this understanding.”

The researchers plan to implement automated acquisition software capable of maintaining their desired parameters to image more giant virus structures and discover common architecture to better understand how the limited structures can be used for multifunctional organisms, Murata said.