Giant viruses that are difficult to study now have a new observation tool – HVEM

Despite their name giant viruses, it is difficult to observe them
in detail.
They are too large for conventional electron microscopes and too small for light microscopes used to study larger samples
.
Now, with the help of ultra-low temperature high-pressure electron microscopy, an international collaboration has revealed for the first time the structure of the Tokyo virus, a giant virus named after the city where it was discovered in 2016
.

They published their findings
in Scientific Reports on December 12 last year.

'Giant viruses' are viruses that are particularly physically sized, larger than small bacteria, and have genomes much larger than other viruses," said co-corresponding author Kazuyoshi Murata, a program professor
at the National Academy of Natural Sciences' Center for Exploration of Life and Living Systems (ExCELLS) and the National Institute of Physiological Sciences.
"Few studies have revealed in detail the capsids of macroicosahedral or 20hedral viruses – the protein shells
that envelop the DNA of double-stranded viruses.
Their size presents special challenges for high-resolution cryo-electron microscopy, which imposes severe limits
on data acquisition.
”

To overcome this challenge, the researchers used one of
the few high-pressure electron microscopy (HVEM) devices in the world for imaging biological specimens.
This type of electron microscope accelerates the voltage, theoretically increasing the power of the microscope, which allows thicker samples to be imaged
at higher resolution.
At Osaka University's UHV Electron Microscopy Research Center, the team imaged the instantaneous Tokyo virion, with the goal of reconstructing individual particles
completely for the first time.

Murata said: "Low-temperature HVEM on biological samples for single-particle analysis
has not been previously reported.
For thicker samples, such as the Tokyo virus, which has a maximum diameter of 250 nanometers, the effect of depth of field causes an internal focus shift, imposing a hard limit
on the achievable resolution.
Accelerating voltages, or shortening the wavelength of emitted electrons, can increase depth of field and improve optical conditions
in thick samples.
”

With these adjustments, the researchers performed detailed imaging of the Tokyo virus to elucidate the structure of
the entire viral particle.
They achieved a 3D reconstruction at a resolution of 7.
7 angstroms, which is only slightly
lower than the resolution that the technology can theoretically achieve.
Murata says the solution's results are limited
by the amount of data they can collect.

"Cryogenic HVEM currently requires manual collection of microscopic images taken by microscopes," Murata said
.
A micrograph is a photograph
taken with a microscope.
"We identified 1182 particles from 160 micrographs, which is a very small number
compared to other reports of giant viruses imaged with low-functioning microscopy.
"

According to Murata, lower magnification increases the number of particles contained in each photomicrograph, but magnification must be high enough to image the particles in detail
.
While automatic acquisition of micrographs (typically used in standard cryo-electron microscopy) promotes a significant increase in the number of images captured at high magnification, manual mode allows researchers to maintain a better particle count
per micrograph while maintaining a higher sampling frequency.

Murata said that even with a limited sample and slightly lower resolution, the researchers gathered enough information to understand the structure of
the giant virus particles more clearly than ever before.

"The frozen HVEM map reveals a new capsid protein network that includes a scaffold protein component network
," Murata said.
He noted that the connection between the apices of icosahedral particles in this scaffold network may determine the size of
the particles.
"icosahedral giant viruses, including Tokyo virus, have large, homogeneous functional cages consisting of limited components to protect the viral genome and infect host cells
.
We are beginning to understand how this works, including the advanced capabilities of the structure, and how we can apply that understanding
.
”

Murata said the researchers plan to implement automated acquisition software that will be able to maintain the parameters they need to image more giant viral structures and discover common structures to better understand how limited structures can be used for multifunctional organisms
.

Akane Chihara, Raymond N.
Burton-Smith, Naoko Kajimura, Kaoru Mitsuoka, Kenta Okamoto, Chihong Song, Kazuyoshi Murata.
A novel capsid protein network allows the characteristic internal membrane structure of Marseilleviridae giant viruses.
Scientific Reports, 2022; 12 (1)