How the virus of COVID-19 covers its tracks and evades the immune system

The COVID-19 pandemic, caused by the SARS CoV-2 virus, continues to threaten populations
around the world after killing more than 1 million Americans.
In recent weeks, the XBB.
1.
5, the most transmissible variant to date, has begun to sweep the country
.

One aspect of what makes the novel coronavirus so contagious and difficult to control is its ability to defeat the body's innate immune defense system
.

A new study examined NendoU, a viral protein responsible for the virus's immune evasion strategy
.
The structure of this key protein is explored in detail, using a technique
called serial femtosecond X-ray crystallography.

This is the first time that the NendoU protein has been imaged to a high resolution
of 2.
5 angstroms at room temperature.
The resulting structure reveals the underlying details
of the protein's flexibility, dynamics, and other features with unprecedented clarity.
This structural information is critical in new drug design and may help advance treatments
for SARS CoV-2.

"Our research focuses on how COVID-19 uses the NendoU protein to hide in
the immune system.
As we better understand the structure and mechanism of NendoU, we have better ideas for
how to design antiviral drugs to fight it.
”

This discovery opens up the possibility of producing drugs that target protein conformational changes, as described in
the new study.
Such therapies will be particularly attractive because they are less prone to drug resistance
.

The Center for Applied Structural Discovery Biodesign has made significant progress in this type of structural research, addressing a variety of complex biological structures
.
The center is directed by Petra Fromme, who was the principal investigator of the study, which was funded
by a RAPID grant from the National Science Foundation and the BioXFEL Center for Science and Technology.

"This work is very exciting because it shows for the first time that differences in protein flexibility play an important role
in functional mechanisms," Fromme said.
"This is critical for the development of anti-NendoU drugs, potentially revealing the presence of the virus to the immune system, which can then respond and stop serious infections
.
"

The conspiracy of the virus

Viruses have evolved sophisticated strategies to evade the body's defense mechanisms
.
Studies point to the tactics used by some of the deadliest coronaviruses, a group of pathogens that include those that cause COVID-19 (SARS CoV-2), severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS
).

The new study explores how the NendoU protein helps SARS CoV-2 evade the immune system in plain sight
.
Once the virus binds to receptors on the cell's surface, it inserts its genetic material into the cell, causing the cell to make multiple copies of the viral genome, made up of
DNA or RNA from a coronavirus.

When viruses like SARS CoV-2 replicate inside cells, their growing RNA sequences produce a tail at the end, called a multi-U tail
.
This tail is unique
to RNA viruses.

Human cells are equipped with sensors that are fine-tuned to detect invading RNA viruses because the multi-tail exposes their identity as foreign invaders, enabling the immune system to target them
.
Studies have shown that SARS CoV-2 uses its NendoU protein to bind and then cut off the poly-U tail
.
When NendoU chews the polyu-tail, this leads to reduced
visibility of the virus to the immune system.

Master of disguise

To block NendoU's ability to hide the virus, researchers needed high-resolution images
of the protein's three-dimensional structure.
Until now, the structure of the NendoU protein could only be done at low temperatures, using a technique called cryo-EM, in which the sample being studied is snap-frozen and imaged
with electron microscopy or X-ray crystallography of large frozen crystals.
This provides important clues to the exact nature of NendoU, but more information
is needed before NendoU can be designed to suppress NendoU and expose the SARS CoV-2 virus to immune targeting.

To achieve this, researchers need to resolve the structure in such detail that they know the location of each atom in the protein, and ideally, the structure will be determined under natural conditions close to room temperature, where dynamics
can be detected.
However, the damage to electrons or X-rays is severe, so data collection is done in most cases under low temperature conditions, where all motion is frozen
.
To achieve such atomic-scale resolution at room temperature, a specialized X-ray device called XFEL (X-ray Free Electron Laser)
is required.

In the current study, the researchers obtained the first snapshot
of the atomic-scale structural pathway.
Known as sequential femtosecond crystallography, the technique involves crystallizing a protein sample into billions of tiny microcrystals and then passing them in jets at room temperature to extremely short bursts of intense X-ray light, producing a series of tens of thousands of diffraction patterns, each derived from a small microcrystal.

This ultrashort X-ray pulse lasts only tens of femtoseconds, exceeding the damage of X-rays to the crystal, allowing data
to be collected at room temperature close to physiological conditions.
To give an idea of how tight the timescale of these X-ray bursts is, 1 femtosecond equals one
quadrillion of a second.
Computers were used to combine large numbers of X-ray snapshots, allowing researchers to construct detailed 3D structures of proteins and examine their dynamic behavior
.

The current study was conducted using LCLS (Linear Accelerator Coherent Light Source), the only X-ray free-electron laser at SLAC in the United States, using macromolecular femtosecond crystallography instruments
.
The researchers used femtosecond X-ray crystallography to unlock the structure
of the NendoU protein as it binds to a substrate.
In living cells, this would be the multi-U-tail of the RNA strand, but in this study, a smaller molecule
called citrate was found at the RNA-binding site.

"It's exciting to be invited to do experiments at LCLS," said
Sabine Botha, co-corresponding author of the study and data analytics project leader.
"They have just been closed for a long time, reopening during the pandemic and calling for a SARS-CoV-2 proposal
.
It was a very challenging experiment that used a completely new X-ray detector, but it was also very beneficial
.
”

Put NendoU in the spotlight

One of the advantages of using XFELs for structural studies is that biological phenomena can be studied
close to their natural physiological state.
The current results show that the room temperature structure of NendoU protein is more flexible
than the low temperature structure.
This may be a more faithful representation
than the previously identified "frozen" structure.

"Like the previous structure, we also saw that NendoU formed a hexamer (six identical NendoU proteins bound together)," said
Debra Hansen, a co-author of the paper and an associate professor at the center.
In addition, the researchers found that half of this protein is more flexible
than the other.

The structural details revealed by XFEL light suggest that NendoU works
through a two-step process.
First, the rigid part of the protein binds
to the active site of the substrate (in this case, the citric acid molecule).
The flexible fraction of the hexamer also binds to citrate (or RNA), but is less tight
.
Once the rigid part has completed the task of cutting the RNA strand, it releases the RNA strand
.
This rigid half then becomes flexible, and the flexible half switches to a rigid state, and the cycle continues
.
This scissors-like movement of NendoU's two main components helps eliminate signals of the presence of virus within cells, rendering the immune response ineffective
.

XFEL snapshots of these movements provide a detailed map
of the final drug design.
Future structures using room temperature conditions will map these different movements, and each plot will allow for the most accurate anti-coronavirus drug calculation design
.