Study reveals clues to how eastern equine encephalitis virus invades brain cells

An atomic-level investigation into how the eastern equine encephalitis virus binds to a key receptor and enters cells has also led to the discovery of a decoy molecule that protects against the potentially fatal brain infection in mice .

The study, led by researchers at Washington University School of Medicine in St. Louis, was published Jan. 3 in the journal Cell. By advancing the understanding of the complex molecular interactions between viral proteins and their receptors on animal cells, the findings lay the foundation for treatments and vaccines against viral infections.

“Understanding how viruses interact with the cells they infect is a critical part of preventing and treating viral diseases,” said co-senior author Michael S. Diamond, MD, Ph.D., Herbert S Professor .Gasser at the University of Washington.

“Once you understand that, you have the foundation to develop vaccines and drugs to block it. In this study, it took us a long time to understand the complexity associated with the particular receptor-virus interaction, but once we gained this knowledge, we were able to design a decoy molecule that was very effective in neutralizing the virus and protecting the mice from disease.”

Although eastern equine encephalitis virus infections in humans are rare – with only a few cases reported worldwide each year – about a third of those infected die, and many survivors suffer lasting neurological problems.

Additionally, scientists predict that as the planet warms and climate change lengthens the seasons and geographic range of mosquito populations, the risk of infection will increase. Currently, there is no approved vaccine against the virus or any specific medication to treat it.

As a first step in finding ways to treat or prevent the deadly virus, Diamond and his co-senior author Daved H. Fremont, Ph.D., professor of pathology and immunology, set out to study how the virus s attaches to one of its key elements. receptors – a molecule called VLDLR, or very low density lipoprotein receptor.

The molecule is found on the surface of cells in the brain and other parts of the body. Co-first author Lucas Adams, MD/Ph.D. student in the Fremont and Diamond laboratories, used cryo-electron microscopy to reconstruct the virus’s binding to the receptor in atomic-level detail.

The results turned out to be surprisingly complex. The molecule is made up of eight repeating segments, called domains, strung together like beads on a string. Usually, a viral protein and its receptor assemble in a very specific way. In this case, however, two or three different spots on the viral surface proteins were capable of attaching to one of the molecule’s five domains.

“What is really striking is that we find multiple binding sites, but the chemistry of each of the binding sites is very similar and also similar to the chemistry of the binding sites of other viruses that interact with related receptors ” said Fremont, who is also a professor of biochemistry & molecular biophysics and of molecular microbiology. “The chemistry works well for how viruses want to attach to cell membranes.”

The domains that make up this molecule are also found in several related cell surface proteins. Similar domains are found in proteins in the animal kingdom.

“Because they use a molecule that naturally has repetitive domains, some alphaviruses have evolved to use the same attachment strategy with several different domains in the same receptor,” said Diamond, who is also a professor of medicine, of molecular biology. microbiology, pathology and immunology.

Alphaviruses include eastern equine encephalitis virus and several other viruses that cause brain or joint diseases. “There are sequence differences in the VLDLR receptor during evolution in different species, but because the virus has this binding flexibility, it is capable of infecting a wide variety of species, including mosquitoes, birds , rodents and humans.”

To block attachment, the researchers created a panel of decoy receptors by combining subsets of all eight domains. The idea was that the virus would mistakenly bind to the decoy instead of the cells’ receptor, and the decoy the virus was attached to could then be eliminated by immune cells.

Co-first author Saravanan Raju, MD, PhD, a postdoctoral researcher in the Diamond lab, evaluated the panel of decoys. He first tested them on cells in dishes. Many have neutralized the virus. Then he turned to the mice.

Raju pretreated the mice with a lure or saline solution, as a control, six hours before injecting the virus under their skin, a mode of infection that mimics natural infection via a mosquito bite. Three lures were tested: one known to be incapable of neutralizing the virus; one made from the complete molecule; and one created from the first two domains only.

All mice given saline, the non-neutralizing lure, or the full lure died within eight days of infection. All mice given the lure made from the first two domains survived without signs of disease.

Certain aspects of its biology give the eastern equine encephalitis virus the potential to be used as a weapon, making it particularly important to find a way to protect against it. In a later experiment in which the mice were infected by inhalation – as would happen if the virus was pulverized and used as a bioweapon – the lure made from the first two domains was still effective, reducing the risk of death of the mice. mouse by 70%.

“Through a combination of structural work and domain deletion work, we were able to determine which domains are most critical and create a highly effective decoy receptor capable of neutralizing viral infection,” Fremont said. “This study expands our knowledge of virus-receptor interactions and could lead to new approaches to preventing viral infections.”