Scientists investigate solutions for building cell membrane defense against COVID-19
Cell membranes are the outermost layer of a cell's defense against SARS-CoV-2, the new coronavirus that causes COVID-19 disease.
Cell membranes are only a few nanometers thick, but they are essential for life. They serve as a barrier between the interior of the cell and the surrounding environment, carrying many of the activities necessary for cellular function.
Researchers from Virginia Tech and the Department of Energy's (DOE) Oak Ridge National Laboratory (ORNL) are using neutron scattering to study how cell membranes and viruses interact and what therapeutic candidates can make cell membranes more resistant to viral entry. This information could help experts design strategies to slow the progression of viral infections and reduce their harmful effects.
"Developing therapies that interfere with the viral infection process could help reduce the severity of COVID-19 disease and allow people to recover more quickly," said John Katsaras, a biophysicist and neutron scattering scientist at ORNL. This, in turn, could reduce hospitalizations and reduce the risk of overwhelming medical facilities.
A membrane-centric mission
Coronaviruses hijack human cells with the help of spike proteins that protrude from their own membranes, giving the virus a coronary appearance. The spike proteins attach to the cell surface and help merge the viral and cell membranes. Once the membranes fuse, the virus can enter the cell and create copies of itself, spreading the infection throughout the body.
By determining how the coronavirus penetrates cell membranes, scientists can develop therapies that impede this process. Many researchers are exploring ways to fight the virus by targeting its spiking proteins, but less attention has been paid to the site where the infection process begins: the cell membrane.
"For a virus to be active, it has to cross the cell membrane," says Rana Ashkar, an assistant professor of physics at Virginia Tech and a former ORNL Shull Fellow, who is leading the research." Much of the work that has been done has focused on inactivating the spike proteins themselves. However, we want to understand how spike proteins interact with membranes and what therapies can indirectly block this interaction by targeting membrane properties."
Ashkar and her research team study the structure and dynamics of biofilm models to gain insight into cell membrane function. When COVID-19 began to spread, she used the resources of her lab to help the scientific community better understand coronavirus infection.
They collaborated with ORNL researchers to establish a molecular understanding of the properties of membranes that allow viral entry, how membranes change when in contact with the virus, and what membrane modifications can inhibit the infection process.
"ORNL is an international leader in the analysis of the nanostructure of biological membranes. Because of our expertise, network of collaborators, and neutron scattering capabilities, we were able to quickly shift our research focus to better understand how coronaviruses and certain other compounds affect membranes," said Katsaras.
This project also leverages the extensive expertise available in the lab." At ORNL, we were able to collaborate with experts in many fields, such as physics, chemistry and biology," said Jessy Labbé, a cellular and molecular fungal geneticist in ORNL's Biological Sciences Division." When the crisis began, we applied this combined knowledge to develop research projects to address some of the biggest challenges associated with epidemics."
Neutron Vision
The team is using ORNL's Liquid Reflectometer for Sputtered Neutron Sources (SNS) (LIQREF) to examine the conformation of membrane and viral spiking proteins, as well as the effects of certain therapeutic candidates. With this instrument, scientists can measure the trajectory of neutrons as they interact with different biological materials. They then use this information to determine how the samples are organized at the molecular level.
"Using this technique, we can capture the structure of the membrane and assess how the molecular details change under physiological conditions when the membrane comes in contact with spiking proteins or therapeutic compounds," said Minh Phan, a postdoctoral research associate at ORNL.
According to Ashkar, biological membranes have nanoscale profiles, which makes it challenging for researchers to investigate their structure and study their behavior. However, neutrons are able to probe biological materials with high resolution without damaging them. Neutron scattering is one of the few techniques to explore how viruses and therapeutic candidates interact with membranes at the nanoscale, she said.
Neutrons are also ideal for this study because they can distinguish between hydrogen and its isotopes, such as deuterium." By selectively replacing the hydrogen atoms in the membrane sample with deuterium atoms, we have this ability to change the way neutrons 'see' things so that we can amplify certain aspects and completely mask others," Ashkar said.
"When we change the levels of hydrogen and deuterium in the sample, we can pinpoint different features within the membrane," added John Ankner, ORNL instrument scientist." By compiling all these measurements, we can create a structural model, like putting together the pieces of a jigsaw puzzle."
Building a molecular map
The researchers conducted experiments with a membrane model that closely mirrors the shape and composition of cell membranes within the human lung, where respiratory viral infections predominantly occur. The samples were prepared by Mary Odom, a scientific associate at SNS. Using LIQREF, the team first characterized the original structure of the membrane. Then, they measured how the properties of the membrane changed when exposed to melatonin or azithromycin-a common product currently being studied by medical experts-as a possible treatment to alleviate COVID-19 symptoms.
Melatonin is a natural hormone produced by the brain that plays a role in regulating the sleep cycle. Synthetic melatonin supplements are primarily used to treat jet lag and sleep disorders, but have also been used to relieve symptoms of various infections. Azithromycin is an antibiotic prescribed to treat a variety of infections, such as bronchitis and pneumonia.
Ashkar is leading additional characterization experiments at Virginia Tech to further study how these products affect membrane tissue. Preliminary results suggest that when melatonin or azithromycin is introduced, the membranes pack more tightly together. This change has the potential to make membrane invasion more challenging for viral spike proteins.
The team will incorporate the viral spike proteins into membrane samples and analyze various factors of the protein-membrane complex. These factors include how the spike protein binds to the membrane, the mechanism of protein insertion, and the membrane's response to the protein, such as changes in membrane compression or stiffness. The team will then examine whether these interactions are disrupted in the presence of the therapeutic candidate.
The neutron scattering work will be performed primarily by Ankner and Phan. In addition, Jacob Kinnun, an ORNL postdoctoral research associate, conducted complementary experiments to explore how these two compounds affect the molecular packing and binding dynamics of membrane samples.
Pat Collier, a staff researcher at ORNL's Center for Nanophase Materials Science, studied membranes formed from lipid-coated water droplets to gain further information about membrane properties.
Going forward, the researchers envision that these methods could be used to build a platform for rapid and systematic screening of various therapeutic approaches for their potential to help mitigate current pandemics and other viral respiratory threats that may emerge in the future.
