The hepatitis C virus, or HCV, causes a chronic liver infection that can lead to permanent liver scarring and, in severe cases, cancer. It affects about 71 million people worldwide and causes about 400,000 deaths each year. While treatments are available for HCV-related infections, they are expensive, difficult to access, and do not protect against re-infection. A vaccine that can help prevent HCV infection is a major unmet medical and public health need.
A major reason there is no HCV vaccine yet is that scientists have yet to identify the correct antigen, otherwise the part of the virus would trigger a protective immune response in the body.
Decades of research have identified HCV E1E2, the only protein on the surface of the virus, as the most promising vaccine candidate. However, developing an HCV vaccine based on that protein is limited by uncertainty about what it looks like. Knowing the structure of the protein is necessary to figure out how the immune system responds to the virus.
So how do researchers pin down the structure of a single protein on a shape-shifting virus?
We are researchers specializing in microscopy and vaccine design. With new technology, we were able to visualize the molecular details of this elusive protein, unlock important insights into how this virus works and provide a potential blueprint for a future vaccine.
This is how we did it.
Challenges in catching a shape-shifting virus
One reason it has been so difficult to pin down the structure of the HCV E1E2 protein is that it is both flexible and fragile. It changes shape so often and is so easy to break that it is a challenge to clean it.
By way of analogy, imagine a bowl of spaghetti soaked in tomato sauce. Now imagine trying to take a picture of each individual piece of spaghetti in the same position over time as the bowl shakes. Hard to do, right? That’s how it was to image the entire E1E2 protein.
There were also technological barriers. Until recently, available imaging techniques were limited in their ability to view microscopic proteins. For example, X-ray crystallography is unable to capture molecules that frequently change and change shape, such as HCV. In addition, other options, such as nuclear magnetic resonance spectroscopy, required cutting large sections of the protein or chemically manipulating it in a way that would alter its physiological state and potentially alter its function.
So to investigate the structure of E1E2, we needed a way to extract and purify, stabilize and trap the entire shape-shifting protein in one configuration.
How to take a picture of protein
Cryo-EM, or cryo-electron microscopy, is a type of imaging technique that views samples at cryogenic temperatures, in this case the boiling point of nitrogen: minus 320.8 degrees Fahrenheit (minus 196 degrees Celsius). At such low temperatures, ice freezes so quickly that it doesn’t have time to crystallize. That creates a beautiful glassy frame around the protein in question, allowing every structural detail to be seen unobstructed. Cryo-EM also needs very few proteins to work, so we need less material to purify.
Cryo-EM, winner of the 2017 Nobel Prize in Chemistry and the 2015 “Method of the Year” award from the journal Nature, is excellent for imaging biological macromolecules in their native or natural state in the aqueous environment of human blood. Cryo-EM was also crucial in characterizing the structure of the COVID-19 virus and its variants.
So how do you take a picture of a protein?
First, we embedded the genetic code to make E1E2 in human cells in a petri dish so that we would have enough proteins to study. After purifying the protein, we immersed it in liquid ethane followed by liquid nitrogen. Liquid ethane is used to freeze the protein because it has a higher boiling point than liquid nitrogen. This means it can trap more heat before turning into a gas, freezing the protein much faster than in liquid nitrogen and preventing structural damage.
Once the protein was vitrified, or in a glassy ice state, we could not only see the overall structure, but also record multiple individual configurations of the protein that it assumes when it changes shape, including the less stable ones.
At this point, our protein was ready for its close-up. We used a microscope that uses a beam of focused, high-energy electrons and a very fancy camera that detects how the elections bounce off the surface of the protein. This created a 2D image that we then mathematically converted into a 3D model. And so we got the coveted ‘close-up’ of HCV’s surface protein.
Our next step then was to determine the location of each amino acid, or building block of the protein, in 3D space. Because each amino acid has a unique shape, we used a computer program that could identify each amino acid in our 3D map. This allowed us to manually reconstruct a high-resolution model of the protein, building block by building block.
A new tool to design an HCV vaccine
Our 3D map and model of the HCV E1E2 protein supports previous research describing its structure, while providing new insights into features paving the way for a long-sought vaccine design against this virus.
For example, our structure shows that the interface between the two main parts of the protein is stabilized by sugars and hydrophobic spots, or areas that push water molecules out. This creates sticky binding hubs along the protein and prevents it from falling apart – a potential site for protective antibodies and new drugs to target.
Researchers now have the resources to design antiviral drugs and vaccines against HCV infection.