Contact: Ian Clark
MADISON, Wis. — Nearly 12,000 photographs and some sophisticated mathematical modeling have led a two-university research team to come up with a picture of a virus that infects millions of people worldwide and kills many in the Third World.
The advance marks a significant leap forward in understanding how the virus replicates – and how to stop it from doing so.
In a collaboration between the University of Wisconsin-Madison and Indiana University at Bloomington, researchers for the first time were able to peer inside the shell, or capsid, of the hepatitis B virus and visually identify structures important for the its gene replication machinery, according to the study published online by the Proceedings of the National Academy of Sciences.
Understanding the internal structures of a virus would help researchers understand mechanistically how the machinery of the virus reproduces its genetic material. That in turn may help researchers eventually to stop the virus in its tracks.
How hepatitis B works
The hepatitis B virus (HBV) is a blood-borne pathogen that causes liver cancer in the great majority of those who suffer from chronic HBV infections. It infects roughly 300 to 400 million people worldwide, primarily in Asia and sub-Saharan Africa.
“Universal vaccination for hepatitis B has worked great in Europe and the US because we have money,” said Dan Loeb, professor of oncology at the UW McArdle Laboratory for Cancer Research. “You have to get three doses of the vaccine — the initial vaccine and additional injections at one and six months — and that makes bringing health care to places like sub-Saharan Africa more difficult because you have to have that level of compliance.”
Among healthy adults who contract hepatitis B, about 95 percent will clear the infection and develop immunity. But for five percent of the population, the immune system only partially reacts and the virus isn’t cleared. Most infections, however, occur between a mother and a newborn. That newborn doesn’t have the immune system to combat the virus, and there is a higher likelihood the infection will become chronic.
Once infection occurs, HBV is actively replicating to maintain the infection, but it’s not the virus that is doing the host harm. In a way, it tries to coexist with the host cell in its preferred site of replication — the liver. Despite its good intentions, there is liver injury, but the damage is mediated by the host’s incomplete immune response. Eventually, after decades of damage, the chronically infected often develop liver cancer.
To maintain a chronic infection, the virus has to actively replicate through a process called reverse transcription, discovered in the 1970s at the McArdle Lab by Howard Temin. He discovered reverse transcriptase, the enzyme that assembles the viral DNA, and later went on to win the Nobel Prize for his discovery in 1975.
Breaking structure to get to function
But the challenge that the HBV particle presented was the capsid’s high degree of symmetry. The capsid had been visualized before, but posed a unique challenge in visualizing its asymmetrical insides.
The protective capsid layer of HBV is an icosahedral. Like a soccer ball or a geodesic dome, you can look at the structure from 60 different angles and see the exact same image.
“One of the big challenges has been breaking the symmetry of the virus to see the asymmetric features on the interior,” said Adam Zlotnick, professor of molecular and cellular biochemistry at IU. “Previously, the only way this had been done is when the virus had a huge external feature attached to it. Here we had no external features to work with, so it was extraordinarily challenging to orient the individual particles.”
Using cryo-electron microscopy, an unstained sample of HBV particles was encased in vitrified ice. Joseph Che-Yen Wang, first author of the paper, took almost 12,000 images of individual particles and reconstructed a single, 3-D image based on the mathematics. The exterior of the particle was symmetrical as expected, but when symmetry was imposed on the images, computationally laying images on top of each other based on their overall symmetry, anything that is asymmetrical was “averaged out.” Over 12,000 images, asymmetrical structures are lost because they are in different locations in different images.
“We had to develop mathematical techniques that allowed us to separate the asymmetrical features of the individual structures from the symmetrical features of the capsid. Joe Wang really led that effort” said Zlotnick.
Once they had a model that worked well, Loeb and Zlotnick saw that the outside was perfectly icosahedral, followed by another shell of asymmetrical mRNA, and inside that, the biggest structure identified was a ring that perfectly matched their expectations for polymerase, an important enzyme that assembles DNA. Reverse transcriptase is a polymerase specific to some viruses.
Imaging can help HBV researchers, virology field
“One of the things that is important about this structure is that the polymerase, which we’re visualizing here for the first time, is the target of all the first-line antivirals that fight hepatitis B,” said Zlotnick. “Those drugs attack that particular molecule, and no one has had the chance to see it before. No one’s had a view of how reverse transcription takes place in hepatitis B, and not having seen it puts huge constraints on understanding that process.”
There are therapies for hepatitis B that target the transcription process, but they’re lifelong, and sometimes resistance arises after prolonged medication.
“This paper demonstrates that it’s now possible to determine asymmetric features inside of a virus particle, and that could have far-reaching implications for a variety of virus studies,” said Loeb. “If we could learn these things about HIV, that would be very insightful. Being able to compliment the biochemistry and genetic studies with 3-dimensional images offers us a major leap forward in understanding how these viruses function.”
This was part of an ongoing collaboration between Loeb and Zlotnick, bringing biological know-how and technical imaging ability together to make visualizing the HBV possible. Joseph Che-Yen Wang, first author of the study, was instrumental to the study, said Zlotnick.
“All of this was possible because Dan and I have this great collaboration and we can bounce ideas off of each other at any point in time, and that allows the best science to happen,” he said.