Viruses have thrived in humans for tens of thousands of years, evolving to take advantage of the machinery of cells to replicate and survive inside us. Some can slip past our defenses and invade without even causing symptoms.
Hepatitis A virus excels at this. This highly contagious virus, which can cause severe liver inflammation, has evolved to spread through close person-to-person contact or through contaminated food and water. Even though an excellent vaccine has been available since the 1990s, there have been 45,000 confirmed cases of hepatitis A, leading to 424 deaths in the United States, since 2016.
Scientists have long been trying to tease apart hepatitis A virus, to understand its inner workings and how it functions in the human body. Stanley M. Lemon, MD, a professor of medicine and microbiology and immunology and a member of the Institute for Global Health and Infectious Diseases at the UNC School Medicine, has been studying positive-strand RNA viruses such as the one that causes hepatitis A since the 1980's.
"You could say that hepatitis A virus knows the insides of a liver cell much better than we do," said Lemon, who is a member of the National Academy of Sciences and UNC Lineberger Comprehensive Cancer Center. "By studying hepatitis A, we have been slowly learning more about cellular responses, the immune system, and even some aspects of cancer that we wouldn't know otherwise."
A Newfound Infection Partner
Lemon's most recent research endeavor focused on how the virus strategically makes its way into liver cells and takes over the cell's inner machinery to replicate its genome.
Working together with colleagues Nat Moorman, PhD, You Li, PhD, Xian Chen, PhD, Jason Whitmire, PhD, and Joseph Alex Duncan, MD, PhD, at the UNC Lineberger Comprehensive Cancer Center and the UNC School of Medicine, Lemon has discovered that a little-known protein, PDGFA-associated protein 1 (PDAP1), is used as a pawn by hepatitis A virus to replicate and infect cells in the liver. Details of the new discovery were published in Science Advances.
The researchers identified PDAP1 through a CRIPSR screen targeting over 19,000 human genes. With assistance from the UNC Gene Therapy Center, Lemon and colleagues ruled out most of these genes as unimportant in hepatitis A infection. They were left with just over 40 genes (and the proteins they encode) that were required for the virus to replicate.
PDAP1 stood out among these genes as having no known function. Curious about what its role in hepatitis A infection might be, Lemon and colleagues dove into current literature, finding just a dozen previous research papers mentioning the protein. These studies suggested an association with gastrointestinal cancers and glioblastoma, but little was known about how the protein functions or how PDAP1 could partner with the virus to promote infection.
One paper, authored by Verónica Delgado-Benito at the Max Delbrück Center for Molecular Medicine in Berlin, Germany, showed that PDAP1 was crucial for upholding cellular stress responses in immune cells that make antibodies to fight infections and diseases. When Delgado-Benito removed PDAP1 from cells in the lab, the immune cells died of stress and were not able to produce their infection-fighting antibodies. This intrigued the research team.
Exploiting the Liver's Stress Response
The "integrated stress response" acts much like a generator in a hurricane, keeping the cell barely functioning until it can recover from the stressor and restore full functionality. Liver cells experience cellular stress when metabolism goes awry or when they become infected. In an effort to conserve their energy and survive, the liver cells shut down production of the majority of their proteins, shifting instead to producing a few special survival proteins to keep the cell alive.
Initial experiments by the research team suggested that hepatitis A virus had learned to leverage this special protein production program to manufacture its own proteins after infecting and inducing stress in liver cells. Lemon and colleagues decided to explore this mechanism further.
Often, researchers can learn a lot about cellular processes by removing components from cells to see what happens (or does not happen). To explore PDAP1's role in hepatitis A and the integrated stress response, the research team first "knocked out," or removed, PDAP1 from cultured liver cells using a molecular pair of "scissors" called CRISPR-Cas9. They also deleted the gene for PDAP1 in a mouse model of hepatitis A.
(Left) Liver cells infected with hepatitis A. The white arrows in show areas with dying liver cells surrounded by inflammatory cells generated by the immune response to the virus. (Right) Liver cells infected with hepatitis A and lacking PDAP1.
When exposed to the virus, the liver cells lacking PDAP1 were unable to support its replication. The mice were also completely resistant to hepatitis A infection. Without PDAP1, the virus was not able to take advantage of the protein's role in the stress response and could not produce its own proteins necessary for its own replication.
Linked to this discovery was a new understanding of the integrated stress response made by the researchers. When exposed to a toxin, mice lacking PDAP1 failed to generate the usual response to stress. The PDAP1-deficient liver cells switched on the genes responsible for the integrated stress response, but could not produce the proteins they encode. These results showed that PDAP1 plays a crucial but previously unknown role in the survival of stressed liver cells.
These two major findings led the researchers to conclude that PDAP1 is not only a pawn used by hepatitis A virus to replicate, but also a key player in maintaining the liver cell's ability to produce cellular proteins essential for survival under severe metabolic or infectious stress.
"We know now that this protein is part of a special program cells use to express a certain subset of proteins under conditions of stress," said Lemon. "Hepatitis A can usurp this program - this protein - to make its own proteins to replicate and successfully infect liver cells. We found that when we knock out PDAP1, the virus can no longer take advantage of this special program and cannot replicate or cause inflammation within the liver."
PDAP1, Infectious Diseases, and Cancer
With more known about PDAP1 and its role in the integrated stress response, Lemon expects that cell biologists who study protein production and cell survival under periods of excessive stress will pick up on the new findings and study PDAP1 more extensively.
Because the integrated stress response is a key regulator of the fate of cells, managing the balance between cell death and survival, it is often exploited by cancer cells to promote their proliferation. This most recent study shows that by studying how a virus works, researchers can learn more about cellular machinery relevant not only to infectious diseases, but also metabolic diseases and cancer.
