Researchers have made important discoveries about human norovirus, which is the primary cause of viral gastroenteritis. This virus leads to an estimated 685 million infections and approximately 212,000 deaths each year worldwide. Their findings could pave the way for developing antiviral medications to prevent, manage, or treat these serious illnesses.
Human norovirus, a positive-strand RNA virus, is the main culprit behind viral gastroenteritis, accounting for about 685 million infections and around 212,000 deaths globally each year. Currently, there are no authorized vaccines or antiviral treatments available. To enhance potential drug therapies, researchers from Baylor College of Medicine and the University of Texas, MD Anderson Cancer Center have identified replication hubs for the human norovirus, as reported in Science Advances. These discoveries may assist in creating antiviral medications aimed at preventing, controlling, or curing these infections.
“When viruses attack cells, they typically create specialized areas known as replication factories, where new viruses are produced, leading to further spread of the disease,” commented Dr. Soni Kaundal, the lead author and a postdoctoral associate in the Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology at Baylor, working under Dr. B.V. Venkataram Prasad, the study’s corresponding author. “However, our understanding of norovirus’s replication factories is limited.”
Recent evidence indicates that these replication factories are often not surrounded by a membrane. Instead, they consist of biomolecular condensates—structures resembling bubbles created by liquid-liquid phase separation. These condensates selectively gather proteins and materials essential for viral replication. Similar liquid-like condensates have been extensively studied in other viruses, such as rabies and measles. Our research aimed to determine if norovirus produces biomolecular condensates that function as replication hubs.
“We recognized that such condensates typically originate from a viral protein that can bind with genetic material, has a flexible segment, and can form oligomers, which are molecules composed of repeating units,” Kaundal explained.
The research team began by using bioinformatic analysis to identify norovirus proteins with qualities that would likely lead to the formation of liquid condensates.
“In our work with the GII.4 strain of human norovirus, which is responsible for most gastroenteritis cases globally, we discovered that the RNA-dependent RNA polymerase has the greatest likelihood of forming biomolecular condensates,” Kaundal shared. “This protein possesses a flexible segment, can create oligomers, binds to RNA (the genetic material of norovirus), and plays a vital role in viral replication by making copies of viral RNA. All these features motivated us to experimentally confirm whether the GII.4 RNA polymerase promotes the generation of biomolecular condensates favorable for viral replication.”
“Our experiments show that GII.4 RNA polymerase indeed forms highly dynamic liquid-like condensates under physiologically relevant conditions in the lab, and the flexible segment of this protein is crucial for this process,” noted Prasad, a professor of molecular virology and microbiology. He also holds the Alvin Romansky Chair in Biochemistry at Baylor. “Moreover, these condensates are very dynamic: they can merge to create larger structures or split into smaller ones, and they move within the cell, exchanging materials with their environment.”
The research team then evaluated whether these liquid-like condensates also exist in human intestinal cells infected with norovirus. For many years, studying norovirus replication within cells has been challenging due to the absence of an effective biological system for growing the virus in laboratory conditions. However, in 2016, Dr. Mary Estes’s lab at Baylor successfully cultivated strains of human norovirus in human intestinal enteroid cultures.
These mini-guts—laboratory models of the human gastrointestinal tract—replicate its cellular complexity, diversity, and function. They mimic strain-specific host-virus interactions, making them an optimal system to investigate human norovirus infection, as seen in this study, to identify specific growth requirements for different strains and to develop and test potential treatments and vaccines.
“We demonstrated that liquid-like condensates are produced in both human norovirus-infected human intestinal enteroid cultures and in the HEK293T human cell line grown in the lab. We propose that these condensates act as replication hubs for norovirus, providing an elegant solution to how ribosome-assisted translation of the viral genome is kept separate from replication by the viral polymerase in positive-strand RNA viruses,” Prasad stated. “Our bioinformatics assessment also revealed that almost all norovirus strain RNA polymerases have a high tendency to create these replication factories, suggesting this could be a widespread phenomenon among noroviruses.”
“This is an impressive study, and I’m thrilled we could confirm the findings in virus-infected cells using our human intestinal enteroid cultivation setup for norovirus,” said Estes, Distinguished Service Professor and Cullen Foundation Endowed Chair of Molecular Virology and Microbiology at Baylor. She also co-directs the Gastrointestinal Experimental Model Systems core at the Texas Medical Center Digestive Diseases Center and is part of Baylor’s Dan L Duncan Comprehensive Cancer Center.
The discoveries not only provide fresh insights into how human norovirus replicates but also unveil potential new targets for developing antiviral treatments against human norovirus infections, which continue to pose significant risks, particularly for children and immunocompromised individuals.
Other contributors to this study include Ramakrishnan Anish, B. Vijayalakshmi Ayyar, Sreejesh Shanker, Gundeep Kaur, Sue E. Crawford, Jeroen Pollet, and Fabio Stossi. The authors are associated with Baylor College of Medicine and the University of Texas, MD Anderson Cancer Center.
Funding for this project came from NIH grant P01 AI057788, Robert Welch Foundation grant Q1279, the Center for Advanced Microscopy and Image Informatics (Cancer Prevention and Research Institute of Texas (CPRIT) grant RP170719), the Integrated Microscopy Core at Baylor College of Medicine (NIH grants: DK56338, CA125123, ES030285, and S10OD030414), and CPRIT grant RR160029.
