Human norovirus, a positive-strand RNA virus that is the leading cause of viral gastroenteritis, causing approximately 685 million cases and approximately 212,000 deaths annually worldwide, has no approved vaccines or antivirals. Paving the way for better drug therapies, researchers at Baylor College of Medicine and the University of Texas MD Anderson Cancer Center report in Scientific advances the discovery of human norovirus replication centers, which could lead to the design of antiviral drugs to prevent, control or treat these infections.
“When viruses infect cells, they usually create specialized compartments (replication factories) where they form new viruses that infect more cells that cause the disease,” said first author Dr. Soni Kaundal, a postdoctoral associate in the Department of Biochemistry at Verna. and Marrs McLean. and Molecular Pharmacology at Baylor in the laboratory of Dr. BV Venkataram Prasad, corresponding author of the work. “However, little is known about norovirus replication factories.
There is growing evidence to show that some replication factories are not normally separated from their environment by a membrane. Instead, they are biomolecular condensates, structures that resemble a bubble formed by liquid-liquid phase separation. These condensates selectively incorporate proteins and other materials necessary for viral replication. Liquid condensates as replication factories have been widely studied in other viruses, including rabies and measles. In this study, the researchers investigated whether norovirus forms biomolecular condensates that serve as replication centers.
“We knew that these condensates are often initiated by a single viral protein capable of binding to genetic material, having a flexible region and forming oligomers, molecules made up of a small number of repeating units,” Kaundal said.
The team began their research by applying bioinformatics analysis to identify norovirus proteins that would exhibit the characteristics that would likely lead to the formation of liquid condensates.
“Working with the pandemic strain of human norovirus GII.4, the one responsible for causing the majority of cases of gastroenteritis worldwide, we found that RNA-dependent RNA polymerase has the highest propensity to form biomolecular condensates,” he said. Kaundal. “This protein has a flexible region, can form oligomers, binds to RNA, the genetic material of norovirus, and plays an essential role during viral replication by making copies of the viral RNA. All these characteristics led us to experimentally test whether RNA “GII.4 polymerase drives the formation of biomolecular condensates conducive to viral replication.”
“Our experimental studies show that RNA polymerase GII.4 forms highly dynamic liquid-like condensates under physiologically relevant conditions in the laboratory and that the flexible region of this protein is critical for this process,” said Prasad, professor of molecular virology. and microbiology. and Alvin Romansky Chair in Biochemistry at Baylor. Prasad is also a member of Baylor’s Dan L Duncan Comprehensive Cancer Center. “In addition, condensates are very dynamic structures: several can fuse forming a larger structure or can divide into smaller ones; they also move within the cell, exchanging materials with their environment.”
The researchers next investigated whether these liquid-like condensates also form in human intestinal cells infected with norovirus. Until recently, studying how norovirus replicates inside cells has been difficult because researchers lacked an effective biological system to grow the virus in the laboratory. But in 2016, Dr. Mary Estes’ lab at Baylor and her colleagues managed to grow strains of human norovirus in cultures of human intestinal enteroids.
Also known as mini-intestines, these cultures are a laboratory model of the human gastrointestinal tract that recapitulates its complexity, diversity, and cellular physiology. Human enteroids mimic the host virus infection patterns of a specific strain, making them an ideal system to dissect human norovirus infection, as in the current study, to identify strain-specific growth requirements and develop and test treatments and vaccines.
“We demonstrate that liquid-like condensates form in cultures of human intestinal enteroids infected with human norovirus, as well as in the human HEK293T cell line grown in the laboratory. We propose that these condensates are centers of human norovirus replication, a elegant solution to the “It is a puzzling question how the ribosome-assisted translation of the viral genome is separated from its replication by the viral polymerase into positive-strand RNA viruses,” Prasad said. “Our bioinformatics analysis also showed that the RNA polymerases of almost all norovirus strains have a high propensity to form these replication factories, suggesting that this may be a common phenomenon of most noroviruses.”
“This is a remarkable paper, and I was pleased that we were able to validate the findings in virus-infected cells using our human intestinal enteroid culture system for human norovirus,” said Estes, distinguished service professor and chair of molecular virology and microbiology at the Cullen Foundation at Baylor. Estes is also co-director of the Gastrointestinal Experimental Model Systems core at the Texas Medical Center Digestive Diseases Center and a member of the Baylor Dan L Duncan Comprehensive Cancer Center.
The findings not only provide new insights into human norovirus replication, but also open new targets for designing antivirals for human norovirus infections, which remain a serious threat in children and immunocompromised patients.
Other contributors to this work include Ramakrishnan Anish, B. Vijayalakshmi Ayyar, Sreejesh Shanker, Gundeep Kaur, Sue E. Crawford, Jeroen Pollet, and Fabio Stossi. The authors are affiliated with Baylor College of Medicine and the University of Texas MD Anderson Cancer Center.
Support for this project was provided by NIH grant P01 AI057788, Robert Welch Foundation grant Q1279, Center for Advanced Microscopy and Imaging 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.