High-resolution images constructed by researchers at Children's Hospital Boston and Harvard Medical School reveal the molecular rearrangements that rotavirus – the most common cause of severe, dehydrating diarrhea and vomiting in children worldwide – uses to break into cells.
The work is a major advance in the understanding of how viruses cause infection, and illustrates how vaccine development can be made "smarter" by probing the physical architecture of viruses and finding the minimum parts needed to prime the immune system, without having to use a whole virus to make a vaccine. The researchers are now collaborating with several other institutions to develop a vaccine based on their discoveries.
Rotavirus infects almost all children, usually between 6 months and 2 years of age, and causes gastroenteritis that is sometimes severe enough to require hospitalization. The virus kills about 440,000 children each year, mainly in developing countries. The only licensed vaccine, RotaShield, was pulled from the U.S. market in 1999 because of reported cases of intestinal intussusception (a condition causing bowel obstruction).
Led by Dr. Philip Dormitzer, a physician and structural virologist in CHB's Laboratory of Molecular Medicine, the researchers used crystallography and electron microscopy to determine the geometric structure and working parts of one of the virus's surface proteins, called VP4.
Rotavirus itself is a large, soccer ball-shaped, 20-sided particle with three layers.
"The outside layer is like a landing apparatus and is stripped off in the course of entry," explains Dormitzer, who also is an assistant professor of Pediatrics at HMS and is affiliated with HMS's Center for Molecular and Cellular Dynamics. "Its job is to get the innermost portions – the genes and the replication machinery -- inside the cell."
From the outer layer project 60 "spikes," each consisting of a cluster of VP4 molecules. Dormitzer and colleagues trimmed the VP4 protein down to two rocklike sub-components that make up the spike's "head" and "body." They crystallized these pieces and used X-ray diffraction to determine their three-dimensional structures, precise down to the atom. Comparison of the crystal structures to electron microscopy images, obtained by colleagues at the Baylor College of Medicine, suggests that VP4 undergoes two consecutive shape changes that allow it to breach the membrane of the cell it's trying to infect.
"This protein goes through some extraordinary gymnastics that are almost certainly related to its function," says Dormitzer.
First, when rotavirus arrives in the intestine, digestive enzymes cause two of the three VP4 molecules in each cluster to form a rigid spike, priming the virus to attack the cell and positioning the spike "head" to bind to the surface of the target cell. Then, in a second rearrangement, the spikes fold back, and VP4 takes on a folded-umbrella structure with three "panels." Dormitzer and colleagues speculate that this folding motion causes the spike's "body" component to break a hole in the cell membrane, allowing the virus to enter.
The key finding for vaccine development is that the "head" and "body" portions of the VP4 protein contain many of the targets that the immune system recognizes when it attacks the virus and protects against infection.
"The work is a clear example of the way in which structural studies can contribute to new good ideas about strategies for vaccines," says Senior Investigator Stephen Harrison, Ph.D., a Howard Hughes Medical Institute investigator and chief of CHB's Laboratory of Molecular Medicine.
Vaccines made from live viruses (such as RotaShield) or killed viruses can present safety concerns and be unstable without refrigeration. In contrast, the "head" of the VP4 protein is very stable at room temperature and easy and relatively cheap to produce, Dormitzer says; he believes the same is true of the "body." A vaccine based on these proteins could be very practical, especially for developing countries where rotavirus causes the most serious illness.
The team's findings may also shed light on how other so-called "non-enveloped" viruses--which lack a fatty outer membrane – enter cells. These viruses include papillomavirus, adenovirus, and rhinovirus. Entry mechanisms for viruses with outer envelopes are much better understood, says Dormitzer.