Directly imaging dynamic biomolecular processes can reveal secrets which scientists have been trying to uncover in indirect ways. The interaction between various virus species and the immune system is one of those topics that would benefit from novel visualization techniques. Now researchers from the Howard Hughes Medical Institute have imaged, with considerable detail, a rotavirus as it is grabbed by an immune system molecule. The technique may allow the development of better vaccines against not only rotavirus, but open a large range of research possibilities in the life sciences.

In the new experiments, Howard Hughes Medical Institute (HHMI) researchers have mapped the structure of an antiviral antibody clamped onto a protein called VP7 that stipples the surface of rotavirus. The structural map reveals intimate new details about how the antibody interferes with VP7, a protein that helps the virus infect cells. The information may be useful in designing a new generation of rotavirus vaccines that could be easier to store and administer than current vaccines, said the researchers.
Rotaviruses replicate mainly in the gut, where they infect cells in the small intestine. The virus has a triple-layered protein coat, which allows it to resist being chewed up by digestive enzymes or the gut’s acidic environment. Rotavirus does not have an envelope covering its protein shell. A virus’ envelope helps it enter host cells, and viruses without envelopes face significant hurdles in penetrating the membrane of the cells they infect. “Since they have no membrane of their own, they must therefore perforate a cellular membrane to gain access to the cytoplasm (the interior of the cell),” [HHMI investigator Stephen C. Harrison] said.
The new research shows that as rotavirus matures inside an infected cell, it assembles a kind of “armor” coating made principally of VP7 and a “spike” protein called VP4. When the mature virus particle exits one cell to infect a new cell, it perforates the endosomal membrane of the target cell by thrusting in its VP4 spike like a grappling hook.
The virus’ ability to infect cells depends on a critical structural change that quickly removes the coat from the interconnected VP7 proteins — an event that unleashes the spike protein. Although researchers still do not know precisely what triggers the uncoating of VP7, they do know that it appears to happen when the virus senses a lowered concentration of calcium in its environment.
“VP7 sort of closes over VP4 locking it in place like the metal grills that surround a tree planted on a city sidewalk,” explained Harrison. “And it is the loss of VP7 in the uncoating step that triggers VP4 to carry out its task.”
To get a closer look at how antibodies latch onto VP7 and neutralize the virus, Harrison and his colleagues used x-ray crystallography to examine the molecular architecture of VP7 in the grasp of a fragment of the antibody. X-ray crystallography is a powerful tool for “seeing” the orientation of atoms and the distances separating them within the molecules.
Before Harrison’s team could use x-ray crystallography, however, they first had to crystallize VP7 in complex with the antibody fragment. Only after that step was completed, could they move on to bombarding those crystallized proteins with x-rays. Computers helped capture the diffraction patterns that emerged as the x-rays scattered from the crystal lattice. By rotating the crystallized protein complexes through multiple exposures, the researchers could record enough data to calculate three-dimensional models, which exposed the underlying architecture of VP7 and the antibody fragment.
The resulting detailed structural map of the VP7-antibody protein complex revealed that the antibody neutralizes the virus by preventing the VP7 proteins from dissociating, said Harrison. “Normally, calcium creates a bridge between VP7 molecules that holds them in place until uncoating,” he said. “Our structure revealed that the antibody makes an additional bridge, cementing the subunits together, making the virus resistant to the uncoating trigger and preventing it from infecting cells.”
Current rotavirus vaccines consist of weakened live virus that triggers the immune system to produce neutralizing antibodies. However, the new structural findings suggest how researchers might engineer a different type of rotavirus vaccine consisting only of immune-triggering protein, said Harrison. This protein-only vaccine could be made of a chemically linked complex of VP7 molecules that would stimulate the immune system more vigorously to produce anti-rotavirus antibodies.
While live-virus-based vaccines have been effective, said Harrison, they have drawbacks that a protein-based vaccine might overcome. The virus-based vaccines are perishable and require refrigeration, but vaccines based on proteins are more stable and can be stored at room temperature. Another benefit, said Harrison, is that protein-based vaccines could be combined with other protein vaccines in a “cocktail” that would cut down on the number of clinic visits since blending cannot be done so readily with virus-based vaccines. These advantages could make protein vaccines especially useful in developing countries that lack an extensive public health infrastructure and where the vast majority of childhood deaths from rotavirus occur, Harrison said.

HHMI press release: New Images May Improve Vaccine Design for Deadly Rotavirus
Abstract in Science: Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

Studying protein folding is a tedious activity that thousands of researchers around the world are working on. Forcing proteins to undergo their unique folding sequence is a difficult process, but now a technique developed at the University of Illinois, that uses rapid pressure changes to induce folding, should help speed up the process.
From a statement issued by University of Illinois:

To induce protein folding, a sample contained in a sapphire cube covered by a small steel diaphragm is pressurized to several thousand atmospheres, causing the biomolecules to unfold. A powerful electrical current then bursts the diaphragm, which releases the pressure and produces a sub-microsecond pressure drop. The proteins re-fold, and are monitored through laser-excited fluorescence.
Gruebele’s electrical-bursting method also allowed for a miniaturization of the apparatus, which improved the speed and sample volume of the diaphragm design. That, in turn, allows for a better comparison between how proteins fold in vitro in the lab versus how a computer algorithm would predict how they would fold.
After the pressure is applied, the proteins were able to re-fold or “spring back” to their native-state structures “much more readily than if we had heated them and cooled them down,” Gruebele said.
Applying pressure to induce protein folding is not a novel laboratory technique. According to Gruebele, previous methods using electrically controlled valves, piezoelectric constriction and burst diaphragms weren’t fast enough or didn’t produce enough pressure to generate viable data on the microsecond timescale.
To reach the realm of simulation-worthy data, “you need hundreds of nanoseconds to a few microseconds worth of data-capture time,” Gruebele said. With the previous methods, “we weren’t close to the timeframe where you could perform computer simulations, right now or in the near future.”

Press release: Faster protein folding achieved through nanosecond pressure jump