After more than a decade of research, Scripps Research Institute scientists have pieced together the structure of a human adenovirus—the largest complex ever determined at atomic resolution. The new findings about the virus, which causes respiratory, eye, and gastrointestinal infections, may lead to more effective gene therapy and to new antiviral drugs. The study was published in the journal Science on Aug. 27, 2010.
"We learned a number of important things about the virus from the structure, including how its key contacts are involved in its assembly," said Scripps Research professor Glen Nemerow, who, together with Scripps Research colleague associate professor Vijay Reddy, led the study. "That's very important if you want to reengineer the virus for gene therapy."
"Even though a number of viral structures have been solved by X-ray crystallography, this is the biggest to date," said Reddy. "The adenovirus is 150 megadaltons, which contains roughly 1 million amino acids—twice as big as PRD1, previously the largest virus ever solved to atomic resolution."
First discovered in the 1950s, adenovirus is a major class of disease-causing agents that include common cold viruses. While the body is usually able to eventually fight off infection, infants and people with compromised immune systems can be susceptible to severe complications. No medications are currently available against adenoviruses, so current treatment focuses on managing symptoms.
While adenovirus has plagued humankind for millennia, more recently scientists have sought to exploit some of its properties—such as its stability and ability to infect many different types of cells—to engineer cures for other diseases. The hope is that modified adenovirus could play a role in gene therapy, used as a vector (carrier) for delivering therapeutic genes to the interior of cells.
"Adenovirus was used early on in pioneering gene therapy trials for the treatment of cystic fibrosis," said Nemerow. "Those trials failed because scientists didn't understand the biology and virus-host cell interactions to be able to use the virus properly."
Despite early setbacks, adenovirus is still being used in about 25 percent of human gene therapy trials, mostly for cancer and cardiovascular disease, according to Nemerow. A better understanding of the virus could help advance those efforts, which, if successful, could have a major impact on a host of conditions.
So, in 1998 Nemerow and Reddy set out to determine the molecular structure of adenovirus—with no idea it would take them 12 years to succeed.
The scientists turned to a technique known as x-ray crystallography, the gold standard of molecular structure determination for large complexes. In this method, scientists produce large quantities of a protein or virus then turn it into crystal form. The crystal is then placed in front of a beam of x-rays, which diffract when they strike the atoms in the crystal. Based on the pattern of diffraction, scientists can reconstruct the shape of the original molecule.
Several steps in this process, however, can be problematic.
The first challenge is producing crystals. Other scientists' attempts at crystallizing adenovirus had failed due to long, spoke-like fibers that stick out of the vertices of the complex. These fibers interfere with crystallization, which requires close packing of the particles.
The Nemerow lab, however, produced an adenovirus lacking these long spindles, sparking hope that these particles could be crystallized. But the scientists found that the new virus was somewhat unstable. In another attempt, the Nemerow lab produced a version of the virus that had short fibers rather than long ones.
"This was a 'best of both' situation," said Reddy. "We had a stable virus and short fibers so we could pack the particles to produce crystals."
However, the production of crystals also required high concentrations of protein in solution. This turned out to be a problem because at high concentrations the adenovirus clumped together, again becoming unstable. After trial and error with various chemical additives and buffers, eventually Nemerow and Reddy arrived at conditions that kept the virus solublized.
The scientists were able to produce crystals at last. But when Nemerow and Reddy took the crystals to the Advanced Light Source synchrotron facility at Lawrence Berkeley National Laboratory, the crystals did not diffract. What the scientists needed was higher quality crystals.
Nemerow and Reddy persevered, continuing to vary the conditions under which the crystals were formed in an attempt to produce diffraction-quality crystals. At this point, Reddy and Nemerow had the idea of turning to robotic crystallization—at that time (around 2002) still somewhat of a novelty and a relatively expensive fee-for-service proposition.
"In the lab, we were limited by the dispensing of small volumes of sample," Reddy explained. "We could not pipette less than one microliter at a time. These robotic trials used very small—50 nanoliter—trials. With these small drops, we could explore a large number of conditions. Crystals also grow faster in these small drops."
After a series of robotic trials, the scientists were able to identify conditions that would produce high-quality crystals. They then reproduced these conditions in the lab and manufactured enough crystals to make another trip to the Berkeley Lab synchrotron. There, Nemerow and Reddy found that the crystals diffracted—but only to 10 angstroms, not enough to solve the structure.
Luckily, around that time (now 2005) a cutting-edge new synchrotron, the 23 ID-D beamline at Advanced Photon Source at Argonne National Laboratory, was coming online in Chicago. Reddy's mentor, Scripps Research professor Jack Johnson, suggested they give that facility a try.
