Volume 1, Issue 1
Hunting the Achilles' Heel of Hepatitis
Jennifer A. Doudna is a Howard Hughes Investigator and Professor of Biochemistry and Molecular Biology, and is affiliated with the California Institute for Quantitative Biomedical Research (QB3).
One way to disrupt a mechanical process is to throw a wrench into the works. This also holds true for viruses, biological parasites that hijack a cell's reproductive mechanisms to replicate themselves. The key though to successful sabotage is knowing precisely where to toss the wrench.
Jennifer A. Doudna, a UC Berkeley professor of Biochemistry and Molecular Biology, is aiding the hunt for this kind of Achilles' Heel in the Hepatitis virus. According to the Center for Disease Control, nearly 4 million Americans and 170 million people worldwide have been infected with Hepatitis C, one of the strains Doudna studies. The failure of available therapies results in 10,000 deaths every year just in this country.
"It's a very serious pathogen and we don't have any good drugs to treat it," says Doudna, whose insights into the virus's structure and reproductive mechanisms could lead to the development of new treatments for the disease.
Doudna's research is focused on ribonucleic acid (RNA), the molecule that carries the genetic blueprint copied from DNA. Inside the cell, ribosomes use the RNA code to assemble proteins, the building blocks of life. Normally, the RNA recruits the ribosomes by using molecular beacons that help bring the two together.
The problem, Doudna explains, is that "viruses have figured out a way to fly under that radar and recruit the ribosome directly" without any molecular signaling. Doudna's efforts to understand the phenomenon began several years ago while she was a professor at Yale University. In 2001, she and collaborators Jeffrey Kieft, a postdoctoral fellow and UC Berkeley alum, and Joachim Frank, a researcher at the Howard Hughes Medical Institute, produced groundbreaking visualizations of the Hepatitis C RNA molecule that recruits ribosomes during a viral infection.
Through cryo-electron microscopy, a low-temperature variant of electron microscopy, the researchers took a close look at a complex structure on one end of the virus's RNA. The researchers determined that the structure, called an internal ribosomal entry site (IRES), physically forces the ribosome to change its shape so that the RNA's protein template is in the perfect location to spur protein synthesis.
"The viral RNA literally clamps around the ribosome," says Doudna, who also holds a faculty position in the College of Chemistry.
The cryo-electron microscopy experiments provided the researchers with "a ten mile view" of the biochemical interactions, Doudna says. Currently, she and her Berkeley colleagues are zooming in even further on the hijacking process. Their tool is x-ray crystallography, a technique made possible by the fact that x-rays are diffracted by crystals. The researchers crystallize the RNA molecule and then bombard it with x-rays generated by the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. The resulting diffraction pattern is then used to reconstruct a three-dimensional image of the molecule.
"With that kind of high-resolution information, drug design becomes a very real possibility," says Doudna.
To that end, the laboratory participates in various scientific interactions with Isis Pharmaceuticals. Doudna hopes that the viral structural information uncovered in her laboratory could guide the design of new drugs to combat Hepatitis C.
In another research effort, Doudna and her colleagues are examining a rarer type of hepatitis virus, called Hepatitis Delta. Prevalent in southeast Asia, the virus only infects individuals who already have Hepatitis B.
Hepatitis Delta's genetic information is encoded in a circular piece of RNA. In this case, an RNA enzyme in an infected cell copies the RNA genome by a "rolling circle" replication mechanism, generating long strings of viral code. Then, in an amazingly efficient bit of biology, a catalytic RNA molecule called a ribozyme acts as a pair of "molecular scissors," chopping the just-duplicated viral genome into unit-length pieces for packaging.
This image, generated by cryo-electron microscopy, depicts the hepatitis C internal ribosome entry site (IRES) bound to a ribosomal subunit (yellow) of a rabbit cell. (visualization from Spahn, C.M.T, Kieft, J.S., Grassucci, R.A., Penczek, P., Zhou, K., Doudna, J.A., and Frank, J., 2001. Science 291:1959—1962)
"It's a neat way that the virus has figured out to use its genetic information to the max," Doudna says. "It has to have RNA to encode viral proteins, but it also uses the RNA itself to catalyze this essential cleavage reaction."
To visualize the nearly instantaneous cleaving process, the researchers are again turning to x-ray crystallography. First, Ailong Ke, a postdoctoral fellow in Doudna's lab, made subtle modifications in the RNA in order to halt the cleavage just before it occurs. Then, using the ALS's x-rays, the researchers grabbed a series of high-resolution snapshots of the molecule mid-cycle. What they observed is that the ribozyme undergoes a change in its molecular shape during the cleaving.
"From a basic science standpoint, we're interested in how enzymes have evolved in biology," Doudna says. "And now what we think is that this virus has evolved this particular ribozyme activity to place a very tight control over when the RNA is cleaved during the viral replication cycle."
As with Hepatitis C, understanding the structure of Hepatitis D's viral ribozyme and its machinations could reveal "a nice target for therapeutic intervention," Doudna says.
"In biology, a picture is worth more than a thousand words."