New Clue to How Viruses Infect Cells
In a development that holds ramifications for gene therapy and infection-fighting drugs, a research collaboration led by Carlos Bustamante of Berkeley Lab's Physical Biosciences Division has discovered the mechanism by which at least some viruses infect the cells of other organisms with their DNA. The mechanism involves one of the most powerful biomolecular motors ever observed.
"This motor pulls with about 57 to 60 piconewtons of force, which scaled up to human dimensions would be enough to lift six aircraft carriers," says Bustamante, a biophysicist who holds a joint appointment with Berkeley Lab and UC Berkeley, and who is also a Howard Hughes Medical Institute investigator.
Biomolecular motors are proteins that undergo shape changes to generate force or torque. Acting like tiny engines, biomolecular motors come in a wide assortment and perform a broad range of tasks, many involving movement and transportation. One such task is the packing of the coiled lengths of DNA into the protective external shell or "capsid" of a number of viruses including those that cause herpes, chicken pox, and shingles. The biomolecular motor that Bustamante and his colleagues observed is the portal motor for the bacteriophage f29 (phi-29), a virus that infects and destroys soil bacteria and is considered an excellent model system for studying viral assembly.
"The portal motor for bacteriophage f29 compresses the DNA 6,000 times its normal volume," says Bustamante. "This generates an internal pressure of about 60 atmospheres, which is about ten times that in a champagne bottle."
Bustamante and his colleagues propose that just as the internal pressure in a champagne bottle will pop a champagne cork, so too does the even greater internal pressure inside the bacteriophage's capsid forcibly inject the viral DNA into an attacked cell. Viruses cannot "live" or reproduce without getting inside a living cell, whether it's a plant, animal, or a bacterium; in the case of f29, the bacteriophage attaches itself to and introduces its DNA into a soil bacterium, which, unlike the virus, can reproduce on its own. The viral DNA takes over the bacterium's reproductive programming and instructs it to reproduce copies of the virus instead. So many copies of the virus are replicated that the bacterium ultimately bursts open, unleashing a mass of new viruses ready to infect other bacteria.
"Understanding how this DNA packing process works could help us design better drugs to interfere with the packing part of the infection cycle of the virus and perhaps halt infection," Bustamante says. "It might also be used in gene therapy as a means of transporting new genetic material into cells."
The results of this research were reported in the October 18 issue of the journal Nature. Coauthoring the paper with Bustamante were Doug Smith, now with UC San Diego; Sander Tans, now at the Institute for Atomic and Molecular Physics in Amsterdam; UC Berkeley's Steven Smith; and Shelley Grimes and Dwight Anderson of the University of Minnesota.
"I would like to emphasize the close collaboration between my laboratory and that of Dwight Anderson that made possible this work," says Bustamante. "It was only because of the excellent complementary expertise of the two laboratories that this phase of the work was successfully completed."
To measure the strength of bacteriophage f29's portal motor, the research team used a unique force-measuring "optical tweezers" setup that was built in Bustamante's laboratory by Steve Smith. Working with capsids that were only partially packed with DNA before the packing process was stalled, the researchers tethered the unpacked end of the DNA and the capsid into which it was being packed between a pair of micron-sized polystyrene beads.
While the capsid-attached bead was held in place by a pipette, the DNA-attached bead was captured by the optical tweezers a laser beam that can be used to grasp and move the beads.
In the presence of adenosine triphosphate (ATP), the fuel that powers many biomolecular motors, the researchers were able to observe virus DNA packing activity in real-time and measure the force being applied by bacteriophage f-29's biomolecular motor. This enabled them to calculate the total amount of work involved, the total internal pressure on the DNA, and the amount of potential energy available for ejecting the DNA out of the capsid and into a bacterium during infection.
"The 57 to 60 piconewtons we calculated as the maximum pull exerted by this motor is an enormous force," Bustamante said. "The question is, then, what happens to all the work done on the DNA during packing? We claim the energy gets stored up inside the head of the bacteriophage and becomes available to initiate rapid injection of the DNA during the next infection phase."
Bustamante and his colleagues next want to answer some fundamental questions about bacteriophage f-29's portal motor, such as whether it is a new class of rotary biomolecular motor, one that can couple rotation to DNA translocation.
Says UCSD's Doug Smith, "The motor, consisting of a 10-nanometer diameter ring of RNA molecules sandwiched between two protein rings, is very intriguing and different from other motors that have been studied. We suspect that rotation of the rings may pull the double helical DNA through the portal similar to the way a rotating nut can pull on a bolt."
This work was funded by the Department of Energy, the National Institutes of Health, and the National Science Foundation.