Study Reveals Structure Of DNA Packaging Motor In Virus

Scientists at Purdue University and the University of Minnesota have solved the three-dimensional structure of the central component of a biological "motor" that powers the DNA packaging system in a virus, providing scientists with their first glimpse of such a motor system.

The study describes atom-by-atom how the core of the tiny motor, just millionths of a millimeter in size, is constructed and suggests how it works to translocate, or pack, long stretches of the virus' genetic material into its outer shell during the process of viral replication.

Details appear in the Dec. 7 issue of the scientific journal Nature.

"This study provides the first knowledge of a DNA packaging motor," says Michael Rossmann, Hanley Distinguished Professor of Biological Sciences at Purdue. "Though other motor systems have been studied in biology, this is the first motor known to translocate genetic material."

Viruses are essentially a simple parasite consisting only of an envelope that contains the genetic material ready for transportation from one host to another. They can reproduce only after infecting a host cell. Once inside a cell, the virus manipulates the cell's machinery to produce all the necessary components, including genetic material, to assemble new viruses. It is here that the biological motor is needed to fill newly assembled envelopes with their genetic material, Rossmann says. The new viruses are then released from the host cell and are free to infect other cells.

The study may provide clues as to how DNA is packaged in similar viruses – including Herpesvirus, which causes human ailments such as Herpes simplex, chicken pox and shingles – and suggest ways for developing drugs that prevent illnesses caused by viral pathogens.

The study also will provide scientists with new insights on how molecular motors work in biology, says Dwight Anderson, professor at the University of Minnesota.

"The beauty of phi29 motor is that it provides a relatively simple system to investigate the mechanism of DNA packaging," Anderson says. "Working in a micro-droplet, or an area the size of a very small drop, the motor packages a DNA about 130 times longer than the viral shell, in just three minutes. The micro-droplet contains 50 billion DNA molecules that end-to-end constitute 200 miles of DNA, and each molecule gets packaged correctly into a protein shell."

In the study, Rossmann, along with Purdue researcher Timothy Baker and others at Purdue, and Anderson and his co-workers at Minnesota, used micro-imaging techniques – including X-ray crystallography and cryo-electron microscopy – to determine the structure of the DNA packaging motor in a virus called Bacteriophage phi29.

Bacteriophages are viruses that only infect bacteria. They are widely used in laboratory research because they are often similar in structure to human viruses. Phi29 infects a type of bacteria known as Bacillus subtilis.

The study shows that DNA packaging motor is comprised of three primary parts: an elongated prohead that serves as the virus shell, a doughnut-shaped connector that is positioned at the entrance to the virus shell and feeds DNA into the shell, and a novel ribonucleic acid (RNA)-enzyme complex that converts chemical energy to mechanical energy needed for packaging.

The scientists analyzed the structure of the connector – the core of the phi29 DNA packaging motor – to a resolution of 3.2 angstroms, or 3.2 hundred-millionths of a centimeter.

Their findings show that the connector is made up of 12 protein subunits that may serve as "cylinders" in the motor system to pull long chains of DNA through the center of the doughnut-shaped system.

Five identical enzymes, called ATPases, are positioned around the connector, just outside the opening in the virus shell. The enzymes break down the cell's chemical fuel, called ATP, to produce the energy needed to power the motor.

The researchers postulate that successive chemical reactions produced by the ATP cause the phi29 connector to oscillate and rotate, pulling the DNA into the shell two base pairs at a time.

"Our results suggest that the prohead and connector comprise a rotary motor, with the head and ATPase complex acting as a stator and the DNA acting as a spindle," Rossmann says.

Rotary-type motor systems are found in two other biological systems, he says, noting that such a motor is used to produce the rotation of flagella of E. coli. "The flagella rotate, and when they rotate synchronously, the bacterium can swim quite rapidly towards a source of food, or carbohydrates, along a concentration gradient."

The enzyme that manufactures ATP, called ATP synthase, also operates as a rotary motor to produce ATP or to pump protons.

The DNA-packaging motor appears to differ from these two known rotary motors, Anderson says, because its apparent spindle, the viral DNA, is translocated or moved to a new position.

"This motor system appears to be novel mechanistically," he says.

The scientists say they believe structural components of the motor and their concerted function can be dissected by further investigations.

The research was funded by the National Institutes of Health, the National Science Foundation and Purdue.

The research team at Purdue included Rossmann; Baker, professor of biological sciences; Alan Simpson, post-doctoral research assistant; Yizhi Tao, who received her doctorate at Purdue in 1999 and is now at Harvard University; Petr Leiman, doctoral student; Yongning He, doctoral student; Norman Olson, senior research electron microscopist; and Marc Morais, post-doctoral research assistant.

In addition to Anderson, the University of Minnesota team included research associates Mohammed Badasso and Shelley Grimes, and Paul Jardine, who was a postdoctoral fellow at the University of New Brunswick, Canada, and is now assistant professor at the University of Minnesota.