A human gene named ATR normally protects people by preventing the replication of cells damaged by radiation or toxic chemicals. Now, Utah and New York researchers have discovered how a gene in the AIDS virus hijacks the human gene and turns it into a weapon that prevents reproduction of immune-system white blood cells, leaving AIDS patients vulnerable to deadly infections and cancer.
The new study "puts us a big step closer to understanding how HIV [human immunodeficiency virus] dismantles the immune system," says molecular biologist Vicente Planelles, an associate professor of pathology at the Ï㽶ÊÓƵ of Utah School of Medicine.
It also raises the prospect for new kinds of treatments for AIDS and cancer.
Researchers already knew that an HIV gene named vpr led to the depletion of immune-system white blood cells named CD4+ lymphocytes. The new study suggests vpr does that by activating the ATR gene, which is found in white blood cells and all human cells.
The ATR gene's normal job is to detect genetic damage to cells caused by radiation, toxic chemicals and chemotherapy, and to stop the damaged cells from replicating until they can repair themselves. Planelles and researchers at the Ï㽶ÊÓƵ of Rochester, N.Y., found evidence that the vpr gene—one of nine genes in the AIDS virus—exploits this normal repair process to stop vital white blood cells from replicating, thus disabling the immune system.
The findings were published last month in The Journal of Biological Chemistry.
The study raises the possibility of treating AIDS-related immune-system damage with medicines that prevent the human ATR gene from being activated by HIV's vpr gene.
"We would like to find a method or a substance that would allow us to interfere with the ability of HIV to kill the white blood cells using this mechanism," Planelles says.
It may take five to 10 years to develop medicines to interfere with the human ATR gene, he says, but they theoretically could offer a big advantage over existing AIDS drugs, which attack HIV but can lose effectiveness when the virus mutates to resist the drugs.
The research also reinforces Planelles' 1999 discovery that the AIDS virus' vpr gene can kill cancer cells in culture. That raises the prospect of developing a drug that mimicks the vpr gene's ability to activate the ATR gene, thus stopping the replication of cancer cells. He says it will take at least five years to find such a drug.
Many existing chemotherapy drugs damage DNA in cancer cells. The ATR gene senses the damage and stops division of the cancer cells, in effect stopping the cancer.
In the new study, Planelles and colleagues did not actually show how the AIDS virus' vpr gene triggers ATR to halt the replication of CD4+ white blood cells, but instead showed how vpr triggers ATR in human cervical cancer cells known as HELA cells, stopping replication of the cancer cells. The cancer cells are easier to use in the laboratory, and vpr works the same in all types of cells, whether white blood cells or cancer cells, Planelles says.
He plans to replicate the study using white blood cells instead of the cancer cells.
Planelles conducted the research with Mikhail Roshal, a medical student at the Ï㽶ÊÓƵ of Rochester; Baek Kim, a Rochester biochemist; Yonghong Zhu, a former Rochester graduate student now at DNAX Research, Inc. in California; and Paul Nghiem, a Harvard Ï㽶ÊÓƵ postdoctoral researcher.
Studying HIV's 'Lethal Weapon'
The vpr gene has been known for years, but in 1995 Planelles discovered that its role was to act as HIV's "lethal weapon" by preventing CD4+ white blood cells from dividing and replicating, thus leaving AIDS patients with a crippled immune system.
Depletion of CD4+ white blood cells is the hallmark of lost immunity in AIDS patients. The number of CD4+ white blood cells in healthy people is about 1,000 cells per microliter of blood, but drops below 200 cells per microliter in untreated AIDS patients and climbs to 500 to 1,000 cells per microliter in AIDS patients receiving antiviral treatment.
"Historically, it was clear that HIV killed white blood cells, but it took a while to figure out how," Planelles says. "We don't know it completely yet. But by 1995, we figured out that the vpr gene was major factor in killing the cells. In fact, we can use vpr alone in the absence of other HIV DNA [genetic material] or proteins to kill white blood cells. We discovered the way vpr disrupts the life of the cell is by first preventing it from dividing or reproducing and then inducing it to die. It's a two-hit mechanism."
The new study showed how vpr triggers the human ATR gene, setting off a "cascade" or "pathway" in which other genes and the proteins they help produce work in a chain reaction to stop white blood cells from dividing.
Planelles worked at the Ï㽶ÊÓƵ of Rochester before moving to Utah in 2002. The Ï㽶ÊÓƵ of Rochester has a patent pending on Planelles' discovery that the AIDS vpr gene activates the human ATR gene, which was discovered in 1996 at Harvard Ï㽶ÊÓƵ.
Demonstrating the ATR Gene's Role
Determining the ATR gene's role was difficult. A common method of learning what a gene does is to breed a "knockout mouse" in which the gene has been disabled. By seeing what goes wrong with the mouse, scientists can determine the gene's normal function. But the method could not be used for ATR because the gene is essential for cell division and development of an organism. If it is knocked out, the organism dies.
So Planelles and colleagues used other methods to block the ATR gene and render it unable to help the AIDS virus vpr gene stop cells from replicating:
- They used a method called "gene silencing" to inhibit the ATR gene.
- They put an inactive mutant version of the ATR gene into the cancer cells, inactivating the ATR gene in those cells.
- They applied drugs to the ATR gene. One was a substance known as LY294002. The other was caffeine, in extremely high doses.
In all three cases, interfering with the ATR gene left it unable to help the AIDS virus' vpr gene. As a result, the cells in the experiment were able to replicate. That demonstrated that vpr activates ATR to block cell replication in cells targeted by the AIDS virus.
Caffeine was used in the study because earlier research found that large concentrations of caffeine blocked the ability of chemotherapy drugs to prevent the division of cancer cells grown in the laboratory, and instead let the cancer cells replicate. Despite the new experiment, Planelles says caffeine is unlikely to be used as an anti-AIDS drug because the necessary doses - equivalent to 140 to 280 cups of coffee per day - would damage patients' nervous system.
To convert the findings into a clinically and commercially useful AIDS treatment, scientists must find a drug that interferes with the ATR gene only in white blood cells and without causing serious side effects, he adds.
Details of the Pathway to Doom
Here is how Planelles outlines the "pathway" by which the AIDS virus vpr gene stops cells from replicating. He describes this chain of events "like a relay race where you pass the baton," except in this case, the proteins produced by genes pass the signal that eventually stops replication of cells:
- The vpr gene activates the ATR gene. Exactly how that happens is not yet known.
- The ATR gene activates a protein named Chk1.
- Chk1 inactivates another protein named Cdc25C.
- Cdc25C inactivates a protein named Cdc2.
- Without active Cdc2, cells that already have duplicated their genetic material are unable to split into two cells.