By measuring DNA produced by a related reverse transcriptase in yeast, the Hopkins team discovered that higher than normal levels of manganese, caused by a defective gene, dramatically lowered the enzyme's activity. The scientists then proved that HIV's reverse transcriptase responds to manganese in the same way.
Hopkins graduate student Eric Bolton determined that the defective gene is PMR1, whose protein carries both manganese and calcium out of cells. Using special yeast developed by others at Hopkins, he discovered that manganese stops reverse transcriptase, the team reports in the April 26 issue of Molecular Cell.
"These results really point to a never-before-proposed way to try to stop HIV in its tracks -- that simply manipulating concentrations of a metal, manganese, can have a profound effect on reverse transcriptase," says Jef Boeke, Ph.D., professor of molecular biology and genetics at the school's Institute for Basic Biomedical Sciences. "We expect the human equivalent of PMR1 could be a good target for developing new drugs against HIV."
Retroviruses like HIV use reverse transcriptase to make copies of their DNA from RNA, the opposite of how genetic information is usually processed in cells. Each retrovirus has a distinct version of the enzyme, identical in function but different in form and sequence, says Boeke, also a professor of oncology.
The scientists found that each reverse transcriptase they studied has at least two places where manganese and the similar metal magnesium can "dock." Having these spots filled with the right metal is crucial for the enzyme's activity -- its ability to read a particular set of RNA, the scientists learned. When the metals' balance is out of whack, the enzyme doesn't work properly, they report.
"Most reverse transcriptases we studied prefer to bind magnesium. At the very least they were more active when magnesium was bound to them," says Boeke. "But a little extra manganese changes the activity of the enzyme."
Normally, charged magnesium ions outnumber those of manganese by the thousands inside cells. Having just three times more manganese than normal can cut the activity of HIV's reverse transcriptase in half, the scientists report, even though there's still much more magnesium.
HIV's ability to adapt and overcome drugs means that current treatments like AZT, which target reverse transcriptase directly, generally stop working over time. Using a combination of drugs helps block the virus on many fronts, but finding new drugs or a new class of drugs is needed to help keep the virus at bay. The new work suggests that targeting a cell's manganese transporter could be an effective way to stop HIV from replicating, without targeting HIV's reverse transcriptase directly.
"We've been working under the idea that studying reverse transcriptase in yeast may help improve understanding of retroviruses and lead to new ways to deal with HIV," says Boeke. "By studying yeast genetics we made an important discovery about how HIV works and have identified a target for a new class of anti-retroviral drug. It was completely unexpected, but very satisfying."
The yeast that were missing PMR1 appeared fine, suggesting that targeting the manganese transporter in humans may be relatively safe, the scientists suggest. It's not known whether targeting manganese levels will have a therapeutic benefit, but the mantra of HIV treatment is to reduce the number of copies of the virus.
The studies were funded by the National Institutes of Health. Albert Mildvan, M.D., professor of biological chemistry, is also an author of the report.
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